Suppression of glycolate oxidase causes glyoxylate accumulation that inhibits photosynthesis through deactivating Rubisco in rice

Physiologia Plantarum 150: 463–476. 2014 © 2013 Scandinavian Plant Physiology Society, ISSN 0031-9317

Suppression of glycolate oxidase causes glyoxylateaccumulation that inhibits photosynthesis throughdeactivating Rubisco in riceYusheng Lua, Yong Lia, Qiaosong Yangb, Zhisheng Zhanga, Yan Chena, Sheng Zhangc and Xin-XiangPenga,∗

aState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, ChinabInstitute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou, ChinacInstitute of Biotechnology, Cornell University, Ithaca, NY, USA

Correspondence*Corresponding author,e-mail: [email protected]

Received 23 July 2013;revised 20 August 2013

doi:10.1111/ppl.12104

Glycolate oxidase (GLO) is a key enzyme for photorespiration in plants.Previous studies have demonstrated that suppression of GLO causesphotosynthetic inhibition, and the accumulated glycolate with the deactivatedRubisco is likely involved in the regulation. Using isolated Rubisco andchloroplasts, it has been found that only glyoxylate can effectively inactivateRubisco and meanwhile inhibit photosynthesis, but little in vivo evidencehas been acquired and reported. In this study, we have generated thetransgenic rice (Oryza sativa) plants with GLO being constitutively silenced,and conducted the physiological and biochemical analyses on these plantsto explore the regulatory mechanism. When GLO was downregulated,the net photosynthetic rate (Pn) was reduced and the plant growth wascorrespondingly stunted. Surprisingly, glyoxylate, as a product of the GLOcatalysis, was accumulated in response to the GLO suppression, like itssubstrate glycolate. Furthermore, the glyoxylate content was found to beinversely proportional to the Pn while the Pn is directly proportional tothe Rubisco activation state in the GLO-suppressed plants. A mathematicalfitting equation using least square method also demonstrated that theRubisco activation state was inversely proportional to the glyoxylate content.Despite that the further analyses we have conducted failed to reveal howglyoxylate was accumulated in response to the GLO suppression, the currentresults do strongly suggest that there may exist an unidentified, alternativepathway to produce glyoxylate, and that the accumulated glyoxylate inhibitsphotosynthesis by deactivating Rubisco, and causes the photorespiratoryphenotype in the GLO-suppressed rice plants.

Abbreviations – BSA, bovine serum albumin; DCT, chloroplast dicarboxylate transporter; GDC, glycine decarboxylase; GDH,glycolate dehydrogenase; GGAT, glutamate glyoxylate aminotransferase; GLO, glycolate oxidase; GLYK, glycerate kinase;GOGAT, glutamate synthase; GR, glyoxylate reductase; GS, glutamine synthetase; HPR, hydroxyl pyruvate reductase; ICL,isocitrate lyase; iTRAQ, isobaric tags for relative and absolute quantitation; LC-MS/MS, liquid chromatography-tandem massspectrometry; MCS, multi-cloning site; PGLP, phosphoglycolate phosphatase; PGR, proton gradient regulation; PLGG, plastidicglycolate glycerate translocator; PMSF, phenylmethanesulfonyl fluoride; PR, photorespiration; RCA, Rubisco activase; RNAi,RNA interference; RT-PCR, real-time-polymerase chain reaction; SGAT, serine glyoxylate aminotransferase; SHMT, serinehydroxymethyltransferase; WT, wild-type.

Physiol. Plant. 150, 2014 463

Introduction

Plant photosynthetic carbon metabolism comprises twocoupled pathways: the reductive photosynthetic carboncycle, also known as the Calvin cycle, and the oxidativephotosynthetic carbon cycle, namely photorespiration(PR). PR used to be regarded as a potentially wastefulprocess limiting plant productivity, as a C3 plant mayphotorespire as much as 25% of the carbon fixed byphotosynthesis under normal circumstances (Sharkey1988, Rachmilevitch et al. 2004). As a result, scientistsonce devoted themselves with high enthusiasm to lookfor ways to abolish this pathway aiming to increase plantproductivity (Ogren 1984). In spite of numerous trialsconducted, the efforts turned out to be unsuccessful.After a series of Arabidopsis PR mutants were screenedand characterized, the essential roles of PR becamewidely accepted (Somerville 2001).

Arabidopsis mutants deficient in various photorespi-ratory enzymes were initially generated by Somervilleand Ogren using a unique approach they developed(Somerville and Ogren 1982b, Somerville 2001). Themutated enzymes include phosphoglycolate phos-phatase (PGLP) (Somerville and Ogren 1979), serineglyoxylate aminotransferase (SGAT) (Somerville andOgren 1980), glycine decarboxylase (GDC) (Somervilleand Ogren 1982a) and serine hydroxymethyltransferase(SHMT) (Somerville and Ogren 1981). After that, similarmutants were also isolated from barley (Blackwellet al. 1988, Kleczkowski et al. 1990, Leegood et al.1995), tobacco (McHale et al. 1988) and other cropplants (Timm and Bauwe 2013). However, the deficientmutants for some PR enzymes such as glycolate oxidase(GLO), glutamate glyoxylate aminotransferase (GGAT)and glycerate kinase (GLYK) did not show up inthis screening process, likely because these enzymesmay either have multiple genes that are functionallycomplementary or play essential roles beyond PR(Somerville and Ogren 1982b). These mutants recentlyemerged by using gene silencing approaches, such asRNA antisense, RNA interference (RNAi), and T-DNAinsertion. Approach of T-DNA insertion generated themutants for GGAT (Igarashi et al. 2003), GLYK (Boldtet al. 2005), SHMT (Voll et al. 2006), PGLP (Schwarteand Bauwe 2007) and HPR (Timm et al. 2008, 2011) inArabidopsis, and for GLO in maize (Zelitch et al. 2009).The plants deficient with GLO were also created throughantisense and co-suppression approaches (Yamaguchiand Nishimura 2000, Xu et al. 2009). Apart from thecore cycle, some enzymes of the photorespiratorypathway-associated processes, such as glutamate syn-thase (GOGAT), glutamine synthetase (GS) and catalase(CAT), were also discovered using the classic screeningprocedures and reverse genetic approaches (Timm and

Bauwe 2013). Most recently, an Arabidopsis mutantdeficient in plastidic glycolate glycerate translocator 1(PLGG1), a photorespiratory transporter, was identified(Pick et al 2013). More interestingly, almost all thesemutants displayed the typical PR phenotype, i.e. theyrequire a higher CO2 concentration (>0.25%) to main-tain the growth and development, otherwise becomechlorotic and even finally lethal in ambient atmosphere.For these plants with PR phenotype photosynthesis iscommonly inhibited, such that it remains a subject ofinterest to understand the mechanism underlying thisphotosynthetic inhibition.

So far two hypotheses have been raised to interpretthe mechanism of the photosynthetic inhibition in thosevarious PR mutants. First, impairment of the photorespi-ratory carbon recycling and/or nitrogen re-assimilationcauses depletion of the C3 cycle intermediates andphotosynthetic proteins leading to the photosyntheticinhibition. Second, disruption of the photorespiratorypathway results in accumulation of the metabolites,which would feedback inhibit the Calvin cycle (Wingleret al. 2000). Evidence from recent years’ studies appearsto better support the second opinion as it was observedthat those downstream metabolites were not signifi-cantly depleted when certain various PR enzymes weredisrupted (Igarashi et al. 2006, Schjoerring et al. 2006,Timm et al. 2008, Xu et al. 2009). In addition, a series ofstudies conducted by Chastain and Ogren (1985, 1989)indicated that the accumulation of glyoxylate, rather thanother metabolites, was the main effector that reduced theactivation level of Rubisco, thereby causing the photo-synthetic inhibition. Logically, however, this mechanismcan be only applied for the mutants deficient in theenzymes downstream of the glyoxylate metabolism,because it has been detected that the pcoA mutationlosing PGLP activity had no negative effect on the acti-vation state of Rubisco (Chastain and Ogren 1985, 1989).As mentioned above, typical PR phenotypes occurred forthe GLO-suppressed plants (Yamaguchi and Nishimura2000, Xu et al. 2009, Zelitch et al. 2009), and more-over, reduced activation state of Rubisco was alsoobserved (Xu et al. 2009). The accumulated glycolatewas proposed to be the candidate molecule causingthe photosynthetic inhibition in these GLO-suppressedplants (Xu et al. 2009, Zelitch et al. 2009). But Chastainand Ogren (1989) detected that only glyoxylate, ratherthan glycolate or other PR metabolites, had the abilityto decrease the activation state of Rubisco. Actually, anumber of studies have also shown that glyoxylate is ableto effectively inactivate Rubisco both in its purified formand within isolated chloroplasts (Lawyer et al. 1983,Mulligan et al. 1983, Chastain and Ogren 1985, Cooket al. 1985, Campbell and Ogren 1990a, Wendler et al.

464 Physiol. Plant. 150, 2014

1992). Theoretically, glyoxylate accumulation less likelyoccurs in these GLO-suppressed plants because it is theproduct of the GLO-catalyzed reaction. Thus, which fac-tor and how it acts to inhibit photosynthesis during GLOsuppression remains to be an intriguing question.

In this study, transgenic rice plants (Oryza sativa)with GLO being constitutively silenced were generatedto further address the mechanism of how GLOregulates photosynthesis. The results obtained in thiswork suggest that there may exist an unidentified,alternative pathway to produce glyoxylate, and that theaccumulated glyoxylate, through deactivating Rubisco,inhibits photosynthesis and causes the photorespiratoryphenotype in the GLO-suppressed rice plants.

Materials and methods

Growth conditions and treatments

Pre-germinated rice seeds were normally grown inKimura B complete nutrient solution (Yoshida et al.1976) under natural growth conditions [temperatureof 30–35/23–26◦C (day/night), maximum photosyn-thetically active radiation of 600–1500 μmol m−2 s−1

depending on weather, and photoperiod of about14 h day/about 10 h night].

Construction of GLO-silencing transgenicrice plants

Rice (O. sativa cv. Zhonghua 11) was used for construct-ing transgenic lines in this study. To generate GLO-silenced lines, the primer pair for amplifying a specificsequence from OsGLO4 (NM_001065444) was care-fully designed to guarantee the specificity of silencing.The upstream and downstream primers for real-time-polymerase chain reaction (RT-PCR) cloning were 5′-CTGGAAGCTTTAGAGCAGCAATGCACGTG-3′ and 5′-AACTGGATCCGAGTGAAGAGCCACGCAAG-3′, res-pectively. Then the amplified fragment product wasligated into the vector pYL-RNAi.5 in a sense orientationat multi-cloning site 1 (MCS1) between HindIII andBamHI. This first round ligated vector was then used asthe template to amplify a second fragment sequence withtwo unique restriction sites in both ends (RNAi-MluI:5′-CACCCTGACGCGTGGTGTTACTT-CTGAAGAGG-3′

and RNAi-PstI: 5′-ACTAGAACTGCAGCCTCAGATCTACCATG-GTCG-3′). The second fragment sequence wassubsequently cloned at MCS2 between PstI and MluI,resulting in an opposite orientation in contrast to thesequence in MCS1. The constructed interference vectorwas then transformed into rice callus via Agrobacterium-mediated infection (strain EHA105). T0 lines wereanalyzed by Southern blot.

Transcript analysis, western blots and enzymeactivity assays

Semi-quantitative and real-time PCR

Total RNA was isolated using TRIZOL reagent. Theisolated total RNA was then treated with DNase Iand used as a template for first-strand cDNA synthesisusing ReverTra Ace (Toyobo, Osaka, Japan) with randomhexamers according to the manufacturer’s instructions.For semi-quantitative RT-PCR analysis, the optimalnumber of PCR cycles was first tested gene by gene.The PCR products were separated on 1% (w/v) agarosegels and visualized by Goldview staining. For real-timequantitative RT-PCR, the PCR reaction consisted of 10 μlof 2×SYBR Green PCR Master Mix (Toyobo), 200 nMprimers, and 2 μl of 1:40-diluted template cDNA in atotal volume of 20 μl. No template controls were set foreach primer pair. The analysis was conducted by a DNAEngine Option 2 Real-Time PCR Detection system andOPTICON MONITOR software (Bio-Rad, Hercules, CA).

Western blot

Equal amount proteins were loaded separately on a12.5% sodium monododecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) gel and electroblotted ontoa nitrocellulose membrane using a Mini Trans-Blot cell(Bio-Rad). Rubisco activase (RCA) protein was detectedusing a monoclonal antibody against the rice RCA. Gly-colate dehydrogenase (GDH) protein was detected usinga rabbit polyclonal antibody raised against the rice GDH.

Enzyme activity assays

GLO activity was assayed according to Hall et al. (1985)with some modifications (Xu et al. 2006). SGAT, GGAT,hydroxyl pyruvate reductase (HPR), isocitrate lyase (ICL)activities were assayed according to Yu et al. (2010).Glyoxylate reductase (GR) activity was determinedaccording to Tolbert et al. (1970). Rubisco activity wasassayed after Ward and Keys (1989). Initial activity andtotal activity were measured before and after incubatingthe crude extract with activation medium. The activationstate of Rubisco was calculated as the relative ratio ofinitial to total Rubisco activities (Perchorowicz et al.1981). Protein concentration was determined accordingto Bradford (1976).

Gas exchange measurements and chlorophyllfluorescence analysis

Gas exchange characteristics including net photosyn-thetic rate (Pn), stomatal conductance (Gs) and internalCO2 concentration (Ci) were analyzed in situ using a


portable photosynthesis system (LI-6400, LI-COR). Theplants were grown in normal natural conditions, and theyoungest fully expanded leaves were used to determinethe photosynthetic parameters. Measurements wereperformed in the morning (9:00–11:00 h.). The othermeasurement conditions were set as follows accordingto the manufacturer’s instructions: leaf temperature at30◦C, relative humidity at 60%, CO2 concentrationof 400 μmol mol−1, photosynthetic photon flux density(PPFD) at 1000 μmol m−2 s−1 (controlled by a LI-CORLED irradiation source). The chlorophyll fluorescencewas measured with a portable chlorophyll fluorometerPAM 2100 (Walz, Effeltrich, Germany). Leaves weredark adapted for at least 20 min prior to the measure-ment. Two measurements were taken from each seedling

to determine the minimum fluorescence yield (Fo) andthe maximum fluorescence yield (Fm), and the maximalphotochemical efficiency of photosystem II (Fv/Fm) wascalculated according to Krause and Weis (1991).

Quantitative proteomic analysis

For protein extraction, 0.1 g rice leaves from bothwild-type (WT) and GLO-silenced plants with biologicalduplicates for each line were ground in liquid N2

following by suspension in 1 ml ice-cold sodium phos-phate buffer (100 mM, pH 7.5) containing 1 mM EDTA,100 μg ml–1 phenylmethanesulfonyl fluoride (PMSF) and0.1% Triton X-100. The homogenate was centrifuged at

Fig. 1. Phenotypes of the GLO-suppressed transgenic plants. Wild-type (WT) and transgenic plants were grown under normal natural conditions[temperature of 30–35/23–26◦C (day/night), maximum photosynthetically active radiation of 600–1500 μmol m−2 s−1 depending on weather andphotoperiod of about 14 h day/about 10 h night]. Photographs of phenotypes were taken at five-leaf stage (C), and the leaves of each group weredetached respectively to analyze the mRNA expression levels (A) and enzyme activities (B) of GLO. Meanwhile both growth rate (D) and biomass (E)were determined for the seedlings. Ri 12 (heterozygote, two copies) and Ri 15 (heterozygote, three copies) represent two independent RNAi lines.The data represent means of six biological replicates ± SD. Different uppercase letters in the same column indicate significant differences at P < 0.01.


12 000 g for 15 min. Protein concentrations were deter-mined by the Bradford assay using bovine serum albumin(BSA) as a standard. The proteins in the supernatantwere lyophilized and reconstituted in 0.2 M triethylam-monium bicarbonate (pH 8.5). An aliquot (100 μg) ofproteins in a total volume of 20 μl was used for isobarictags for relative and absolute quantitation (iTRAQ 4)-plexlabeling. Specifically 114-tag and 115-tag were usedfor labeling two samples of WT plants while 116-tagand 117-tag were labeled for GLO suppressed plants,respectively. The detailed labeling procedures andsubsequent 2D liquid chromatography-tandem massspectrometry (LC-MS/MS) analysis using a LTQ-OrbitrapVelos (Thermo-Fisher Scientific, San Jose, CA) massspectrometer were performed as described by Yanget al. (2012).

Quantification of organic acids

Glyoxylate and glycolate were determined as reportedby Xu et al. (2006).

Results

We previously used an inducible antisense approachto downregulate GLO expression in rice and observed

that growth of the antisense plants was heavily inhibited(Xu et al. 2009). In this study, the constitutively GLO-interfered plants (Ri) were further generated. The similargrowth-inhibited phenotypes were observed for theGLO constitutively suppressed plants as found for theinducible antisense plants (Fig. 1, Xu et al. 2009). Pn waspreviously observed to be linearly correlated with GLOactivity in the GLO inducible antisense plants (Xu et al.2009). Zelitch et al. (2009) also noticed that the GLOmutated maize had much lower Pn. Here, for the GLOconstitutively interfered plants, the Pn was remarkablyreduced (Fig. 2A), responding to GLO suppression ina threshold-dependent manner, rather than in a linearcorrelation as previously noticed in the inducible case(Xu et al. 2009). Stomatal conductance (Gs) was reducedwhereas internal CO2 concentration (Ci) was increasedin the GLO-suppressed plants (Fig. 2B), indicating thatthe photosynthetic inhibition is less likely to be causedby the stomatal factor (Farquhar et al. 1980). Fv/Fm wasonly slightly decreased in this constitutive case (Fig. 2C),much less than that previously detected in the induciblecase (Xu et al. 2009).

In order to understand the mechanism underlying thephotosynthetic inhibition in response to GLO suppres-sion, quantitative proteomic analysis was conducted. All

Fig. 2. Response of photosynthetic parameters to regulation of GLO. WT and transgenic plants were grown under normal natural conditions asdescribed in Fig. 1. At five-leaf stage, net photosynthesis rate (Pn) (A); Gs and Ci (B) and Fv/Fm (C) were determined onto the youngest fully expandedleaf of each plant between 9:00 h and 11:00 h of the day (for details, see Materials and methods), and meanwhile the same leaf was detached forGLO activity assay, respectively.


Table 1. Relative expressional levels of photosynthesis-related proteins in GLO-suppressed plants. WT and transgenic plants were grown undernormal natural conditions as described in Fig. 1. At five-leaf stage, Pn was determined onto the youngest fully expanded leaf, and then according tothe Pn determined, WT plants and those individuals with about 80% of Pn downregulated were used for iTRAQ analysis. The data represent meansof two biological replicates ± SD. *Indicates significant differences at P < 0.05.

Protein name Accession Ri/WT ratio Function

Rubisco activase gi|115461056 1.039 ± 0.025 Calvin cycleRubisco large subunit gi|11466795 0.857 ± 0.054 Calvin cycleRubisco small subunit gi|297606119 0.803 ± 0.032 Calvin cycleRubisco small subunit gi|115487804 1.216 ± 0.034 Calvin cycleRubisco small subunit gi|115488144 0.900 ± 0.051 Calvin cycleRubisco small subunit gi|115488236 0.992 ± 0.004 Calvin cyclePhosphoglycerate kinase gi|115444481 1.064 ± 0.051 Calvin cyclePhosphoglycerate kinase gi|115469436 0.925 ± 0.012 Calvin cyclePhosphoglycerate kinase gi|297720489 0.839 ± 0.045 Calvin cycleGlyceraldehyde-3-phosphate dehydrogenase gi|115450493 0.964 ± 0.040 Calvin cycleGlyceraldehyde-3-phosphate dehydrogenase gi|115458768 0.838 ± 0.028 Calvin cycleTriosephosphate isomerase gi|115434516 0.984 ± 0.023 Calvin cycleTriosephosphate isomerase gi|115480367 1.118 ± 0.007 Calvin cycleAldolase gi|29759814 1.128 ± 0.024 Calvin cycleFructose-1,6-bisphosphatase gi|115441253 1.014 ± 0.049 Calvin cycleFructose-1,6-bisphosphatase gi|115452127 0.937 ± 0.056 Calvin cycleFructose-1,6-bisphosphatase gi|115469396 0.914 ± 0.004 Calvin cycleTransketolase gi|115457470 0.922 ± 0.002 Calvin cycleTransketolase gi|115466224 1.001 ± 0.049 Calvin cycleSedoheptulose-bisphosphatase gi|115457386 0.941 ± 0.010 Calvin cycleRibulose-phosphate 3-epimerase gi|115450991 1.097 ± 0.036 Calvin cycleRibose 5-phosphate isomerase A gi|115457638 1.351 ± 0.014 Calvin cycleRibose 5-phosphate isomerase A gi|115470849 1.069 ± 0.057 Calvin cyclePhosphoribulokinase gi|115448091 0.954 ± 0.025 Calvin cyclePhosphoribulokinase gi|297723423 0.790 ± 0.025 Calvin cycleChlorophyll a–b binding protein gi|115472785 1.090 ± 0.083 Light harvestingChlorophyll a–b binding protein gi|115472983 0.920 ± 0.068 Light harvestingChlorophyll a–b binding protein 3 gi|297598774 0.792 ± 0.016 Light harvestingChloroplast protease gi|115470052 1.031 ± 0.009 Light harvestingPhotosystem I P700 apoprotein a2 gi|11466786 0.642 ± 0.030* PS IPhotosystem I subunit VII gi|11466848 0.968 ± 0.006 PS IPhotosystem I light harvesting complex protein 5 gi|115448873 0.797 ± 0.030 PS IPhotosystem I reaction centre subunit N family protein gi|115455127 1.094 ± 0.180 PS IPhotosystem I psah protein gi|115465409 0.747 ± 0.008 PS IThylakoid membrane phosphoprotein gi|115472001 0.787 ± 0.028 PS IPhotosystem II 10 kDa polypeptide gi|115475227 0.762 ± 0.091 PS IPhotosystem I reaction center subunit ii gi|115477831 0.843 ± 0.011 PS IPhotosystem I reaction center subunit v gi|115479799 0.717 ± 0.035 PS IPhotosystem I reaction center subunit n gi|115487694 0.652 ± 0.038* PS IPhotosystem I reaction center subunit XI gi|115488344 0.740 ± 0.040 PS IPhotosystem I reaction center subunit III gi|297601775 0.746 ± 0.011 PS IPhotosystem I reaction center subunit IV a gi|297607127 1.027 ± 0.052 PS IATP synthase delta chain gi|115448701 0.876 ± 0.042 PS IPGR5-like protein 1a gi|115456709 0.576 ± 0.018* PS IThylakoid membrane phosphoprotein gi|115472001 0.787 ± 0.028 PS IElectron carrier electron transporter iron ion binding protein gi|115472141 1.040 ± 0.099 PS IPhotosystem II protein D1 gi|11466764 0.857 ± 0.046 PS IIPhotosystem II protein D2 gi|11466770 0.940 ± 0.136 PS IIPhotosystem II CP43 chlorophyll apoprotein gi|11466771 0.887 ± 0.106 PS IIPhotosystem II cytochrome b559 alpha subunit gi|11466807 0.995 ± 0.098 PS IIPhotosystem II 22 kDa protein gi|115441299 0.936 ± 0.093 PS IIPhotosystem II reaction center w protein gi|115442119 0.971 ± 0.033 PS IIPhotosystem II oxygen-evolving enhancer protein gi|115446893 0.924 ± 0.016 PS IIPhotosystem II kDa protein gi|115452847 1.032 ± 0.085 PS II


Table 1. Continued.

Protein name Accession Ri/WT ratio Function

Chloroplast photosystem II 22 kDa component gi|115461508 1.342 ± 0.111 PS IIOxygen-evolving enhancer protein 3–1 gi|115470199 0.986 ± 0.054 PS II23 kDa polypeptide of photosystem II gi|115470529 0.978 ± 0.067 PS IIPhotosystem II reaction center family protein gi|297722003 1.050 ± 0.028 PS IICytochrome b6 gi|11466819 1.164 ± 0.052 PS IITetratricopeptide repeat-containing protein gi|115436512 0.876 ± 0.015 PS IIFused signal recognition particle receptor gi|115442343 0.882 ± 0.102 PS IILow PS II accumulation 3 protein gi|115443809 0.984 ± 0.004 PS IISerine-type peptidase gi|115465521 0.849 ± 0.004 PS IIATP synthase gamma chain gi|115472339 0.824 ± 0.011 PS IIProtease do-like 8 gi|297723211 0.970 ± 0.004 PS IIProtease do-like chloroplastic-like gi|297723707 1.078 ± 0.021 PS IIGlycolate oxidase (GLO4)(the protein to be silenced) gi|115470621 0.364 ± 0.035* PhotorespirationGlycolate oxidase (GLO1) gi|115455773 0.034 ± 0.011* PhotorespirationGlycolate oxidase (GLO5) gi|115473355 0.932 ± 0.017 PhotorespirationPhosphoglycolate phosphatase gi|115459134 0.913 ± 0.050 PhotorespirationPhosphoglycolate phosphatase gi|297609126 1.160 ± 0.062 PhotorespirationSerine:glyoxylate aminotransferase gi|115477148 1.060 ± 0.059 PhotorespirationAlanine-2-oxoglutarate aminotransferase 2 gi|115470235 0.910 ± 0.004 PhotorespirationGlycine decarboxylase P protein gi|115468926 1.123 ± 0.087 PhotorespirationGlycine decarboxylase P protein gi|115439533 1.108 ± 0.001 PhotorespirationGlycine decarboxylase T protein gi|115460656 0.924 ± 0.001 PhotorespirationGlycine decarboxylase H protein gi|115482934 1.326 ± 0.028 PhotorespirationSerine hydroxymethyltransferase gi|115455221 1.008 ± 0.026 PhotorespirationSerine hydroxymethyltransferase gi|297611783 1.073 ± 0.044 PhotorespirationSerine hydroxymethyltransferase gi|115488306 1.063 ± 0.004 PhotorespirationHydroxypyruvate reductase gi|115443619 0.903 ± 0.013 PhotorespirationHydroxypyruvate reductase gi|115435442 1.183 ± 0.021 PhotorespirationGlycerate kinase gi|115439213 0.931 ± 0.006 Photorespiration

the 2087 proteins commonly identified in the two biolog-ical replicates were used to compute the internal error,which was defined as the value of the log2 iTRAQ ratio(silenced plant/WT), at which 95% of all proteins hadno deviation from each other, where the deviation wasthe absolute value of the difference in iTRAQ log2 ratiosbetween the x-axis and y-axis (Redding et al. 2006, Ganet al. 2007). The internal error 0.47 (corresponding to a±1.385-fold change) was used as a threshold of signifi-cance in the present work. As shown in Table 1, a totalof 83 photosynthesis-related proteins were confidentlyidentified by iTRAQ-based shotgun analysis, including25 for Calvin cycle proteins, 41 for light reaction proteinsand 17 for photorespiratory proteins. To the best of ourknowledge, all the Calvin cycle and photorespiratoryproteins were identified by this quantitative proteomicsapproach. The result showed that all the Calvin cycleproteins, including Rubisco and its activase, were notsignificantly altered in the silenced plants over WT lines(Table 1). Similarly, except the target protein GLO, theother photorespiratory proteins were not affected at allfor the silenced plants. Overall, only three light reac-tion related proteins were significantly downregulated

in the silenced plants, which are P700 apoprotein a2,photosystem I (PSI) reaction center subunit n, proton gra-dient regulation-5 (PGR-5) like protein 1a. Interestingly,we noticed that the specific silencing of GLO4 simul-taneously caused more suppression of GLO1. We haveearlier demonstrated that GLO1 and GLO4 are coordi-nately regulated at the transcriptional level (Zhang et al.2012). Such coordinated suppression also commonlyoccurred for other genes, such as RBCS and RBCL,which encode the Rubisco small and large subunits,respectively (Suzuki et al. 2001, Ogawa et al. 2012).

In the GLO-silenced plants, as Pn suppression wasincreased, the initial Rubisco activity was decreasedin parallel while its total activity was not affected.Consequently, the activation state of Rubisco wasdecreased accordingly (Fig. 3A). The Pn was directlyproportional to the initial Rubisco activity (P < 0.01)(Fig. 3B). We previously noticed that the transcriptionalexpression of RCA was repressed as GLO was induciblysuppressed (Xu et al. 2009). While similar transcriptionalexpression patterns occurred in these constitutivelyGLO-silenced plants (Fig. 3C), the RCA proteins were


Fig. 3. The relationship between Pn and Rubisco activity and its activase (RCA). WT and transgenic plants were grown under normal natural conditionas described in Fig. 1. At five-leaf stage, Pn was determined onto the youngest fully expanded leaf. According to the Pn determined, those individualswith about 40, 60 and 80% of Pn decreased were respectively grouped, and then Rubisco activity and Rubisco activation state (A), transcripts (C) andproteins (D) of RCA were determined (the data represent means of three biological replicates ± SD). For correlation analysis, the same leaf of eachplant was detached immediately for assaying initial Rubisco activities after measuring Pn, then the Pn data and corresponding Rubisco initial activitieswere collected for correlation analysis (B).

not altered (Fig. 3D), which is nicely consistent with theresults from the proteomic analysis (Table 1).

GLO is a well-known enzyme in catalyzing theproduction of glyoxylate from glycolate. Surprisingly,we found that when GLO was suppressed the contentof glyoxylate was even increased. This unexpectedfinding was firmly validated by various independentexperiments and different researchers in our group.More interestingly, glyoxylate was first linearly increasedand then leveled off as glycolate was continuouslyaccumulated (Fig. 4A). Meanwhile, Pn was first linearlydecreased and subsequently leveled off when glycolatewas increasingly accumulated (Fig. 4B). Correlationanalysis showed that Pn was inversely proportionalto the glyoxylate content (P < 0.01) (Fig. 4C). Ourprevious study showed, in the inducible antisense plants,glyoxylate was unchanged in response to the GLOsuppression (Xu et al. 2009). At present, we are not clearabout how such inconsistency occurred. As a matterof fact, we also generated the GLO over-expressed ricelines, and analyses on these plants turned out that boththe Pn and the glyoxylate content were little alteredunder normal growth conditions despite the fact that its

GLO activity was increased up to 220% relative to thatof WT (data not shown).

In order to further understand how glyoxylate isaccumulated in response to GLO suppression, weextended investigation of other relevant enzymeswhich are known to be involved in metabolism ofglyoxylate. Cleavage of isocitrate was a well-knownsource producing glyoxylate, which is catalyzed by ICL(Hayashi et al. 1995). We have previously noticed thatthe transcriptional expression of ICL was upregulatedin response to GLO suppression (Xu et al. 2009). Inthis study, however, it was found that the ICL catalyticactivity was not upregulated in the silenced plants (Fig.5A). In addition, a GDH was reported in mitochondriaof Arabidopsis and rice (Bari et al. 2004, Niessenet al. 2007, 2012), but our current analyses showedno detectable GDH catalytic activity in rice and thatthe expressions of OsGDH were not changed at bothtranscriptional and translational levels in response toGLO suppression (Fig. 5B). Furthermore, a glycolate-oxidizing activity was reported in chloroplasts of algaeand spinach (Murai and Katoh 1975, Goyal and Tolbert1996, Goyal 2002). Nevertheless in our efforts, no such


Fig. 4. Correlation between Pn and glycolate and glyoxylate. WT and RNAi plants were grown under normal natural condition as described in Fig.1. At five-leaf stage, Pn was determined onto the youngest fully expanded leaf. After the measurement, the same leaf was immediately detachedfor determination of glycolate and glyoxylate contents. The correlation analysis was respectively made between glycolate and glyoxylate (A); Pn andglycolate (B); and Pn and glyoxylate (C).

activity has been detected in rice chloroplasts. There areseveral enzymes responsible for glyoxylate downstreammetabolism, such as GR, HPR, SGAT and GGAT.Activity measurement revealed that these enzymes wereeven slightly upregulated in the GLO-silenced plants(Fig. 5C, D, E).

Discussion

Lines of evidence have shown that, when GLO wassuppressed in plants, photosynthesis was inhibited andsubsequently photoinhibition occurred (Yamaguchi andNishimura 2000, Zelitch et al. 2009, Xu et al. 2009, Fig.2). The question is that how does the reduced GLOinhibit photosynthesis? Our previous work showed thatRubisco activation state was correspondingly reducedas Pn was inhibited in the GLO-antisensed plants (Xuet al. 2009), and in this study, it is further revealed thatreduction in Pn is directly proportional to the Rubiscoactivation state in the GLO-suppressed plants (Fig. 3B).It appears that Rubisco activation state is involved in theGLO-regulated photosynthesis. It is easily believed thatRCA should be the most possible target to be regulated byGLO in this case. We have previously reported that the

transcriptional expression of RCA was downregulatedin response to GLO suppression (Xu et al. 2009).Even though the same transcriptional patterns are alsodetected in this current study (Fig. 3C), our proteomicsdata and western blot assay show that the RCA proteinabundance is not altered (Table 1, Fig. 3D). This resultdoes not support the possibility that RCA is involvedin the GLO-mediated regulation of photosynthesis, eventhough it is still possible that the catalytic activity of RCAcould be involved. Yamori and Caemmer (2009) alsonoticed that temperature response of Rubisco activationwas not intimately dependent on the RCA content intobacco plants under heat stress, and they proposed thatthere could exist other alternative factors modulatingRubisco activation (Yamori and Caemmer 2009). Morethan 20 years ago, Campbell and Ogren (1990a) used tosuggest that there could be a component in the in vivoRubisco activation system that is not yet identified andcould be inhibited by glyoxylate.

It is unexpected that glyoxylate accumulation alsooccurred in the GLO-suppressed plants (Fig. 4A).Pick et al. (2013) recently showed that glyoxylate wasalso somewhat accumulated albeit less than glycolate,


Fig. 5. Response of some other enzymes for glyoxylate metabolism to GLO suppression. WT and RNAi plants were grown under normal naturalcondition as described in Fig. 1. At five-leaf stage, 2–3 cm tips from the youngest fully expanded leaf was first detached to determine GLO activity,and then according to the activity determined, those individuals with about 15 and 25% GLO activities were respectively grouped, and the expressionlevels or activities of the enzymes were determined. (A): ICL; (B): GDH; (C): SGAT/GGAT; (D): GR; (E): HPR. The data represent means of threebiological replicates ± SD. Different lowercase letters in the same column indicate significant differences at P < 0.05.

when a chloroplastic glycolate transporter (PLGG1) wasmutated. More interestingly, we found that the Pn isinversely proportional to the glyoxylate concentrationwhile directly proportional to the Rubisco activationstate (P < 0.01, Figs 3B and 4C). On the basis of theabove two function equations experimentally estab-lished (Figs 3B and 4C), a fitting equation was con-structed using least square method through MATLAB soft-ware (MathWorks Inc., Natick, MA). The result indicatesthat it is highly likely that Rubisco activation state isinversely correlated with glyoxylate concentrations inrice (y = −439.6x + 477.1, P < 0.01). It has been welldocumented that glyoxylate has the ability to inhibitCO2 fixation in chloroplasts (Oliver and Zelitch 1977,Enser and Heber 1980, Flugge et al. 1980, Lawyer et al.

1983, Mulligan et al. 1983, Chastain and Ogren 1989,Campbell and Ogren 1990a). In the mutants, wheredefects occurred in the photorespiratory GDC, SGAT,SHMT, chloroplast dicarboxylate transporter (DCT) andGOGAT, glyoxylate was accumulated, and both Pn andRubisco activation state were simultaneously decreased(Chastain and Ogren 1985). Chastain and Ogren (1989)further demonstrated that, among the photorespiratoryintermediates such as glycolate, glyoxylate, glycine,α-oxoglutarate and glutamate, only glyoxylate can effec-tively inhibit Rubisco activation in chloroplasts. Glyoxy-late appears to inactivate Rubisco in an indirect mannerdependent of photosynthetic electron transport system(Campbell and Ogren 1990a, 1990b). The mechanismhas been proposed in several aspects. It may directly


inhibit Rubisco by inducing reductions under stromalpH conditions (Enser and Heber 1980, Flugge et al.1980, Mulligan et al. 1983) and in appropriate NADPHconcentration as mediated by the GR-catalyzed reaction(Oliver and Zelitch 1977, Lawyer et al. 1983, Mulliganet al. 1983, Cook et al. 1985). But a subsequent analysisby Campbell and Ogren (1990a) seemingly negated theabove possibilities and the author considered that a yet-unidentified factor(s) was present in intact chloroplastwhich may be involved in activation of Rubisco in vivo,and that glyoxylate may interact with this factor(s) undercertain conditions and act as a metabolic regulator forRubisco activation in vivo (Campbell and Ogren 1990a).Overall, more data are still needed to fully explain howglyoxylate regulates Rubisco, but our current data firmlysupport that glyoxylate is involved in the GLO-regulatedphotosynthesis in rice.

Another question is how glyoxylate is accumulated inresponse to GLO suppression. On the basis of the avail-able knowledge, glyoxylate content should be decreasedwhen GLO is suppressed. That is because glyoxylate isthe product from the GLO-catalyzed oxidation of gly-colate. Though it is hard to understand this currentoutcome, it can be reasonable to suspect that there mayexist alternative reactions which could complement gly-oxylate, and that these reactions would be localizedto organelles beyond the peroxisome, where glyoxylatecould not be easily metabolized like within the perox-isome. To the best of our knowledge, in addition tothe GLO-catalyzed reaction, there are two additionalsources. The first is isocitrate cleavage catalyzed by ICL(Hayashi et al. 1995). However, we found that the ICLcatalytic activity was not changed in response to GLOsuppression (Fig. 5A). Thus it seems less likely for theisocitrate cleavage reaction to compensate for glyoxy-late formation in response to GLO suppression. A GDHwas reported in mitochondria of Arabidopsis and rice(Bari et al. 2004, Niessen et al. 2007, 2012). In fact, wefailed to detect the GDH activity in rice and also nodifferences were found in its both mRNA and proteinlevels after GLO was suppressed (Fig. 5B). Actually, itwas later on demonstrated that this GDH much preferredto use D-lactate as its substrate and should be a D-lactatedehydrogenase (Engqvist et al. 2009, 2011). Thus, its realfunction is still not certain. In addition, we have demon-strated that the enzymes responsible for metabolizingglyoxylate, such as SGAT, GGAT, GR1, GR2, HPR1,HPR2, were not suppressed and even increased in theGLO-suppressed plants (Fig. 5C, D, E). A chloroplasticglycolate-oxidizing system was reported in spinach andalgae (Murai and Katoh 1975, Goyal and Tolbert 1996,Goyal 2002), which was indirectly supported by thework of Cegelski and Schaefer (2006) as they found that

the rate of photorespiratory CO2 release was significantlyless than the oxygenation activity of Rubisco. Morerecent data still implicate that chloroplasts are capableof actively producing glyoxylate from glycolate (Kebeishet al. 2007, Blume et al. 2013). We have also tried manytimes to detect both glycolate-oxidizing activities andglyoxylate abundance in rice chloroplasts, but no reli-able data could be achieved. The failure is likely due tothe difficulty in isolating well integrated rice chloroplasts.Thus, how glyoxylate is accumulated in response to GLOsuppression remains to be further investigated.

Acknowledgements – This work was supported by theNational Natural Science Foundation of China (U1201212,31170222). The authors would like to thank Dr De-An Jiang(College of Life Science, Zhejiang University) for providingthe RCA monoclonal antibody, and Dr Chang-hong Guo(College of Science, South China Agricultural University)for helping establish the fitting equation. The proteomicssample labeling and 2D-LC-MS/MS data acquisitions havebeen conducted in Proteomics and Mass SpectrometryFacility at Cornell University.

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Suppression of glycolate oxidase causes glyoxylate accumulation that inhibits photosynthesis through deactivating Rubisco in rice