Research ArticleOverexpression of a Vesicle Trafficking Gene OsRab7 EnhancesSalt Tolerance in Rice

Xiaojue Peng1 Xia Ding1 Tianfang Chang1 Zhoulong Wang1 Rong Liu1 Xin Zeng1

Yaohui Cai2 and Youlin Zhu1

1 Key Laboratory ofMolecular Biology andGene Engineering of Jiangxi Province College of life science NanchangUniversity Nanchang330031 China

2 Jiangxi Super-rice Research and Development Center Nanchang 330200 China

Correspondence should be addressed to Xiaojue Peng xiaojuepenggmailcom

Received 3 November 2013 Accepted 24 December 2013 Published 12 February 2014

Academic Editors L R Ceci and V Niknam

Copyright copy 2014 Xiaojue Peng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

High soils salinity is amain factor affecting agricultural production Studying the function of salt-tolerance-related genes is essentialto enhance crop tolerance to stress Rab7 is a small GTP-binding protein that is distributed widely among eukaryotes Endocytictrafficking mediated by Rab7 plays an important role in animal and yeast cells but the current understanding of Rab7 in plants isstill very limited Herein we isolated a vesicle trafficking geneOsRab7 from rice Transgenic rice over-expressingOsRab7 exhibitedenhanced seedling growth and increased proline content under salt-treated conditions Moreover an increased number of vesicleswas observed in the root tip ofOsRab7 transgenic riceTheOsRab7 over-expression plants showed enhanced tolerance to salt stresssuggesting that vacuolar trafficking is important for salt tolerance in plants

1 Introduction

High soil salinity is a major environmental stress inhibitingplant growth and development and it results in severe yieldloss in agricultural crops Plants exhibit various responses toincrease their capability to resist salt stress Recent studiesdemonstrated that proteins involved in intracellular mem-brane trafficking in the secretory and endocytic pathwaysplay an important role in plant defense against pathogens andstress adaptation [1]

Vesicle trafficking has been traditionally viewed as aconstitutive housekeeping process but recent reports havedescribed the involvement of intracellular vesicle traffickingin the resistance to environmental stresses In E coli muta-tions that cause increased vesiculation enhanced bacterialsurvival upon challenge with stressing agents or the accumu-lation of toxicmisfolded proteins [2] In yeast vesicle traffick-ing between the cytosoll and plasmamembranewas inhibitedby oxidative stress [3] In Arabidopsis thaliana the transgenicplants in which the expression of an autophagy-related gene

AtATG18awas knocked down were more sensitive to salt andosmotic stress [4]

The Rab family is conserved from yeast to animals andhas been implicated in intracellular vesicle trafficking andmembrane organization [5] In Arabidopsis it is reportedthat the Rab7-related protein appears to localize to thevacuolar membrane and regulate the vesicle fusion with thevacuole [6] In soybean however the presence of Rab7-related protein was found on both endosomes and tonoplastsindicating that Rab7 multivesicular bodies are participatedin endocytosis pathway In addition many plant Rab7 geneswere shown to be affected by environmental stressors andtransgenic experiments demonstrated that the overexpres-sion of theAtRab7 andPgRab7 could increase stress tolerancein transgenic Arabidopsis thaliana and transgenic tobaccorespectively [6 7]

OsRab7 transcripts were found to accumulated slightlyunder salt stress in rice [8] but direct evidence showingthat the overexpression of OsRab7 can increase tolerance tosalt stress has not been reported In this paper we prepared

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 483526 7 pageshttpdxdoiorg1011552014483526

2 The Scientific World Journal

transgenic rice plants that overexpressed OsRab7 and testedtheir tolerance to salt stress The transgenic plants exhibitedenhanced tolerance to salt stress and increased numerousdistributed vesicles in the root tips

2 Materials and Methods

21 PlantMaterials andGrowthConditions The japonica ricevariety Zhonghua 11 was used in this study T

0 T1 and T

2

progeny transgenic plants were grown at 25∘C under 16 h ofdaylight in a greenhouse For the physiological analysis theplants were grown under a controlled temperature of 23∘C at70 relative humidity

22 Gene Cloning and Rice Transformation Total RNA wasisolated from rice seedlings with Trizol reagent (Invitro-gen) Reverse transcription was performed using MMLVReverse Transcriptase (Invitrogen) according to the man-ufacturerrsquos directions To obtain the full length sequenceof OsRab7 a pair of primers was designed accordingto the OsRab7 cDNA sequence The primer sequencewas forward 51015840-ATGGCTTCGCGCCGC-31015840 Reverse 51015840-CTAGCAGCAGCCTGATGATCTTG-31015840TheOsRab7 cDNAwas digested with BamHI and ligated to the modified binaryvector plasmid pCU [9] The resulting constructs weredesignated pOsRab7 The japonica rice variety zhonghua 11was transformed using Agrobacterium tumefaciens EHA-105following the published procedures published by Hiei et al[10]

23 Semiquantitative RT-PCR and Real-Time RT-PCRAnalysis Total RNAwas isolated and cDNAwas synthesizedfrom transgenic and wild-type rice as described aboveThe actin gene was used as an internal control for RT-PCR analysis General PCR was performed using thefollowing program an initial denaturation at 94∘C for3min followed by 25 cycles of 94∘C for 45 s 60∘C for45 s and 72∘C for 1min and a final extension at 72∘C for10min The RT-PCR experiments were repeated three timesand the PCR products were detected in a 1 agarose gelwith 1 times TAE and EtBr Real-time PCR was performedwith a Rotor-Gene 2000 real-time thermal cycling system(Corbett Research) using the SYBR Green Real-time PCRMaster Mix (TOYOBO) The reaction for OsRab7 usingspecific primers (sense 51015840-AGCCGTGTGGTCTCTGAG-31015840 antisense 51015840-AATGGGTCCTTGAGAGTCAC-31015840) wasperformed at 95∘C for 10 s followed by 40 cycles of 95∘Cfor 10 s 55∘C for 10 s and 72∘C for 10 s The reaction foractin as internal standard using specific primers (sense51015840-AGCATGAAGATCAAGGTGGTC-31015840 antisense 51015840-GCCTTGGCAATCCACATC-31015840) was performed in thesame as that for OsRab7

24 Preparation of Total Protein Extract From Rice Totalprotein was extracted from transgenic seedling andwild-typeseedlingsThe tissueswere ground into a fine powder in liquidnitrogen and then 400ndash500mg of tissue was extracted in1mL of extraction buffer (66mM Tris-Cl pH 68 2 SDS

2 vv 2-mercaptoethanol) at room temperature followed bycentrifugation for 20min at 40000timesg to remove the insolublefractionThe proteins were precipitated atminus20∘C for 1 h by theaddition of 1mL of an 8 1 mixture of ice-cold acetone andtrichloroacetic acid with 01 vv 2-mercaptoethanol Thesupernatantwas discarded after centrifugation at 18000timesg for15min at 4∘C and the pellet was washed in 1mL of ice-coldacetoneThe remaining acetone was removed by air drying atroom temperatureThe resulting pellet was redissolved in theprotein extraction solution

25 Western Blotting Analysis A polyclonal antibody againstOsRab7 was obtained by the injection of a purified OsRab7peptide into rabbits Equal amounts of protein from indepen-dent transgenic rice and wild-type rice plants were separatedon a 12 SDS-PAGE gel at 4∘C and then transferred ontoa PVDF transfer membrane at 80V for 2 h The membranewas incubated in 5 wv and nonfat milk 005 vv Tween-20 in phosphate buffered saline (PBS) for 1 h washed threetimes in PBST (PBS 005 Tween-20) for 10min eachand incubated in a 1 1000 dilution of rabbit antiserumovernight at 4∘CAfter four washes with PBST themembranewas incubated with goat anti-rabbit IgG conjugated withalkaline phosphatase (AP) in PBST solution for 2 h Afterfour 10min washes in PBST the signal was visualized bychemiluminescent detection (Pierce Rockford IL) accordingto the manufacturerrsquos protocol

26 Evaluation of Transgenic Rice Lines during Salt Stress T2

generation seeds of OsRab7 homozygous plants were used toassess the relative salinity tolerance Six-day-old transgenicand wild-type seedlings grown in 12 MS solid medium weretransferred and placed with roots pointing downward intoliquid medium supplemented with 200mMNaCl for 10 daysto induce salt stress The Data from 10 plants each of thewild-type and T1 transgenic lines were collected for eachexperiment and the mean values and standard deviationswere calculated and compared in phenotypic changes thetreated plants were carefully investigated and photographedEach treatment was repeated three times

27 Determination of Proline Content To test the defenseresponse triggered by OsRab7 overexpression in transgenicrice T

2homozygous plants (trans3 trans5) and wild-type

plants were assayed for their proline content Tow-week-old seedlings of both wild-type and transgenic rice weresalt-treated (250mM) for 12 h The determination of thefree proline content was performed according to Bates etal [11] The absorbance of chromophore containing toluenewas recorded at 520 nm in a Shimadzu UV-1700 spectropho-tomerter (Columbia USA) L-proline (Sigma USA)was usedfor the preparation of a standard curve

28 Observation of Vesicle Fluorescence Root tips from T2

homozygous plants and wild-type plant were respectivelyincubated with 5120583gmL FM4-64 (Sigma) for 12min on iceAfter being washed twice the root tips were examined

The Scientific World Journal 3

Figure 1 Expression of the OsRab7 gene in transgenic rice plants (a) Real-time quantitative PCR analysis in transgenic plants Total RNAwas extracted from transgenic plant and wild-type plants Actin was used as an internal control (b) OsRab7 expression in transgenic plantswas determined by semiquantitative RT-PCR analysis (c) a SDS-PAGE showed the protein content in the samples Equal concentrations ofprotein extracts from the rice samples were separated by SDS-PAGE (d) Western blot analysis of transgenic rice plants All lanes were runwith equal amounts of the protein extracts from the rice samples

with a scanning confocal microscope (Olympus FV 1000)Excitation was 514 nm for the visualization of FM4-64

3 Results

31 Molecular Characterization of Transgenic Rice LinesTo study the function of the OsRab7 gene in plants thegene was cloned downstream of a constitutive ubiquitinpromoter and introduced into rice plants via Agrobacteriumtumefaciens-mediated transformation Overall 25 indepen-dent events were obtained and 23 transgenic events wereconfirmed by PCR firstly (Supplementary Material availableonline at httpdxdoiorg1011552014483526) Analysis ofthe OsRab7 gene expression in the T

2progeny transgenic

rice by semiquantitative RT-PCR and real-time RT-PCRshowed that two homozygous lines (designated Trans3 andTrans5) exhibited a high level of transgene overexpressionAs shown in Figure 1(a) the transcript levels of OsRab7increased to 42- and 78-fold respectively compared withthe wild-type Semiquantitative RT-PCR result also showedthat the expression ofOsRab7 increased in Trans3 and Trans5(Figure 1(b)) Moreover expression of the transgene at theprotein level was assayed by western blot Equal amounts of

protein from transgenic and wild-type plant were separatedby SDS-PAGE gel (Figure 1(c)) An increased amount ofOsRab7 protein was obviously viable in Trans3 and Trans5compared with the wild-type plants (Figure 1(d))

32 Evaluation of OsRab7 Transgenic Rice Plants in SaltStress To analyze the impact of constitutive high OsRab7expression on salt stress tolerance the wild-type and thetransgenic plants (Tans3 and Tran5) were subjected to saltstress by irrigation with 200mM NaCl The treatment inhib-ited growth in both the wild-type and transgenic plantsbut the transgenic plants were much less sensitive to thetreatment compared with the wild-type plants (Figure 2(a))The seedling growth of the transgenic plant andwild-typewasinvestigated inmore detailThe seedling length of Trans5 wason average 018 cm 022 cm and 081 cm longer than that ofwild-type seedlings growing at 4 7 and 10 days respectivelyin 200mM of NaCl We also measured the seedling lengthof Trans3 at these stages and obtained the same results thatthe growth of OsRab7-overexpression transgenic rice wasrapid compared with that of wild-type rice under salt stress(Figure 2(b)) Furthermore the development of the lateralroot in wild-type rice was delayed compared with that in

4 The Scientific World Journal

(a) (b)

(c) (d)

(e)

Figure 2 Salt stress tolerance in wild-type and transgenic plants (a) Morphology of wild-type and transgenic seedlings after 12 days under200mM salt stress (b) Growth rate compared between the transgenic rice and WT plants under salt stress (c) Root morphology undersalt stress (d) Analysis of the number of lateral roots at 4 7 and 10 days The data represents the mean plusmn SD derived from three separateexperiments The bars represent the average data collected from 10 plants of each of the WT Trans3 and Trans5 lines (e) Morphology ofwild-type and transgenic seedlings transplanted from salt liquid medium to soil The wild-type and transgenic seedlings subjected to saltstress for 12 days were transplanted to soil and the transgenic seedling growth was rescued after 8 days

The Scientific World Journal 5

(a) (b)

Figure 3 Overexpression of OsRab7 in rice increased the proline level under salt stress (a) Level of proline in transgenic rice and WT riceunder normal conditions and salt treatment (b) Increasing rate of proline production after salt stress between the transgenic and WT riceThese data are the averages of triplicate assays and the error bars indicate the SDs

(a) (b)

Figure 4 Vesicles in the transgenic plant and WT rice (a) Vesicle morphology in the wild-type rice root tip (b) Vesicle morphology in thetransgenic plant root tipThe root tips were incubated in 5 120583gmL FM4-64 (Sigma) for 12min and images of the vesicles were captured usingconfocal microscopy Bar 20120583m

the transgenic plants under the salt stress (Figure 2(c)) Thetotal number of lateral roots in the transgenic seedlingswas higher than that of the wild-type seedling at 4 7 and10 days under salt stress However the number of lateralroots per centimeter was almost equal to that of the wild-type (Figure 4(d)) Almost all of the wild-type plants died12 days after the beginning of the treatment with 200mMNaCl whereas many of the transformed plants still had greenleaves (Figure 4(a)) To address the question of whether theseedling growth would resume after removing the salt stressthe wild-type and transgenic seedlings subjected to salt stressfor 12 days were transplanted to soil As shown in Figure 2(e)transgenic seedling growth was rescued after 8 days but thewild-type seedling growth was not recovered at the sametime (Figure 2(e)) Actually transgenic rice plants producedseeds normally 4 weeks later whereas the wild-type almost

died (data not shown) These findings demonstrated that theoverexpression of OsRab7 enhanced salt tolerance in rice

33 The Amount of Proline Increased in OsRab7 Trans-genic Rice Plant in Salt Stress Proline a widely distributedosmolyte plays an important role in enhancing abiotic stresstolerance To investigate the physiological changes in thetransgenic plants under salt stress 2-week-old seedlings weretreated with 200mM NaCl for 24 h and then used to deter-mine the free proline content Under normal conditions andsalt-treated conditions increased proline levels were found inboth transgenic and wild-type plants and the proline contentin the transgenic rice being slightly lower than that in thewild-type (Figure 3(a)) However upon salt treatment thetransgenic seedlings exhibited a 35-fold increased proline

6 The Scientific World Journal

content level compared with those under normal condi-tions whereas the proline content in the wild-type plantsonly increased 15-fold after the salt treatment (Figure 3(b))Therefore the results indicated that the overexpression ofOsRab7 increased the proline content under salt-treatedconditions which helped transgenic seedlings to better adaptto the salt stress

34TheVesicles Increased inOsRab7 Transgenic Rice Plant Inplants Rab7 plays an important role in the vesicle traffickingprocess and thus the vesicle distribution in transgenic ricewas investigated using the vesicle fluorescent dye FM4-64As shown in Figure 4 a strong aggregated fluorescenceintensity of FM4-64 was observed in the transgenic rice roottips whereas a much weaker diffuse fluorescence intensitywas detected in the wild-type root tip This result sug-gested the acceleration of vesicles trafficking in the OsRab7transgenic rice

4 Discussion

Thephysiological response of plants to salt stress is a complexprocess requiring the coordinated function of many genesAlthoughOsRab7 transcript levels were to be found increasedunder salt stress direct evidence for the association of thisgene with salt tolerance was lacking In this paper ourexperiments showed that the overexpression ofOsRab7 couldconfer salinity tolerance in transgenic rice

Vacuoles play a critical role in salt tolerance by accu-mulating sodium which is then removed from the cytosolwhere it is toxic [12 13] A connection between salt sensitivityand vacuolar trafficking has been well established in animalcells and yeast Post-Golgi organelles involved in vacuolartrafficking were shown to be targets of salt stress resulting ininefficient vacuolar trafficking in yeast and mammalian cells[14ndash16] In plants Leshem et al reported that the endocyticpathway to the vacuole was also important for salt tolerancein Arabidopsis [17 18] The Rab7 proteins are importantcomponents of the vesicle trafficking system in all eukaryotes[5] In animals and yeast the Rab7 protein localizes to lateendosomes and to lysosomesvacuoles [19ndash22] In plantsthe Rab7 proteins have been localized to the tonoplast inbothArabidopsis and rice [8 23] Moreover enhanced vesicletrafficking and endocytic trafficking were also observed inRab7 overexpression transgenic soybean and in animal cellscontaining constitutive active forms of Rab7 [24 25] respec-tively In our studies an increased number of distributed vesi-cles was observed in OsRab7 overexpression transgenic ricesuggesting that the overexpression of OsRab7 may activatethe vesicle trafficking in rice Together with the results of salttolerance displayed by the OsRab7 overexpression transgenicrice we infer that the overexpression of OsRab7 increasesplant salt tolerance via enhanced vesicle trafficking in riceThus our results provide additional evidence that vacuolartrafficking is important for salt tolerance

5 Conclusion

In summary we prepared transgenic rice plants that over-expressed OsRab7 and tested their tolerance to salt stressThe transgenic plants exhibited enhanced tolerance to saltstress and increased numerous distributed vesicles in the roottips These results indicate that the overexpression ofOsRab7increased plant salt tolerance via enhanced vesicle traffickingin rice

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

X Peng and X Ding have contributed equally in this work

Acknowledgments

This work was supported by the Natural Science Foundationof China (nos 31160270 31160019) the YouthNatural ScienceFoundation of Jiangxi (No 20114BAB214009) the Project ofJiangxi Provincial Department of Education (no GJJ13102)and the earmarked fund for Modern Agro-industry Technol-ogy Research System (no CARS-01-04)

References

[1] C J Carter S Y Bednarek and N V Raikhel ldquoMembranetrafficking in plants new discoveries and approachesrdquo CurrentOpinion in Plant Biology vol 7 no 6 pp 701ndash707 2004

[2] A J McBroom and M J Kuehn ldquoRelease of outer membranevesicles by Gram-negative bacteria is a novel envelope stressresponserdquo Molecular Microbiology vol 63 no 2 pp 545ndash5582007

[3] A Levine B Belenghi H Damari-Weisler and D GranotldquoVesicle-associated membrane protein of arabidopsis sup-presses bax-induced apoptosis in yeast downstream of oxidativeburstrdquo Journal of Biological Chemistry vol 276 no 49 pp46284ndash46289 2001

[4] Y Liu Y Xiong and D C Bassham ldquoAutophagy is required fortolerance of drought and salt stress in plantsrdquoAutophagy vol 5no 7 pp 954ndash963 2009

[5] M Zerial and H McBride ldquoRab proteins as membrane orga-nizersrdquo Nature Reviews Molecular Cell Biology vol 2 no 2 pp107ndash117 2001

[6] A Mazel Y Leshem B S Tiwari and A Levine ldquoInductionof salt and osmotic stress tolerance by overexpression of anintracellular vesicle trafficking protein AtRab7 (AtRabG3e)rdquoPlant Physiology vol 134 no 1 pp 118ndash128 2004

[7] P K Agarwal P Agarwal P Jain B Jha M K Reddy and SK Sopory ldquoConstitutive overexpression of a stress-induciblesmall GTP-binding protein PgRab7 from Pennisetum glaucumenhances abiotic stress tolerance in transgenic tobaccordquo PlantCell Reports vol 27 no 1 pp 105ndash115 2008

[8] M Y Nahm S W Kim D Yun S Y Lee M J Cho and J DBahk ldquoMolecular and biochemical analyses of OsRab7 a riceRab7 homologrdquo Plant and Cell Physiology vol 44 no 12 pp1341ndash1349 2003

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

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Microbiology

2 The Scientific World Journal

transgenic rice plants that overexpressed OsRab7 and testedtheir tolerance to salt stress The transgenic plants exhibitedenhanced tolerance to salt stress and increased numerousdistributed vesicles in the root tips

2 Materials and Methods

21 PlantMaterials andGrowthConditions The japonica ricevariety Zhonghua 11 was used in this study T

0 T1 and T

2

progeny transgenic plants were grown at 25∘C under 16 h ofdaylight in a greenhouse For the physiological analysis theplants were grown under a controlled temperature of 23∘C at70 relative humidity

22 Gene Cloning and Rice Transformation Total RNA wasisolated from rice seedlings with Trizol reagent (Invitro-gen) Reverse transcription was performed using MMLVReverse Transcriptase (Invitrogen) according to the man-ufacturerrsquos directions To obtain the full length sequenceof OsRab7 a pair of primers was designed accordingto the OsRab7 cDNA sequence The primer sequencewas forward 51015840-ATGGCTTCGCGCCGC-31015840 Reverse 51015840-CTAGCAGCAGCCTGATGATCTTG-31015840TheOsRab7 cDNAwas digested with BamHI and ligated to the modified binaryvector plasmid pCU [9] The resulting constructs weredesignated pOsRab7 The japonica rice variety zhonghua 11was transformed using Agrobacterium tumefaciens EHA-105following the published procedures published by Hiei et al[10]

23 Semiquantitative RT-PCR and Real-Time RT-PCRAnalysis Total RNAwas isolated and cDNAwas synthesizedfrom transgenic and wild-type rice as described aboveThe actin gene was used as an internal control for RT-PCR analysis General PCR was performed using thefollowing program an initial denaturation at 94∘C for3min followed by 25 cycles of 94∘C for 45 s 60∘C for45 s and 72∘C for 1min and a final extension at 72∘C for10min The RT-PCR experiments were repeated three timesand the PCR products were detected in a 1 agarose gelwith 1 times TAE and EtBr Real-time PCR was performedwith a Rotor-Gene 2000 real-time thermal cycling system(Corbett Research) using the SYBR Green Real-time PCRMaster Mix (TOYOBO) The reaction for OsRab7 usingspecific primers (sense 51015840-AGCCGTGTGGTCTCTGAG-31015840 antisense 51015840-AATGGGTCCTTGAGAGTCAC-31015840) wasperformed at 95∘C for 10 s followed by 40 cycles of 95∘Cfor 10 s 55∘C for 10 s and 72∘C for 10 s The reaction foractin as internal standard using specific primers (sense51015840-AGCATGAAGATCAAGGTGGTC-31015840 antisense 51015840-GCCTTGGCAATCCACATC-31015840) was performed in thesame as that for OsRab7

24 Preparation of Total Protein Extract From Rice Totalprotein was extracted from transgenic seedling andwild-typeseedlingsThe tissueswere ground into a fine powder in liquidnitrogen and then 400ndash500mg of tissue was extracted in1mL of extraction buffer (66mM Tris-Cl pH 68 2 SDS

2 vv 2-mercaptoethanol) at room temperature followed bycentrifugation for 20min at 40000timesg to remove the insolublefractionThe proteins were precipitated atminus20∘C for 1 h by theaddition of 1mL of an 8 1 mixture of ice-cold acetone andtrichloroacetic acid with 01 vv 2-mercaptoethanol Thesupernatantwas discarded after centrifugation at 18000timesg for15min at 4∘C and the pellet was washed in 1mL of ice-coldacetoneThe remaining acetone was removed by air drying atroom temperatureThe resulting pellet was redissolved in theprotein extraction solution

25 Western Blotting Analysis A polyclonal antibody againstOsRab7 was obtained by the injection of a purified OsRab7peptide into rabbits Equal amounts of protein from indepen-dent transgenic rice and wild-type rice plants were separatedon a 12 SDS-PAGE gel at 4∘C and then transferred ontoa PVDF transfer membrane at 80V for 2 h The membranewas incubated in 5 wv and nonfat milk 005 vv Tween-20 in phosphate buffered saline (PBS) for 1 h washed threetimes in PBST (PBS 005 Tween-20) for 10min eachand incubated in a 1 1000 dilution of rabbit antiserumovernight at 4∘CAfter four washes with PBST themembranewas incubated with goat anti-rabbit IgG conjugated withalkaline phosphatase (AP) in PBST solution for 2 h Afterfour 10min washes in PBST the signal was visualized bychemiluminescent detection (Pierce Rockford IL) accordingto the manufacturerrsquos protocol

26 Evaluation of Transgenic Rice Lines during Salt Stress T2

generation seeds of OsRab7 homozygous plants were used toassess the relative salinity tolerance Six-day-old transgenicand wild-type seedlings grown in 12 MS solid medium weretransferred and placed with roots pointing downward intoliquid medium supplemented with 200mMNaCl for 10 daysto induce salt stress The Data from 10 plants each of thewild-type and T1 transgenic lines were collected for eachexperiment and the mean values and standard deviationswere calculated and compared in phenotypic changes thetreated plants were carefully investigated and photographedEach treatment was repeated three times

27 Determination of Proline Content To test the defenseresponse triggered by OsRab7 overexpression in transgenicrice T

2homozygous plants (trans3 trans5) and wild-type

plants were assayed for their proline content Tow-week-old seedlings of both wild-type and transgenic rice weresalt-treated (250mM) for 12 h The determination of thefree proline content was performed according to Bates etal [11] The absorbance of chromophore containing toluenewas recorded at 520 nm in a Shimadzu UV-1700 spectropho-tomerter (Columbia USA) L-proline (Sigma USA)was usedfor the preparation of a standard curve

28 Observation of Vesicle Fluorescence Root tips from T2

homozygous plants and wild-type plant were respectivelyincubated with 5120583gmL FM4-64 (Sigma) for 12min on iceAfter being washed twice the root tips were examined

The Scientific World Journal 3

Figure 1 Expression of the OsRab7 gene in transgenic rice plants (a) Real-time quantitative PCR analysis in transgenic plants Total RNAwas extracted from transgenic plant and wild-type plants Actin was used as an internal control (b) OsRab7 expression in transgenic plantswas determined by semiquantitative RT-PCR analysis (c) a SDS-PAGE showed the protein content in the samples Equal concentrations ofprotein extracts from the rice samples were separated by SDS-PAGE (d) Western blot analysis of transgenic rice plants All lanes were runwith equal amounts of the protein extracts from the rice samples

with a scanning confocal microscope (Olympus FV 1000)Excitation was 514 nm for the visualization of FM4-64

3 Results

31 Molecular Characterization of Transgenic Rice LinesTo study the function of the OsRab7 gene in plants thegene was cloned downstream of a constitutive ubiquitinpromoter and introduced into rice plants via Agrobacteriumtumefaciens-mediated transformation Overall 25 indepen-dent events were obtained and 23 transgenic events wereconfirmed by PCR firstly (Supplementary Material availableonline at httpdxdoiorg1011552014483526) Analysis ofthe OsRab7 gene expression in the T

2progeny transgenic

rice by semiquantitative RT-PCR and real-time RT-PCRshowed that two homozygous lines (designated Trans3 andTrans5) exhibited a high level of transgene overexpressionAs shown in Figure 1(a) the transcript levels of OsRab7increased to 42- and 78-fold respectively compared withthe wild-type Semiquantitative RT-PCR result also showedthat the expression ofOsRab7 increased in Trans3 and Trans5(Figure 1(b)) Moreover expression of the transgene at theprotein level was assayed by western blot Equal amounts of

protein from transgenic and wild-type plant were separatedby SDS-PAGE gel (Figure 1(c)) An increased amount ofOsRab7 protein was obviously viable in Trans3 and Trans5compared with the wild-type plants (Figure 1(d))

32 Evaluation of OsRab7 Transgenic Rice Plants in SaltStress To analyze the impact of constitutive high OsRab7expression on salt stress tolerance the wild-type and thetransgenic plants (Tans3 and Tran5) were subjected to saltstress by irrigation with 200mM NaCl The treatment inhib-ited growth in both the wild-type and transgenic plantsbut the transgenic plants were much less sensitive to thetreatment compared with the wild-type plants (Figure 2(a))The seedling growth of the transgenic plant andwild-typewasinvestigated inmore detailThe seedling length of Trans5 wason average 018 cm 022 cm and 081 cm longer than that ofwild-type seedlings growing at 4 7 and 10 days respectivelyin 200mM of NaCl We also measured the seedling lengthof Trans3 at these stages and obtained the same results thatthe growth of OsRab7-overexpression transgenic rice wasrapid compared with that of wild-type rice under salt stress(Figure 2(b)) Furthermore the development of the lateralroot in wild-type rice was delayed compared with that in

4 The Scientific World Journal

(a) (b)

(c) (d)

(e)

Figure 2 Salt stress tolerance in wild-type and transgenic plants (a) Morphology of wild-type and transgenic seedlings after 12 days under200mM salt stress (b) Growth rate compared between the transgenic rice and WT plants under salt stress (c) Root morphology undersalt stress (d) Analysis of the number of lateral roots at 4 7 and 10 days The data represents the mean plusmn SD derived from three separateexperiments The bars represent the average data collected from 10 plants of each of the WT Trans3 and Trans5 lines (e) Morphology ofwild-type and transgenic seedlings transplanted from salt liquid medium to soil The wild-type and transgenic seedlings subjected to saltstress for 12 days were transplanted to soil and the transgenic seedling growth was rescued after 8 days

The Scientific World Journal 5

(a) (b)

Figure 3 Overexpression of OsRab7 in rice increased the proline level under salt stress (a) Level of proline in transgenic rice and WT riceunder normal conditions and salt treatment (b) Increasing rate of proline production after salt stress between the transgenic and WT riceThese data are the averages of triplicate assays and the error bars indicate the SDs

(a) (b)

Figure 4 Vesicles in the transgenic plant and WT rice (a) Vesicle morphology in the wild-type rice root tip (b) Vesicle morphology in thetransgenic plant root tipThe root tips were incubated in 5 120583gmL FM4-64 (Sigma) for 12min and images of the vesicles were captured usingconfocal microscopy Bar 20120583m

the transgenic plants under the salt stress (Figure 2(c)) Thetotal number of lateral roots in the transgenic seedlingswas higher than that of the wild-type seedling at 4 7 and10 days under salt stress However the number of lateralroots per centimeter was almost equal to that of the wild-type (Figure 4(d)) Almost all of the wild-type plants died12 days after the beginning of the treatment with 200mMNaCl whereas many of the transformed plants still had greenleaves (Figure 4(a)) To address the question of whether theseedling growth would resume after removing the salt stressthe wild-type and transgenic seedlings subjected to salt stressfor 12 days were transplanted to soil As shown in Figure 2(e)transgenic seedling growth was rescued after 8 days but thewild-type seedling growth was not recovered at the sametime (Figure 2(e)) Actually transgenic rice plants producedseeds normally 4 weeks later whereas the wild-type almost

died (data not shown) These findings demonstrated that theoverexpression of OsRab7 enhanced salt tolerance in rice

33 The Amount of Proline Increased in OsRab7 Trans-genic Rice Plant in Salt Stress Proline a widely distributedosmolyte plays an important role in enhancing abiotic stresstolerance To investigate the physiological changes in thetransgenic plants under salt stress 2-week-old seedlings weretreated with 200mM NaCl for 24 h and then used to deter-mine the free proline content Under normal conditions andsalt-treated conditions increased proline levels were found inboth transgenic and wild-type plants and the proline contentin the transgenic rice being slightly lower than that in thewild-type (Figure 3(a)) However upon salt treatment thetransgenic seedlings exhibited a 35-fold increased proline

6 The Scientific World Journal

content level compared with those under normal condi-tions whereas the proline content in the wild-type plantsonly increased 15-fold after the salt treatment (Figure 3(b))Therefore the results indicated that the overexpression ofOsRab7 increased the proline content under salt-treatedconditions which helped transgenic seedlings to better adaptto the salt stress

34TheVesicles Increased inOsRab7 Transgenic Rice Plant Inplants Rab7 plays an important role in the vesicle traffickingprocess and thus the vesicle distribution in transgenic ricewas investigated using the vesicle fluorescent dye FM4-64As shown in Figure 4 a strong aggregated fluorescenceintensity of FM4-64 was observed in the transgenic rice roottips whereas a much weaker diffuse fluorescence intensitywas detected in the wild-type root tip This result sug-gested the acceleration of vesicles trafficking in the OsRab7transgenic rice

4 Discussion

Thephysiological response of plants to salt stress is a complexprocess requiring the coordinated function of many genesAlthoughOsRab7 transcript levels were to be found increasedunder salt stress direct evidence for the association of thisgene with salt tolerance was lacking In this paper ourexperiments showed that the overexpression ofOsRab7 couldconfer salinity tolerance in transgenic rice

Vacuoles play a critical role in salt tolerance by accu-mulating sodium which is then removed from the cytosolwhere it is toxic [12 13] A connection between salt sensitivityand vacuolar trafficking has been well established in animalcells and yeast Post-Golgi organelles involved in vacuolartrafficking were shown to be targets of salt stress resulting ininefficient vacuolar trafficking in yeast and mammalian cells[14ndash16] In plants Leshem et al reported that the endocyticpathway to the vacuole was also important for salt tolerancein Arabidopsis [17 18] The Rab7 proteins are importantcomponents of the vesicle trafficking system in all eukaryotes[5] In animals and yeast the Rab7 protein localizes to lateendosomes and to lysosomesvacuoles [19ndash22] In plantsthe Rab7 proteins have been localized to the tonoplast inbothArabidopsis and rice [8 23] Moreover enhanced vesicletrafficking and endocytic trafficking were also observed inRab7 overexpression transgenic soybean and in animal cellscontaining constitutive active forms of Rab7 [24 25] respec-tively In our studies an increased number of distributed vesi-cles was observed in OsRab7 overexpression transgenic ricesuggesting that the overexpression of OsRab7 may activatethe vesicle trafficking in rice Together with the results of salttolerance displayed by the OsRab7 overexpression transgenicrice we infer that the overexpression of OsRab7 increasesplant salt tolerance via enhanced vesicle trafficking in riceThus our results provide additional evidence that vacuolartrafficking is important for salt tolerance

5 Conclusion

In summary we prepared transgenic rice plants that over-expressed OsRab7 and tested their tolerance to salt stressThe transgenic plants exhibited enhanced tolerance to saltstress and increased numerous distributed vesicles in the roottips These results indicate that the overexpression ofOsRab7increased plant salt tolerance via enhanced vesicle traffickingin rice

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

X Peng and X Ding have contributed equally in this work

Acknowledgments

This work was supported by the Natural Science Foundationof China (nos 31160270 31160019) the YouthNatural ScienceFoundation of Jiangxi (No 20114BAB214009) the Project ofJiangxi Provincial Department of Education (no GJJ13102)and the earmarked fund for Modern Agro-industry Technol-ogy Research System (no CARS-01-04)

References

[1] C J Carter S Y Bednarek and N V Raikhel ldquoMembranetrafficking in plants new discoveries and approachesrdquo CurrentOpinion in Plant Biology vol 7 no 6 pp 701ndash707 2004

[2] A J McBroom and M J Kuehn ldquoRelease of outer membranevesicles by Gram-negative bacteria is a novel envelope stressresponserdquo Molecular Microbiology vol 63 no 2 pp 545ndash5582007

[3] A Levine B Belenghi H Damari-Weisler and D GranotldquoVesicle-associated membrane protein of arabidopsis sup-presses bax-induced apoptosis in yeast downstream of oxidativeburstrdquo Journal of Biological Chemistry vol 276 no 49 pp46284ndash46289 2001

[4] Y Liu Y Xiong and D C Bassham ldquoAutophagy is required fortolerance of drought and salt stress in plantsrdquoAutophagy vol 5no 7 pp 954ndash963 2009

[5] M Zerial and H McBride ldquoRab proteins as membrane orga-nizersrdquo Nature Reviews Molecular Cell Biology vol 2 no 2 pp107ndash117 2001

[6] A Mazel Y Leshem B S Tiwari and A Levine ldquoInductionof salt and osmotic stress tolerance by overexpression of anintracellular vesicle trafficking protein AtRab7 (AtRabG3e)rdquoPlant Physiology vol 134 no 1 pp 118ndash128 2004

[7] P K Agarwal P Agarwal P Jain B Jha M K Reddy and SK Sopory ldquoConstitutive overexpression of a stress-induciblesmall GTP-binding protein PgRab7 from Pennisetum glaucumenhances abiotic stress tolerance in transgenic tobaccordquo PlantCell Reports vol 27 no 1 pp 105ndash115 2008

[8] M Y Nahm S W Kim D Yun S Y Lee M J Cho and J DBahk ldquoMolecular and biochemical analyses of OsRab7 a riceRab7 homologrdquo Plant and Cell Physiology vol 44 no 12 pp1341ndash1349 2003

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 3

Figure 1 Expression of the OsRab7 gene in transgenic rice plants (a) Real-time quantitative PCR analysis in transgenic plants Total RNAwas extracted from transgenic plant and wild-type plants Actin was used as an internal control (b) OsRab7 expression in transgenic plantswas determined by semiquantitative RT-PCR analysis (c) a SDS-PAGE showed the protein content in the samples Equal concentrations ofprotein extracts from the rice samples were separated by SDS-PAGE (d) Western blot analysis of transgenic rice plants All lanes were runwith equal amounts of the protein extracts from the rice samples

with a scanning confocal microscope (Olympus FV 1000)Excitation was 514 nm for the visualization of FM4-64

3 Results

31 Molecular Characterization of Transgenic Rice LinesTo study the function of the OsRab7 gene in plants thegene was cloned downstream of a constitutive ubiquitinpromoter and introduced into rice plants via Agrobacteriumtumefaciens-mediated transformation Overall 25 indepen-dent events were obtained and 23 transgenic events wereconfirmed by PCR firstly (Supplementary Material availableonline at httpdxdoiorg1011552014483526) Analysis ofthe OsRab7 gene expression in the T

2progeny transgenic

rice by semiquantitative RT-PCR and real-time RT-PCRshowed that two homozygous lines (designated Trans3 andTrans5) exhibited a high level of transgene overexpressionAs shown in Figure 1(a) the transcript levels of OsRab7increased to 42- and 78-fold respectively compared withthe wild-type Semiquantitative RT-PCR result also showedthat the expression ofOsRab7 increased in Trans3 and Trans5(Figure 1(b)) Moreover expression of the transgene at theprotein level was assayed by western blot Equal amounts of

protein from transgenic and wild-type plant were separatedby SDS-PAGE gel (Figure 1(c)) An increased amount ofOsRab7 protein was obviously viable in Trans3 and Trans5compared with the wild-type plants (Figure 1(d))

32 Evaluation of OsRab7 Transgenic Rice Plants in SaltStress To analyze the impact of constitutive high OsRab7expression on salt stress tolerance the wild-type and thetransgenic plants (Tans3 and Tran5) were subjected to saltstress by irrigation with 200mM NaCl The treatment inhib-ited growth in both the wild-type and transgenic plantsbut the transgenic plants were much less sensitive to thetreatment compared with the wild-type plants (Figure 2(a))The seedling growth of the transgenic plant andwild-typewasinvestigated inmore detailThe seedling length of Trans5 wason average 018 cm 022 cm and 081 cm longer than that ofwild-type seedlings growing at 4 7 and 10 days respectivelyin 200mM of NaCl We also measured the seedling lengthof Trans3 at these stages and obtained the same results thatthe growth of OsRab7-overexpression transgenic rice wasrapid compared with that of wild-type rice under salt stress(Figure 2(b)) Furthermore the development of the lateralroot in wild-type rice was delayed compared with that in

4 The Scientific World Journal

(a) (b)

(c) (d)

(e)

Figure 2 Salt stress tolerance in wild-type and transgenic plants (a) Morphology of wild-type and transgenic seedlings after 12 days under200mM salt stress (b) Growth rate compared between the transgenic rice and WT plants under salt stress (c) Root morphology undersalt stress (d) Analysis of the number of lateral roots at 4 7 and 10 days The data represents the mean plusmn SD derived from three separateexperiments The bars represent the average data collected from 10 plants of each of the WT Trans3 and Trans5 lines (e) Morphology ofwild-type and transgenic seedlings transplanted from salt liquid medium to soil The wild-type and transgenic seedlings subjected to saltstress for 12 days were transplanted to soil and the transgenic seedling growth was rescued after 8 days

The Scientific World Journal 5

(a) (b)

Figure 3 Overexpression of OsRab7 in rice increased the proline level under salt stress (a) Level of proline in transgenic rice and WT riceunder normal conditions and salt treatment (b) Increasing rate of proline production after salt stress between the transgenic and WT riceThese data are the averages of triplicate assays and the error bars indicate the SDs

(a) (b)

Figure 4 Vesicles in the transgenic plant and WT rice (a) Vesicle morphology in the wild-type rice root tip (b) Vesicle morphology in thetransgenic plant root tipThe root tips were incubated in 5 120583gmL FM4-64 (Sigma) for 12min and images of the vesicles were captured usingconfocal microscopy Bar 20120583m

the transgenic plants under the salt stress (Figure 2(c)) Thetotal number of lateral roots in the transgenic seedlingswas higher than that of the wild-type seedling at 4 7 and10 days under salt stress However the number of lateralroots per centimeter was almost equal to that of the wild-type (Figure 4(d)) Almost all of the wild-type plants died12 days after the beginning of the treatment with 200mMNaCl whereas many of the transformed plants still had greenleaves (Figure 4(a)) To address the question of whether theseedling growth would resume after removing the salt stressthe wild-type and transgenic seedlings subjected to salt stressfor 12 days were transplanted to soil As shown in Figure 2(e)transgenic seedling growth was rescued after 8 days but thewild-type seedling growth was not recovered at the sametime (Figure 2(e)) Actually transgenic rice plants producedseeds normally 4 weeks later whereas the wild-type almost

died (data not shown) These findings demonstrated that theoverexpression of OsRab7 enhanced salt tolerance in rice

33 The Amount of Proline Increased in OsRab7 Trans-genic Rice Plant in Salt Stress Proline a widely distributedosmolyte plays an important role in enhancing abiotic stresstolerance To investigate the physiological changes in thetransgenic plants under salt stress 2-week-old seedlings weretreated with 200mM NaCl for 24 h and then used to deter-mine the free proline content Under normal conditions andsalt-treated conditions increased proline levels were found inboth transgenic and wild-type plants and the proline contentin the transgenic rice being slightly lower than that in thewild-type (Figure 3(a)) However upon salt treatment thetransgenic seedlings exhibited a 35-fold increased proline

6 The Scientific World Journal

content level compared with those under normal condi-tions whereas the proline content in the wild-type plantsonly increased 15-fold after the salt treatment (Figure 3(b))Therefore the results indicated that the overexpression ofOsRab7 increased the proline content under salt-treatedconditions which helped transgenic seedlings to better adaptto the salt stress

34TheVesicles Increased inOsRab7 Transgenic Rice Plant Inplants Rab7 plays an important role in the vesicle traffickingprocess and thus the vesicle distribution in transgenic ricewas investigated using the vesicle fluorescent dye FM4-64As shown in Figure 4 a strong aggregated fluorescenceintensity of FM4-64 was observed in the transgenic rice roottips whereas a much weaker diffuse fluorescence intensitywas detected in the wild-type root tip This result sug-gested the acceleration of vesicles trafficking in the OsRab7transgenic rice

4 Discussion

Thephysiological response of plants to salt stress is a complexprocess requiring the coordinated function of many genesAlthoughOsRab7 transcript levels were to be found increasedunder salt stress direct evidence for the association of thisgene with salt tolerance was lacking In this paper ourexperiments showed that the overexpression ofOsRab7 couldconfer salinity tolerance in transgenic rice

Vacuoles play a critical role in salt tolerance by accu-mulating sodium which is then removed from the cytosolwhere it is toxic [12 13] A connection between salt sensitivityand vacuolar trafficking has been well established in animalcells and yeast Post-Golgi organelles involved in vacuolartrafficking were shown to be targets of salt stress resulting ininefficient vacuolar trafficking in yeast and mammalian cells[14ndash16] In plants Leshem et al reported that the endocyticpathway to the vacuole was also important for salt tolerancein Arabidopsis [17 18] The Rab7 proteins are importantcomponents of the vesicle trafficking system in all eukaryotes[5] In animals and yeast the Rab7 protein localizes to lateendosomes and to lysosomesvacuoles [19ndash22] In plantsthe Rab7 proteins have been localized to the tonoplast inbothArabidopsis and rice [8 23] Moreover enhanced vesicletrafficking and endocytic trafficking were also observed inRab7 overexpression transgenic soybean and in animal cellscontaining constitutive active forms of Rab7 [24 25] respec-tively In our studies an increased number of distributed vesi-cles was observed in OsRab7 overexpression transgenic ricesuggesting that the overexpression of OsRab7 may activatethe vesicle trafficking in rice Together with the results of salttolerance displayed by the OsRab7 overexpression transgenicrice we infer that the overexpression of OsRab7 increasesplant salt tolerance via enhanced vesicle trafficking in riceThus our results provide additional evidence that vacuolartrafficking is important for salt tolerance

5 Conclusion

In summary we prepared transgenic rice plants that over-expressed OsRab7 and tested their tolerance to salt stressThe transgenic plants exhibited enhanced tolerance to saltstress and increased numerous distributed vesicles in the roottips These results indicate that the overexpression ofOsRab7increased plant salt tolerance via enhanced vesicle traffickingin rice

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

X Peng and X Ding have contributed equally in this work

Acknowledgments

This work was supported by the Natural Science Foundationof China (nos 31160270 31160019) the YouthNatural ScienceFoundation of Jiangxi (No 20114BAB214009) the Project ofJiangxi Provincial Department of Education (no GJJ13102)and the earmarked fund for Modern Agro-industry Technol-ogy Research System (no CARS-01-04)

References

[1] C J Carter S Y Bednarek and N V Raikhel ldquoMembranetrafficking in plants new discoveries and approachesrdquo CurrentOpinion in Plant Biology vol 7 no 6 pp 701ndash707 2004

[2] A J McBroom and M J Kuehn ldquoRelease of outer membranevesicles by Gram-negative bacteria is a novel envelope stressresponserdquo Molecular Microbiology vol 63 no 2 pp 545ndash5582007

[3] A Levine B Belenghi H Damari-Weisler and D GranotldquoVesicle-associated membrane protein of arabidopsis sup-presses bax-induced apoptosis in yeast downstream of oxidativeburstrdquo Journal of Biological Chemistry vol 276 no 49 pp46284ndash46289 2001

[4] Y Liu Y Xiong and D C Bassham ldquoAutophagy is required fortolerance of drought and salt stress in plantsrdquoAutophagy vol 5no 7 pp 954ndash963 2009

[5] M Zerial and H McBride ldquoRab proteins as membrane orga-nizersrdquo Nature Reviews Molecular Cell Biology vol 2 no 2 pp107ndash117 2001

[6] A Mazel Y Leshem B S Tiwari and A Levine ldquoInductionof salt and osmotic stress tolerance by overexpression of anintracellular vesicle trafficking protein AtRab7 (AtRabG3e)rdquoPlant Physiology vol 134 no 1 pp 118ndash128 2004

[7] P K Agarwal P Agarwal P Jain B Jha M K Reddy and SK Sopory ldquoConstitutive overexpression of a stress-induciblesmall GTP-binding protein PgRab7 from Pennisetum glaucumenhances abiotic stress tolerance in transgenic tobaccordquo PlantCell Reports vol 27 no 1 pp 105ndash115 2008

[8] M Y Nahm S W Kim D Yun S Y Lee M J Cho and J DBahk ldquoMolecular and biochemical analyses of OsRab7 a riceRab7 homologrdquo Plant and Cell Physiology vol 44 no 12 pp1341ndash1349 2003

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 The Scientific World Journal

(a) (b)

(c) (d)

(e)

Figure 2 Salt stress tolerance in wild-type and transgenic plants (a) Morphology of wild-type and transgenic seedlings after 12 days under200mM salt stress (b) Growth rate compared between the transgenic rice and WT plants under salt stress (c) Root morphology undersalt stress (d) Analysis of the number of lateral roots at 4 7 and 10 days The data represents the mean plusmn SD derived from three separateexperiments The bars represent the average data collected from 10 plants of each of the WT Trans3 and Trans5 lines (e) Morphology ofwild-type and transgenic seedlings transplanted from salt liquid medium to soil The wild-type and transgenic seedlings subjected to saltstress for 12 days were transplanted to soil and the transgenic seedling growth was rescued after 8 days

The Scientific World Journal 5

(a) (b)

Figure 3 Overexpression of OsRab7 in rice increased the proline level under salt stress (a) Level of proline in transgenic rice and WT riceunder normal conditions and salt treatment (b) Increasing rate of proline production after salt stress between the transgenic and WT riceThese data are the averages of triplicate assays and the error bars indicate the SDs

(a) (b)

Figure 4 Vesicles in the transgenic plant and WT rice (a) Vesicle morphology in the wild-type rice root tip (b) Vesicle morphology in thetransgenic plant root tipThe root tips were incubated in 5 120583gmL FM4-64 (Sigma) for 12min and images of the vesicles were captured usingconfocal microscopy Bar 20120583m

the transgenic plants under the salt stress (Figure 2(c)) Thetotal number of lateral roots in the transgenic seedlingswas higher than that of the wild-type seedling at 4 7 and10 days under salt stress However the number of lateralroots per centimeter was almost equal to that of the wild-type (Figure 4(d)) Almost all of the wild-type plants died12 days after the beginning of the treatment with 200mMNaCl whereas many of the transformed plants still had greenleaves (Figure 4(a)) To address the question of whether theseedling growth would resume after removing the salt stressthe wild-type and transgenic seedlings subjected to salt stressfor 12 days were transplanted to soil As shown in Figure 2(e)transgenic seedling growth was rescued after 8 days but thewild-type seedling growth was not recovered at the sametime (Figure 2(e)) Actually transgenic rice plants producedseeds normally 4 weeks later whereas the wild-type almost

died (data not shown) These findings demonstrated that theoverexpression of OsRab7 enhanced salt tolerance in rice

33 The Amount of Proline Increased in OsRab7 Trans-genic Rice Plant in Salt Stress Proline a widely distributedosmolyte plays an important role in enhancing abiotic stresstolerance To investigate the physiological changes in thetransgenic plants under salt stress 2-week-old seedlings weretreated with 200mM NaCl for 24 h and then used to deter-mine the free proline content Under normal conditions andsalt-treated conditions increased proline levels were found inboth transgenic and wild-type plants and the proline contentin the transgenic rice being slightly lower than that in thewild-type (Figure 3(a)) However upon salt treatment thetransgenic seedlings exhibited a 35-fold increased proline

6 The Scientific World Journal

content level compared with those under normal condi-tions whereas the proline content in the wild-type plantsonly increased 15-fold after the salt treatment (Figure 3(b))Therefore the results indicated that the overexpression ofOsRab7 increased the proline content under salt-treatedconditions which helped transgenic seedlings to better adaptto the salt stress

34TheVesicles Increased inOsRab7 Transgenic Rice Plant Inplants Rab7 plays an important role in the vesicle traffickingprocess and thus the vesicle distribution in transgenic ricewas investigated using the vesicle fluorescent dye FM4-64As shown in Figure 4 a strong aggregated fluorescenceintensity of FM4-64 was observed in the transgenic rice roottips whereas a much weaker diffuse fluorescence intensitywas detected in the wild-type root tip This result sug-gested the acceleration of vesicles trafficking in the OsRab7transgenic rice

4 Discussion

Thephysiological response of plants to salt stress is a complexprocess requiring the coordinated function of many genesAlthoughOsRab7 transcript levels were to be found increasedunder salt stress direct evidence for the association of thisgene with salt tolerance was lacking In this paper ourexperiments showed that the overexpression ofOsRab7 couldconfer salinity tolerance in transgenic rice

Vacuoles play a critical role in salt tolerance by accu-mulating sodium which is then removed from the cytosolwhere it is toxic [12 13] A connection between salt sensitivityand vacuolar trafficking has been well established in animalcells and yeast Post-Golgi organelles involved in vacuolartrafficking were shown to be targets of salt stress resulting ininefficient vacuolar trafficking in yeast and mammalian cells[14ndash16] In plants Leshem et al reported that the endocyticpathway to the vacuole was also important for salt tolerancein Arabidopsis [17 18] The Rab7 proteins are importantcomponents of the vesicle trafficking system in all eukaryotes[5] In animals and yeast the Rab7 protein localizes to lateendosomes and to lysosomesvacuoles [19ndash22] In plantsthe Rab7 proteins have been localized to the tonoplast inbothArabidopsis and rice [8 23] Moreover enhanced vesicletrafficking and endocytic trafficking were also observed inRab7 overexpression transgenic soybean and in animal cellscontaining constitutive active forms of Rab7 [24 25] respec-tively In our studies an increased number of distributed vesi-cles was observed in OsRab7 overexpression transgenic ricesuggesting that the overexpression of OsRab7 may activatethe vesicle trafficking in rice Together with the results of salttolerance displayed by the OsRab7 overexpression transgenicrice we infer that the overexpression of OsRab7 increasesplant salt tolerance via enhanced vesicle trafficking in riceThus our results provide additional evidence that vacuolartrafficking is important for salt tolerance

5 Conclusion

In summary we prepared transgenic rice plants that over-expressed OsRab7 and tested their tolerance to salt stressThe transgenic plants exhibited enhanced tolerance to saltstress and increased numerous distributed vesicles in the roottips These results indicate that the overexpression ofOsRab7increased plant salt tolerance via enhanced vesicle traffickingin rice

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

X Peng and X Ding have contributed equally in this work

Acknowledgments

This work was supported by the Natural Science Foundationof China (nos 31160270 31160019) the YouthNatural ScienceFoundation of Jiangxi (No 20114BAB214009) the Project ofJiangxi Provincial Department of Education (no GJJ13102)and the earmarked fund for Modern Agro-industry Technol-ogy Research System (no CARS-01-04)

References

[1] C J Carter S Y Bednarek and N V Raikhel ldquoMembranetrafficking in plants new discoveries and approachesrdquo CurrentOpinion in Plant Biology vol 7 no 6 pp 701ndash707 2004

[2] A J McBroom and M J Kuehn ldquoRelease of outer membranevesicles by Gram-negative bacteria is a novel envelope stressresponserdquo Molecular Microbiology vol 63 no 2 pp 545ndash5582007

[3] A Levine B Belenghi H Damari-Weisler and D GranotldquoVesicle-associated membrane protein of arabidopsis sup-presses bax-induced apoptosis in yeast downstream of oxidativeburstrdquo Journal of Biological Chemistry vol 276 no 49 pp46284ndash46289 2001

[4] Y Liu Y Xiong and D C Bassham ldquoAutophagy is required fortolerance of drought and salt stress in plantsrdquoAutophagy vol 5no 7 pp 954ndash963 2009

[5] M Zerial and H McBride ldquoRab proteins as membrane orga-nizersrdquo Nature Reviews Molecular Cell Biology vol 2 no 2 pp107ndash117 2001

[6] A Mazel Y Leshem B S Tiwari and A Levine ldquoInductionof salt and osmotic stress tolerance by overexpression of anintracellular vesicle trafficking protein AtRab7 (AtRabG3e)rdquoPlant Physiology vol 134 no 1 pp 118ndash128 2004

[7] P K Agarwal P Agarwal P Jain B Jha M K Reddy and SK Sopory ldquoConstitutive overexpression of a stress-induciblesmall GTP-binding protein PgRab7 from Pennisetum glaucumenhances abiotic stress tolerance in transgenic tobaccordquo PlantCell Reports vol 27 no 1 pp 105ndash115 2008

[8] M Y Nahm S W Kim D Yun S Y Lee M J Cho and J DBahk ldquoMolecular and biochemical analyses of OsRab7 a riceRab7 homologrdquo Plant and Cell Physiology vol 44 no 12 pp1341ndash1349 2003

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 5

(a) (b)

Figure 3 Overexpression of OsRab7 in rice increased the proline level under salt stress (a) Level of proline in transgenic rice and WT riceunder normal conditions and salt treatment (b) Increasing rate of proline production after salt stress between the transgenic and WT riceThese data are the averages of triplicate assays and the error bars indicate the SDs

(a) (b)

Figure 4 Vesicles in the transgenic plant and WT rice (a) Vesicle morphology in the wild-type rice root tip (b) Vesicle morphology in thetransgenic plant root tipThe root tips were incubated in 5 120583gmL FM4-64 (Sigma) for 12min and images of the vesicles were captured usingconfocal microscopy Bar 20120583m

the transgenic plants under the salt stress (Figure 2(c)) Thetotal number of lateral roots in the transgenic seedlingswas higher than that of the wild-type seedling at 4 7 and10 days under salt stress However the number of lateralroots per centimeter was almost equal to that of the wild-type (Figure 4(d)) Almost all of the wild-type plants died12 days after the beginning of the treatment with 200mMNaCl whereas many of the transformed plants still had greenleaves (Figure 4(a)) To address the question of whether theseedling growth would resume after removing the salt stressthe wild-type and transgenic seedlings subjected to salt stressfor 12 days were transplanted to soil As shown in Figure 2(e)transgenic seedling growth was rescued after 8 days but thewild-type seedling growth was not recovered at the sametime (Figure 2(e)) Actually transgenic rice plants producedseeds normally 4 weeks later whereas the wild-type almost

died (data not shown) These findings demonstrated that theoverexpression of OsRab7 enhanced salt tolerance in rice

33 The Amount of Proline Increased in OsRab7 Trans-genic Rice Plant in Salt Stress Proline a widely distributedosmolyte plays an important role in enhancing abiotic stresstolerance To investigate the physiological changes in thetransgenic plants under salt stress 2-week-old seedlings weretreated with 200mM NaCl for 24 h and then used to deter-mine the free proline content Under normal conditions andsalt-treated conditions increased proline levels were found inboth transgenic and wild-type plants and the proline contentin the transgenic rice being slightly lower than that in thewild-type (Figure 3(a)) However upon salt treatment thetransgenic seedlings exhibited a 35-fold increased proline

6 The Scientific World Journal

content level compared with those under normal condi-tions whereas the proline content in the wild-type plantsonly increased 15-fold after the salt treatment (Figure 3(b))Therefore the results indicated that the overexpression ofOsRab7 increased the proline content under salt-treatedconditions which helped transgenic seedlings to better adaptto the salt stress

34TheVesicles Increased inOsRab7 Transgenic Rice Plant Inplants Rab7 plays an important role in the vesicle traffickingprocess and thus the vesicle distribution in transgenic ricewas investigated using the vesicle fluorescent dye FM4-64As shown in Figure 4 a strong aggregated fluorescenceintensity of FM4-64 was observed in the transgenic rice roottips whereas a much weaker diffuse fluorescence intensitywas detected in the wild-type root tip This result sug-gested the acceleration of vesicles trafficking in the OsRab7transgenic rice

4 Discussion

Thephysiological response of plants to salt stress is a complexprocess requiring the coordinated function of many genesAlthoughOsRab7 transcript levels were to be found increasedunder salt stress direct evidence for the association of thisgene with salt tolerance was lacking In this paper ourexperiments showed that the overexpression ofOsRab7 couldconfer salinity tolerance in transgenic rice

Vacuoles play a critical role in salt tolerance by accu-mulating sodium which is then removed from the cytosolwhere it is toxic [12 13] A connection between salt sensitivityand vacuolar trafficking has been well established in animalcells and yeast Post-Golgi organelles involved in vacuolartrafficking were shown to be targets of salt stress resulting ininefficient vacuolar trafficking in yeast and mammalian cells[14ndash16] In plants Leshem et al reported that the endocyticpathway to the vacuole was also important for salt tolerancein Arabidopsis [17 18] The Rab7 proteins are importantcomponents of the vesicle trafficking system in all eukaryotes[5] In animals and yeast the Rab7 protein localizes to lateendosomes and to lysosomesvacuoles [19ndash22] In plantsthe Rab7 proteins have been localized to the tonoplast inbothArabidopsis and rice [8 23] Moreover enhanced vesicletrafficking and endocytic trafficking were also observed inRab7 overexpression transgenic soybean and in animal cellscontaining constitutive active forms of Rab7 [24 25] respec-tively In our studies an increased number of distributed vesi-cles was observed in OsRab7 overexpression transgenic ricesuggesting that the overexpression of OsRab7 may activatethe vesicle trafficking in rice Together with the results of salttolerance displayed by the OsRab7 overexpression transgenicrice we infer that the overexpression of OsRab7 increasesplant salt tolerance via enhanced vesicle trafficking in riceThus our results provide additional evidence that vacuolartrafficking is important for salt tolerance

5 Conclusion

In summary we prepared transgenic rice plants that over-expressed OsRab7 and tested their tolerance to salt stressThe transgenic plants exhibited enhanced tolerance to saltstress and increased numerous distributed vesicles in the roottips These results indicate that the overexpression ofOsRab7increased plant salt tolerance via enhanced vesicle traffickingin rice

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

X Peng and X Ding have contributed equally in this work

Acknowledgments

This work was supported by the Natural Science Foundationof China (nos 31160270 31160019) the YouthNatural ScienceFoundation of Jiangxi (No 20114BAB214009) the Project ofJiangxi Provincial Department of Education (no GJJ13102)and the earmarked fund for Modern Agro-industry Technol-ogy Research System (no CARS-01-04)

References

[1] C J Carter S Y Bednarek and N V Raikhel ldquoMembranetrafficking in plants new discoveries and approachesrdquo CurrentOpinion in Plant Biology vol 7 no 6 pp 701ndash707 2004

[2] A J McBroom and M J Kuehn ldquoRelease of outer membranevesicles by Gram-negative bacteria is a novel envelope stressresponserdquo Molecular Microbiology vol 63 no 2 pp 545ndash5582007

[3] A Levine B Belenghi H Damari-Weisler and D GranotldquoVesicle-associated membrane protein of arabidopsis sup-presses bax-induced apoptosis in yeast downstream of oxidativeburstrdquo Journal of Biological Chemistry vol 276 no 49 pp46284ndash46289 2001

[4] Y Liu Y Xiong and D C Bassham ldquoAutophagy is required fortolerance of drought and salt stress in plantsrdquoAutophagy vol 5no 7 pp 954ndash963 2009

[5] M Zerial and H McBride ldquoRab proteins as membrane orga-nizersrdquo Nature Reviews Molecular Cell Biology vol 2 no 2 pp107ndash117 2001

[6] A Mazel Y Leshem B S Tiwari and A Levine ldquoInductionof salt and osmotic stress tolerance by overexpression of anintracellular vesicle trafficking protein AtRab7 (AtRabG3e)rdquoPlant Physiology vol 134 no 1 pp 118ndash128 2004

[7] P K Agarwal P Agarwal P Jain B Jha M K Reddy and SK Sopory ldquoConstitutive overexpression of a stress-induciblesmall GTP-binding protein PgRab7 from Pennisetum glaucumenhances abiotic stress tolerance in transgenic tobaccordquo PlantCell Reports vol 27 no 1 pp 105ndash115 2008

[8] M Y Nahm S W Kim D Yun S Y Lee M J Cho and J DBahk ldquoMolecular and biochemical analyses of OsRab7 a riceRab7 homologrdquo Plant and Cell Physiology vol 44 no 12 pp1341ndash1349 2003

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 The Scientific World Journal

content level compared with those under normal condi-tions whereas the proline content in the wild-type plantsonly increased 15-fold after the salt treatment (Figure 3(b))Therefore the results indicated that the overexpression ofOsRab7 increased the proline content under salt-treatedconditions which helped transgenic seedlings to better adaptto the salt stress

34TheVesicles Increased inOsRab7 Transgenic Rice Plant Inplants Rab7 plays an important role in the vesicle traffickingprocess and thus the vesicle distribution in transgenic ricewas investigated using the vesicle fluorescent dye FM4-64As shown in Figure 4 a strong aggregated fluorescenceintensity of FM4-64 was observed in the transgenic rice roottips whereas a much weaker diffuse fluorescence intensitywas detected in the wild-type root tip This result sug-gested the acceleration of vesicles trafficking in the OsRab7transgenic rice

4 Discussion

Thephysiological response of plants to salt stress is a complexprocess requiring the coordinated function of many genesAlthoughOsRab7 transcript levels were to be found increasedunder salt stress direct evidence for the association of thisgene with salt tolerance was lacking In this paper ourexperiments showed that the overexpression ofOsRab7 couldconfer salinity tolerance in transgenic rice

Vacuoles play a critical role in salt tolerance by accu-mulating sodium which is then removed from the cytosolwhere it is toxic [12 13] A connection between salt sensitivityand vacuolar trafficking has been well established in animalcells and yeast Post-Golgi organelles involved in vacuolartrafficking were shown to be targets of salt stress resulting ininefficient vacuolar trafficking in yeast and mammalian cells[14ndash16] In plants Leshem et al reported that the endocyticpathway to the vacuole was also important for salt tolerancein Arabidopsis [17 18] The Rab7 proteins are importantcomponents of the vesicle trafficking system in all eukaryotes[5] In animals and yeast the Rab7 protein localizes to lateendosomes and to lysosomesvacuoles [19ndash22] In plantsthe Rab7 proteins have been localized to the tonoplast inbothArabidopsis and rice [8 23] Moreover enhanced vesicletrafficking and endocytic trafficking were also observed inRab7 overexpression transgenic soybean and in animal cellscontaining constitutive active forms of Rab7 [24 25] respec-tively In our studies an increased number of distributed vesi-cles was observed in OsRab7 overexpression transgenic ricesuggesting that the overexpression of OsRab7 may activatethe vesicle trafficking in rice Together with the results of salttolerance displayed by the OsRab7 overexpression transgenicrice we infer that the overexpression of OsRab7 increasesplant salt tolerance via enhanced vesicle trafficking in riceThus our results provide additional evidence that vacuolartrafficking is important for salt tolerance

5 Conclusion

In summary we prepared transgenic rice plants that over-expressed OsRab7 and tested their tolerance to salt stressThe transgenic plants exhibited enhanced tolerance to saltstress and increased numerous distributed vesicles in the roottips These results indicate that the overexpression ofOsRab7increased plant salt tolerance via enhanced vesicle traffickingin rice

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

X Peng and X Ding have contributed equally in this work

Acknowledgments

This work was supported by the Natural Science Foundationof China (nos 31160270 31160019) the YouthNatural ScienceFoundation of Jiangxi (No 20114BAB214009) the Project ofJiangxi Provincial Department of Education (no GJJ13102)and the earmarked fund for Modern Agro-industry Technol-ogy Research System (no CARS-01-04)

References

[1] C J Carter S Y Bednarek and N V Raikhel ldquoMembranetrafficking in plants new discoveries and approachesrdquo CurrentOpinion in Plant Biology vol 7 no 6 pp 701ndash707 2004

[2] A J McBroom and M J Kuehn ldquoRelease of outer membranevesicles by Gram-negative bacteria is a novel envelope stressresponserdquo Molecular Microbiology vol 63 no 2 pp 545ndash5582007

[3] A Levine B Belenghi H Damari-Weisler and D GranotldquoVesicle-associated membrane protein of arabidopsis sup-presses bax-induced apoptosis in yeast downstream of oxidativeburstrdquo Journal of Biological Chemistry vol 276 no 49 pp46284ndash46289 2001

[4] Y Liu Y Xiong and D C Bassham ldquoAutophagy is required fortolerance of drought and salt stress in plantsrdquoAutophagy vol 5no 7 pp 954ndash963 2009

[5] M Zerial and H McBride ldquoRab proteins as membrane orga-nizersrdquo Nature Reviews Molecular Cell Biology vol 2 no 2 pp107ndash117 2001

[6] A Mazel Y Leshem B S Tiwari and A Levine ldquoInductionof salt and osmotic stress tolerance by overexpression of anintracellular vesicle trafficking protein AtRab7 (AtRabG3e)rdquoPlant Physiology vol 134 no 1 pp 118ndash128 2004

[7] P K Agarwal P Agarwal P Jain B Jha M K Reddy and SK Sopory ldquoConstitutive overexpression of a stress-induciblesmall GTP-binding protein PgRab7 from Pennisetum glaucumenhances abiotic stress tolerance in transgenic tobaccordquo PlantCell Reports vol 27 no 1 pp 105ndash115 2008

[8] M Y Nahm S W Kim D Yun S Y Lee M J Cho and J DBahk ldquoMolecular and biochemical analyses of OsRab7 a riceRab7 homologrdquo Plant and Cell Physiology vol 44 no 12 pp1341ndash1349 2003

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 7

[9] R Chen X Zhao Z Shao et al ldquoRice UDP-glucose pyrophos-phorylase1 is essential for pollen callose deposition and itscosuppression results in a new type of thermosensitive genicmale sterilityrdquo Plant Cell vol 19 no 3 pp 847ndash861 2007

[10] Y Hiei S Ohta T Komari and T Kumashiro ldquoEfficient trans-formation of rice (Oryza sativa L) mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNArdquo PlantJournal vol 6 no 2 pp 271ndash282 1994

[11] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[12] M P Apse G S AharonWA Snedden and E Blumwald ldquoSalttolerance conferred by overexpression of a vacuolar Na+H+antiport in Arabidopsisrdquo Science vol 285 no 5431 pp 1256ndash1258 1999

[13] L Xiong H Lee M Ishitani et al ldquoRepression of stress-re-sponsive genes by FIERY2 a novel transcriptional regulator inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 16 pp 10899ndash109042002

[14] C Bachert T H Lee and A D Linstedt ldquoLumenal endosomaland Golgi-retrieval determinants involved in pH-sensitive tar-geting of an early Golgi proteinrdquo Molecular Biology of the Cellvol 12 no 10 pp 3152ndash3160 2001

[15] J M Pardo B Cubero E O Leidi and F J Quintero ldquoAlkalication exchangers roles in cellular homeostasis and stresstolerancerdquo Journal of Experimental Botany vol 57 no 5 pp1181ndash1199 2006

[16] A Hernandez X Jiang B Cubero et al ldquoMutants of the ar-abidopsis thaliana cationH+ antiporter AtNHX1 conferringincreased salt tolerance in yeast the endosomeprevacuolarcompartment is a target for salt toxicityrdquo Journal of BiologicalChemistry vol 284 no 21 pp 14276ndash14285 2009

[17] Y Leshem N Melamed-Book O Cagnac et al ldquoSuppressionof Arabidopsis vesicle-SNARE expression inhibited fusion ofH2O2-containing vesicles with tonoplast and increased salt

tolerancerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 47 pp 18008ndash180132006

[18] Y Leshem L Seri and A Levine ldquoInduction of phosphatidyli-nositol 3-kinase-mediated endocytosis by salt stress leads tointracellular production of reactive oxygen species and salttolerancerdquo Plant Journal vol 51 no 2 pp 185ndash197 2007

[19] F Schimmoller andH Riezman ldquoInvolvement of Ypt7p a smallGTPase in traffic from late endosome to the vacuole in yeastrdquoJournal of Cell Science vol 106 part 3 pp 823ndash830 1993

[20] F Bruckert O Laurent and M Satre ldquoRab7 a multifacetedGTP-binding protein regulating access to degradative compart-ments in eukaryotic cellsrdquo Protoplasma vol 210 no 3-4 pp108ndash116 2000

[21] C Bucci P Thomsen P Nicoziani J McCarthy and B vanDeurs ldquoRab7 a key to lysosome biogenesisrdquo Biology of the Cellvol 11 no 2 pp 467ndash480 2000

[22] H R Pelham ldquoInsights from yeast endosomesrdquoOpinion in CellBiology vol 14 no 4 pp 454ndash462 2002

[23] C Saito T Ueda H Abe et al ldquoA complex andmobile structureforms a distinct subregion within the continuous vacuolarmembrane in young cotyledons of Arabidopsisrdquo Plant Journalvol 29 no 3 pp 245ndash255 2002

[24] E Limpens S Lvanov W van Esse G Voets E Fedorovaand T Bisseling ldquoMedicago N

2-Fixing symbiosomes acquire

the endocytic identity marker Rab7 but delay the acquisition ofvacuolar identityrdquo Plant Cell vol 21 no 9 pp 2811ndash2828 2009

[25] S Meresse J-P Gorvel and P Chavrier ldquoThe rab7 GTPaseresides on a vesicular compartment connected to lysosomesrdquoJournal of Cell Science vol 108 no 11 pp 3349ndash3358 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology