2005 Plant Cell Enviromnent

Plant Cell and Environment

(2005)

28

898ndash905

898

copy 2005 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKPCEPlant Cell and Environment0016-8025Blackwell Science Ltd 2005 2005

28898905Original Article

Development of kudzu isoprene emissionA E Wiberley

et al

Correspondence Thomas Sharkey Fax

+

1 (608) 262 7509 e-mailtsharkeywiscedu

Development of the capacity for isoprene emission in kudzu

AMY E WIBERLEY AUTUMN R LINSKEY TANYA G FALBEL amp THOMAS D SHARKEY

Department of Botany University of Wisconsin-Madison 430 Lincoln Drive Madison WI 53706 USA

ABSTRACT

Isoprene is a biogenic hydrocarbon that has significanteffects on tropospheric chemistry It is emitted by a num-ber of plant species including kudzu a leguminous vinethat grows profusely in the south-eastern United StatesThis study investigated development of the capacity forisoprene emission in kudzu Previous studies examinedisoprene emission during leaf development but a molecu-lar explanation for the observed developmental delay inemission was lacking This study found that kudzu leavesgrown at a high temperature could emit isoprene at leasta week before they were fully expanded and 1 d afterbecoming photosynthetically competent When grown atlow temperature however leaves did not emit isopreneuntil 1 week after they became fully expanded and 2weeks after the onset of photosynthetic competence Lev-els of mRNA and protein for isoprene synthase whichcatalyses the final step in isoprene biosynthesis wereinvestigated it was found that transcription and transla-tion of this gene began at the same developmental stageas onset of emission in both growth conditions Thereforeplant growth conditions not leaf developmental stagehave primary control over expression of isoprene synthaseand onset of kudzu isoprene emission This finding maybe useful in modelling early season isoprene emissionrates

Key-words

atmospheric chemistry isoprene isoprenesynthase kudzu photosynthesis temperature

INTRODUCTION

Isoprene (C

5

H

8

) is an abundant biogenic hydrocarbon inthe atmosphere (Fehsenfeld

et al

1992) Some plant spe-cies including many trees and the leguminous vinekudzu can emit large amounts of isoprene others emitnone (Sharkey amp Yeh 2001) As global isoprene emissionrates are very large [400 Tg per year (Guenther

et al

1995 Fuentes

et al

2000)] and isoprene is quite reactivewith hydroxyl radicals and nitrogen oxides in the atmo-sphere isoprene plays a significant role in atmosphericchemistry (Thompson 1992 Monson amp Holland 2001) As

such the ability to predict isoprene emission rates isquite important and for such predictions an understand-ing of the mechanisms that control isoprene emission isnecessary

Isoprene emission from plants is correlated with toler-ance of short high-temperature episodes (Sharkey Chen ampYeh 2001 Pentildeuelas

et al

2005 Velikova amp Loreto 2005Sharkey 2005) as well as with oxidative stress (Loreto ampVelikova 2001 Affek amp Yakir 2002) The role of isoprenein thermotolerance is consistent with the observation thatisoprene emission capacity increases when damaging hightemperatures are more likely such as when the ambienttemperature exceeds 30

infin

C or in high light Mature leavesof emitting species can be induced to emit isoprene byexposure to high temperatures when grown in a coolgreenhouse in winter kudzu does not emit isoprene but agreenhouse-grown leaf can be induced to emit if held at30

infin

C for 6 h (Sharkey amp Loreto 1993) Isoprene emissionis lower in the spring and autumn than in midsummer(Monson

et al

1994 Goldstein

et al

1998) and isopreneemission capacity varies throughout the season (SchnitzlerLehning amp Steinbrecher 1997 Fuentes amp Wang 1999 Shar-key

et al

1999 Fuentes Wang amp Gu 1999 Geron

et al

2000) The lack of emission in the spring is believed toresult from a delay in isoprene emission capacity duringleaf development (Grinspoon Bowman amp Fall 1991Kuzma amp Fall 1993 Sharkey amp Loreto 1993 Harley

et al

1994 Monson

et al

1994) and most subsequent studies ofisoprene emission have been done on fully expandedleaves (eg Wildermuth amp Fall 1996) since developingleaves had been found to emit considerably less if anyisoprene

Isoprene is synthesized from dimethylallyl diphosphate(DMAPP) made by the chloroplastic methylerythritol 4-phosphate pathway (Schwender

et al

1997) Isoprene syn-thase (IspS) converts DMAPP to isoprene (Silver amp Fall1991) The activity of isoprene synthase can control iso-prene emission rate (Kuzma amp Fall 1993 Schnitzler

et al

1997 Sharkey

et al

2005) IspS activity can in turndepend on transcriptional translational or post-transla-tional regulatory mechanisms This study was conducted toelucidate the influence of leaf developmental stage on thecapacity for isoprene emission from kudzu Isoprene emis-sion rates and IspS mRNA and protein quantities weremeasured for leaves developing in 20 or 30

infin

C greenhouserooms IspS protein was found in both soluble and insolu-ble leaf fractions so these were measured separately and

Development of kudzu isoprene emission

899



28

898ndash905

correlated with isoprene emission rates throughout leafdevelopment

MATERIALS AND METHODS

Plant growth conditions

Kudzu plants (

Pueraria montana

var

lobata

(Willd) Mae-sen amp S Almeida) were grown from stem cuttings in 10-Lpots containing a vermiculitepeat moss-based growthmedium (Metro-Mix 360 The Scotts Company Marys-ville OH USA) The plants were grown in temperature-controlled greenhouses at the Biotron facility of theUniversity of Wisconsin-Madison Three plants weregrown at 2016

infin

C daynight temperature with noextended day-length whereas two were grown at 3020

infin

Cdaynight temperature and day-length extended to 16 hwith high pressure sodium vapour lamps Plants werewatered with varying strength Hoaglandrsquos solution (Hoag-land amp Arnon 1938) and fertilized with 14-14-14 N-P-KOsmocote according to the manufacturerrsquos instructions(The Scotts Company)

Leaf measurement and sample collection

Leaves near the tops of the plants were tagged when theywere 1ndash2 cm in length and were measured daily thereafteruntil they were fully expanded Leaf length was measuredfrom the junction of the terminal leaflet blade and petiolealong the midrib to the tip of the blade Samples werecollected from mid-July up to the end of August 2004 andleaf measurements and collections were carried outbetween 1030 and 1230 h All tissue samples were collectedclose to the tips of the lateral leaflet blades and were frozenimmediately in dry ice and then stored at

-

80

infin

C until useSamples were collected in leaf lengths of 3ndash4 cm 4ndash5 cm

5ndash6 cm 6ndash7 cm and 7ndash8 cm also 24 h past full expansion1 week past full expansion and 2 weeks past full expansionThese categories were selected because healthy kudzuleaves can have a final length ranging from 8 to 20 cm andrarely stop growing before reaching 8 cm (unpublishedobservation) Collected leaf punches were 933 mm

2

inarea

Gas exchange and isoprene emission measurement

Gas exchange measurements were conducted as describedby Wolfertz

et al

(2003) all measurements were made at30

infin

C and 1000

m

mol m

-

2

s

-

1

The air source for the Li-Cor6400 (Li-Cor Inc Lincoln NE USA) was a cylinder ofcompressed air Ten millilitres of the air exiting the leafcuvette were collected by syringe and analysed for isoprenecontent by gas chromatography as described by Loreto ampSharkey (1993) with the following modifications the col-umn was maintained at 52

infin

C and the liquid isoprene stan-dard was serially diluted to 128 nmol mol

-

1

in N

2

Leafisoprene emission rates were calculated as described bySingsaas

et al

(1997)

RNA extraction and quantitative polymerase chain reaction analysis

Total RNA was extracted from leaf samples as follows Aleaf punch was ground in liquid nitrogen with a mortar andpestle Extraction buffer [01

M

Tris-HCl (Fisher ScientificPittsburgh PA) pH 80 01

M

LiCl 02 m

M

ethylenedi-aminetetraacetic acid(EDTA) 1 wv sodium dodecyl shl-phate (SDS) 50 vv phenol] heated to 80

infin

C was added(500

m

L per 10 mg tissue) and then homogenized The mix-ture was transferred to a polypropylene tube and shaken vig-orously 100

m

L 24 1 chloroform isoamyl alcohol wereadded and the tube was shaken again and then centrifugedat 12 000

yen

g

at 4

infin

C for 5 min The upper phase solution wascollected and precipitated with an equal volume of 4

M

LiClat 4

infin

C for 2 h This solution was centrifuged at 12000

yen

g at4

infin

C for 10 min the supernatant was removed and the pelletwas resuspended in 700

m

L diethylpyrocarbonate (DEPC)-treated H

2

O by pipetting This was extracted with 25 24 1phenol chloroform isoamyl alcohol twice and with 24 1chloroform isoamyl alcohol once Three molar sodium ace-tate (110 volume) and isopropanol (1 volume) were addedto the resulting solution and RNA was precipitated over-night at

-

20

infin

C This solution was centrifuged at 12000

yen

g

for 10 min the supernatant was removed and the pellet waswashed once with 70 ethanol and once with 100 ethanolThe pellet was air-dried and redissolved in 30ndash50

m

L DEPC-treated H

2

O All chemicals were obtained from Sigma-Aldrich (St Louis MO USA) unless otherwise noted

RNA concentration was determined using a BeckmanDUcopy 640 spectrophotometer (Beckman Coulter Inc Ful-lerton CA USA) and 0125

m

g from each sample werereverse transcribed using oligo(dT)15 primer and M-MLVreverse transcriptase according to the manufacturerrsquosinstructions (Promega Corp Madison WI

lt

USA) Quan-titative polymerase chain reaction (PCR) was then carriedout on 1 or 2

m

L of the reverse transcriptase (RT) productusing an Mx3000P

TM

real-time PCR system with Brilliant

reg

SYBR

reg

green QPCR master mix (Stratagene La JollaCA USA) according to the manufacturerrsquos instructionsThe primers used were forward 5

cent

-TGGCGAGTTATTTGTGCT-3

cent

reverse 5

cent

-CCTCTAGCTTTGTTGCCTT-3

cent

these span the first intron of the gene which is 357 bp long(Sharkey

et al

2005) and therefore prevented amplifica-tion of any contaminating genomic DNA The thermal pro-file was 95

infin

C for 10 min 40 cycles of 95

infin

C for 30 s 51

infin

Cfor 1 min and 72

infin

C for 30 s 95

infin

C for 1 min 45 cycles of30 s each with temperature beginning at 51

infin

C and increas-ing 1

infin

C per cycle The target sequence had previously beenamplified from a kudzu RNA preparation and reverse tran-scription with concentration determined as described forRNA extracts Dilutions of this each containing a knownnumber of copies of the target amplicon were used toprepare a standard curve that was used to quantify thePCR product in the plant samples To prevent the forma-tion of primer dimers which would give inaccurate quanti-tation each plant samplersquos reaction mixture was spikedwith 1000 copies of the amplicon this number was then

900

A E Wiberley

et al



28

898ndash905

subtracted from the copy number calculated by theMx3000P software

Protein extraction and western blot analysis

Total soluble protein was extracted from tissue samples asdescribed by Heck

et al

(1995) The extract was centrifugedfor 5 min at 12 000

yen

g

The supernatant from this extractionwas separated from the pellet and the pellet was mixed witha volume of SDS sample buffer (1

M

Tris-HCl pH 68 10SDS 10

a

-mercaptoethanol 20 glycerol 0004 bro-mphenol blue) equal to the volume of the pellet vortexedand heated at 70

infin

C for 20 min This was centrifuged brieflyat 12 000

yen

g to bring down cell wall fragments leaving mem-brane-associated proteins in the supernatant Soluble andmembrane protein fractions were separated on NuPAGE

TM

NOVEX 4ndash12 Bis-Tris gels (Invitrogen Corp CarlsbadCA USA) and transferred to Hybond-P PVDF membranes(Amersham Biosciences Piscataway NJ USA) using theNOVEX XCellII

TM

Mini-Cell system as directed by themanufacturer (Invitrogen Corp) Immunoreactive proteinwas detected using the ECL western blotting system accord-ing to manufacturerrsquos instructions (Amersham Biosciences)A polyclonal primary antibody for IspS was generated usingkudzu IspS cDNA expressed in a pET15b vector in

Escher-ichia coli

and subjected to 10 denaturing PAGE (Sharkey

et al

2005) The primary antibody was diluted 1 1250 andthe secondary donkey antirabbit antibody coupled to horse-radish peroxidase was diluted 1 5000 Blots were exposedto Kodak (Rochester NY USA) Biomax MS X-ray film for5ndash20 min Membranes were stained with Coomassie blue tovisualize all of the protein and check for equal loading Theisoprene synthase used as a standard was purified from

Ecoli

as previously described (Sharkey

et al

2005) and its con-centration determined by Bradford assay (Bradford 1976)Western blot films were scanned and bands were quantitatedusing I

MAGE

J (US National Institutes of Health httprsbinfonihgovij)

IspS activity assays

A number of different extraction protocols were attemptedfor extraction of isoprene synthase activity from kudzu leafsamples In general leaf punches were ground in extractionbuffer and centrifuged briefly at 12 000

yen

g to bring downcell wall fragments and the supernatants were assayedSeveral buffers were tested EB [50 m

M

Tris-HCl pH 8020 m

M

MgCl

2

5 glycerol with 10 polyvinyl polypyrroli-done 20 m

M

dithiothreitol (DTT) 1 m

M

phenylmethylsul-phonyl fluoride (PMSF) and 1 m

M

benzamidine-HCladded immediately before use (Silver amp Fall 1991)] EBT[EB with 05 Triton X-100 added (Schnitzler

et al

1996)]and EBC [extraction buffer with 15 m

M

3-[(3-cholami-dopropyl) dimethyl amino]-l-propanesulfate (CHAPS)added] In some cases the supernatants were concentratedwith a 30ndash60 (NH

4

)

2

SO

4

precipitation the pellet from the60 cut was re-suspended in an equal volume of assaybuffer and then assayed The assay buffer was 50 m

M

bicine

pH 80 50 m

M

MgCl

2

5 m

M

KCl 2 m

M

NaF and 5 glyc-erol Assay reaction mixtures were 20

m

L assay buffer 1

m

L1

M

DTT 10

m

L 50 mM DMAPP 1 mL (22024 mg) IspSpurified from E coli and 9 mL extract extraction buffer orwater DMAPP was synthesized as described by DavissonWoodside amp Poulter (1985) These mixtures were incubatedat 35 infinC for 15 min in 55-mL sealed vials after which 3 mLof headspace were removed by syringe with water addedto the vial simultaneously to equalize pressure and analysedfor isoprene in the manner described for plant gas samples

RESULTS

Development of isoprene emission and photosynthesis capacities

Leaves grown in inductive conditions (30 infinC daytime tem-perature) emitted isoprene about a week before theybecame fully expanded while leaves grown in non-inductive conditions (20 infinC daytime temperature) did notemit isoprene until after full expansion when they emittedsubstantially less than their 30 infinC counterparts (Fig 1)Leaves grown at 30 infinC began to emit when they were 5ndash6 cm long since kudzu leaves grown in these conditionstypically lengthen about 1 cm per day until they near theirfinal size and then grow more slowly for about 3 d beforegrowth stops (unpublished observation) 5ndash6 cm leaveswere at least a week away from reaching their final size ofge8 cm Emission rates for 30 infinC-grown leaves increased

Figure 1 Basal isoprene emission rates for kudzu leaves of different developmental stages grown at 20 and 30 infinC Emission rates were measured at 30 infinC and 1000 mmol m-2 s-1 light Each value is the average of three leavesrsquo emission rates plusmn standard error Light bars 20 infinC leaves dark bars 30 infinC leaves The detection limit was 5 nmol m-2 s-1 lower rates were indistinguishable from signal noise

Development of kudzu isoprene emission 901

copy 2005 Blackwell Publishing Ltd Plant Cell and Environment 28 898ndash905

during the first week past full expansion and then nearlydoubled at 2 weeks past full expansion Leaves grown at20 infinC however did not emit significant amounts of iso-prene (ge 5 nmol m-2 s-1 lower quantities were indistin-guishable from noise) until after they became fullyexpanded even then their emission rates were only about13 nmol m-2 s-1 which is less than the 30 infinC-grown leavesemitted when they were 6ndash7 cm long and at least 5 d awayfrom full expansion

Photosynthesis rates were substantially greater thanzero for 30 infinC-grown leaves 4ndash5 cm long and larger andfor 20 infinC-grown leaves 6ndash7 cm long and larger (Fig 2) In30 infinC-grown leaves the development of photosyntheticcompetence preceded the onset of isoprene emission byabout 1 d whereas in 20 infinC-grown leaves photosyntheticcompetence preceded isoprene emission by about 2weeks

IspS mRNA copy numbers

When quantitative RT-PCR was performed on samplesexpected to have low IspS copy numbers [based on lack ofIspS amplification in non-quantitative RT-PCR (data notshown)] the QPCR software calculated very large copynumbers The dissociation curves from these reactionsshowed peaks at slightly higher and lower temperaturesthan 775 infinC the melting point of this amplicon (Fig 3) soit appeared that the SYBRreg green dye being used for detec-tion was binding to amplified primer dimers and these werebeing reported as copies of the IspS amplicon This wasobserved only in samples with few or no IspS copies so it

seemed that the lack of sufficient target template was allow-ing dimer formation All reactions were therefore spikedwith 1000 copies of the amplicon eliminating the formationof primer dimers (Fig 3) and allowing accurate templatequantification

IspS mRNA first appeared in 30 infinC-grown leaves whenthey were 5ndash6 cm long the same stage at which they beganto emit With the exception of the samples taken at 1 weekpast full expansion mRNA levels increased from 5ndash6 up to7ndash8 cm leaves and then levelled off (Fig 4) The 20 infinC-grown leaves however did not have significant IspS mRNApresent until 2 weeks after full expansion at which timethey had as much message as the 30 infinC-grown leaves(Fig 4)

IspS protein quantities

For 30 infinC-grown leaves small amounts of IspS protein firstappeared in the membrane fraction of 5ndash6 cm leaves inleaves 7ndash8 cm and larger it was detected in both membraneand soluble fractions (Fig 5) Both membrane-bound andsoluble IspS levels increased throughout leaf development(Table 1) The IspS from the soluble fraction ran slightlyfurther on the gel than that of the membrane fraction(Fig 5) In 20 infinC-grown leaves IspS protein appeared inthe membrane and soluble fractions of leaves that were 1and 2 weeks past full expansion As was observed for 30 infinC-grown leaves the soluble IspS ran to a slightly smallermolecular weight than did the membrane-bound IspS(Fig 5) For all leaves levels of membrane-bound solubleand total IspS correlated well with isoprene emission rates(Fig 6)

Figure 2 Photosynthesis rates for kudzu leaves of different developmental stages grown at 20 and 30 infinC Rates were measured at 30 infinC and 1000 mmol m-2 s-1 light Each value is the average of three leavesrsquo photosynthesis rates plusmn standard error Light bars 20 infinC leaves dark bars 30 infinC leaves

Figure 3 Dissociation curves for quantitative PCR reactions of samples with low IspS copy numbers with and without a spike of 1000 copies of the amplicon Dotted line unspiked reaction solid line spiked reaction Template was from 20 infinC-grown 3ndash4 cm leaves Melting temperature of the IspS amplicon = 775 infinC

902 A E Wiberley et al


IspS enzyme activity

Attempts were made to extract IspS activity in several buff-ers but it was found that some compound present in kudzuwhole-leaf extracts inhibited IspS activity Reactions run

with purified IspS added showed a decrease in IspS activitywhen leaf extracts were added (Fig 7) and reactions runwithout added IspS showed no significant IspS activity(data not shown) EB itself did not inhibit added IspS activ-ity but EBT did inhibit EBC on the other hand increasedthe activity of added IspS Kudzu crude extracts in EB andEBC both inhibited the added IspS activity as did ammo-nium sulphate precipitates of these crude extracts (Fig 7)

DISCUSSION

Kudzu leaves are capable of emitting isoprene well beforethey reach full expansion when they are grown in inducingconditions whereas growth in non-inducing conditionsdelays the onset of emission by about 2 weeks (Fig 1)Emission from 30 infinC-grown leaves begins about 1 d afterthe leaves become photosynthetically competent whereasemission from 20 infinC-grown leaves does not begin untilabout 2 weeks after photosynthesis rates rise above zero(Figs 1 amp 2) Grinspoon et al (1991) found that velvet beanisoprene emission begins about 3 d after photosyntheticcompetence in plants grown at 28 infinC at about the sametime as leaves become fully expanded these findings weresupported by Harley et al (1994) with velvet bean plantsgrown at 30 infinC Thus the onset of isoprene emission followsthe onset of photosynthetic competence but the amount oftime between the two events varies This is also consistentwith the observations of Monson et al (1994) who foundthat poplar leaves that developed in cool spring tempera-tures had a delay of about 2 weeks between the onsetsof photosynthetic competence and isoprene emissionwhereas for leaves that developed in the summer the delaywas about 2 d In that study data was collected based on

Figure 4 IspS mRNA copy numbers for different developmental stages and growth temperatures Each value is the number of copies in 1 pg total RNA bars represent standard error n = 3 Light bars 20 infinC leaves dark bars 30 infinC leaves The detection limit was 02 copies pg-1

Figure 5 Western blots showing IspS protein in leaves of different developmental stages and growth temperatures The detected proteins were about 65 kDa (a) 20 infinC-grown leaves (b) 30 infinC-grown leaves M membrane fraction S soluble fraction (+) positive control M lanes were loaded with protein from 0112 cm2 of leaf S lanes with protein from 00280 cm2 of leaf Both positive control lanes were loaded with 1875 ng IspS

Table 1 IspS protein found in emitting leaves

20 infinC membrane 20 infinC soluble 30 infinC membrane 30 infinC soluble

3ndash4 cm 0 0 0 04ndash5 cm 0 0 0 05ndash6 cm 0 0 028 plusmn 009 06ndash7 cm 0 0 050 plusmn 007 163 plusmn 0307ndash8 cm 0 0 024 plusmn 011 113 plusmn 04624 h 0 0 048 plusmn 026 223 plusmn 0711 w 004 plusmn 000 011 plusmn 000 105 plusmn 006 439 plusmn 0462 w 007 plusmn 004 027 plusmn 009 135 plusmn 016 552 plusmn 014

Values are in mg protein per square metre of leaf plusmn standard error n = 3



days since leaf emergence so it was unclear whether themore rapid onset of emission in the summer was due todirect induction of emission by high temperature or to aheat-induced increase in the rate of leaf development witha concomitant decrease in the delay between photosyn-thetic competence and emission The present study demon-strates that the former is the case in kudzu Kudzu leavesdo develop more rapidly when grown at higher tempera-tures (unpublished observation) but when leaves are anal-ysed based on developmental stage rather than time pastemergence it is clear that the onset of isoprene emission isgoverned more by plant growth conditions than by leafdevelopmental stage

IspS mRNA and protein appear in kudzu leaves at thesame developmental stage as the onset of emission (Figs 4amp 5) gene expression does not significantly precedeenzyme activity This indicates that much control over IspSactivity is exerted at the level of IspS transcription There

does not seem to be any specific developmental stage atwhich IspS transcription is always turned on in kudzuleaves However this does not rule out post-translationalregulation of IspS activity Both membrane-bound and sol-uble forms of IspS were found in kudzu as have been foundin willow (Wildermuth amp Fall 1998) Wildermuth (1997)suggested that the membrane-bound willow IspS may bepalmitoylated associating it with the thylakoid membraneand increasing its activity The membrane-bound form inkudzu ran to a slightly higher molecular weight than thesoluble form in SDS-PAGE analysis indicating a possiblepost-translational modification However membrane-bound and soluble protein levels both correlated well withisoprene emission rates (Fig 6) so it does not seem thatany post-translational modification that might occur has aneffect on IspS activity The high correlation between iso-prene emission rate and IspS protein levels could providea basis for predictions of isoprene emissions in mixed for-ests In such forests measuring the isoprene emission ratesof leaves at the top of the canopy is often physically impos-sible but leaf samples could be obtained and their IspScontent determined and used to predict emission rate if anantibody that cross-reacted with the IspS of many specieswere available The range of cross reactivity of our antibodyhas not yet been determined

Numerous attempts were made to measure IspS activityin kudzu leaf extracts but none was successful First it wasfound that Triton X-100 a component of some IspS extrac-

Figure 6 Correlation of membrane-bound soluble and total IspS protein quantities in emitting leaves with basal isoprene emission rates (emission rates at 30 infinC and 1000 mmol m-2 s-1 light)

Figure 7 Suppression of IspS activity by kudzu whole-leaf extracts lsquoCrudersquo indicates a crude extract in the specified buffer lsquopptrsquo the re-suspended 60 (NH4)2SO4 pellet from a crude extract



tion buffers (eg Schnitzler et al 1996) inhibits the activityof kudzu IspS Other buffer components did not themselvesinhibit activity but whenever kudzu leaf extract was addedto reaction mixtures containing purified IspS the activity ofthat IspS decreased by 29ndash99 It therefore seems thatkudzu whole-leaf extracts contain some compound thatinhibits kudzu IspS activity This compound could not beremoved by ammonium sulphate precipitation indeedsuppression of activity became more pronounced after pre-cipitation It would probably be possible to obtain IspSactivity from isolated kudzu chloroplasts since if the inhib-itor were present in chloroplasts kudzu would probably notemit as much isoprene as it does but activity obtained fromthis isolation would not be sufficiently quantitative to fitwith the aims of the present study Therefore it was notpossible within this study to determine how in vitro IspSactivity changed over the course of leaf development orwhether the membrane-bound or soluble form was moreactive

The observation that heat-grown leaves develop thecapacity for isoprene emission much sooner than their cool-grown counterparts is consistent with the thermotolerancehypothesis for isoprene emission This hypothesis statesthat plants emit isoprene to protect against damage causedby brief high-temperature episodes (Sharkey amp Yeh 2001)Such episodes are more likely to be experienced when aplant is already exposed to high temperatures as were the30 infinC-grown plants in this study Plants grown in such con-ditions commonly have a higher isoprene emission capacitythan cool-grown plants (Sharkey amp Loreto 1993) It hasbeen observed that temperatures experienced during the2 d preceding measurement of a leafrsquos isoprene emissioncapacity exert considerable influence over the emissioncapacity (Sharkey et al 1999) this study indicates that thisis the case in developing as well as full-grown leaves

The results of this study show that early season isopreneemission depends on growth conditions mediated by tran-scriptional control of IspS Kudzu leaves grown at hightemperature express IspS and emit much sooner thanleaves grown at low temperature This information may beuseful in predictions of early season isoprene emissionWork is currently underway to determine how variation ofisoprene emission in the middle of a growing season isregulated

ACKNOWLEDGMENTS

This research was supported by the National Science Foun-dation grant IBN-0212204

REFERENCES

Affek HP amp Yakir D (2002) Protection by isoprene against sin-glet oxygen in leaves Plant Physiology 129 269ndash277

Bradford MM (1976) A rapid and sensitive method for the quan-titation of microgram quantities of protein utilizing the principleof protein-dye binding Analytical Biochemistry 72 248ndash254

Davisson VJ Woodside AB amp Poulter CD (1985) Synthesis ofallylic and homoallylic isoprenoid pyrophosphates Methods inEnzymology 110 130ndash144

Fehsenfeld FC Calvert J Fall R et al (1992) Emissions ofvolatile organic compounds from vegetation and the implica-tions for atmospheric chemistry Global Biogeochemical Cycles6 389ndash430

Fuentes JD amp Wang D (1999) On the seasonality of isopreneemission from a mixed temperate forest Ecological Applications9 1118ndash1131

Fuentes JD Lerdau M Atkinson R et al (2000) Biogenichydrocarbons in the atmospheric boundary layer a review Bul-letin of the American Meteorological Society 81 1537ndash1575

Fuentes JD Wang D amp Gu L (1999) Seasonal variations inisoprene emissions from a boreal aspen forest Journal ofApplied Meteorology 38 855ndash869

Geron C Guenther A Sharkey T amp Arnts RR (2000) Temporalvariability in basal isoprene emission factor Tree Physiology 20799ndash805

Goldstein AH Goulden ML Munger JW Wofsy SC amp GeronCD (1998) Seasonal course of isoprene emissions from a mid-latitude deciduous forest Journal of Geophysical Research-Atmospheres 103 31045ndash31056

Grinspoon J Bowman WD amp Fall R (1991) Delayed onset ofisoprene emission in developing velvet bean (Mucuna sp)leaves Plant Physiology 97 170ndash174

Guenther A Hewitt CN Erickson D et al (1995) A globalmodel of natural volatile organic compound emissions Journalof Geophysical Research ndash Atmospheres 100 8873ndash8892

Harley PC Litvak ME Sharkey TD amp Monson RK (1994)Isoprene emission from velvet bean leaves Interactions amongnitrogen availability growth photon flux density and leaf devel-opment Plant Physiology 105 279ndash285

Heck GR Perry SE Nichols KW amp Fernandez DE (1995)AGL15 a MADS domain protein expressed in developingembryos Plant Cell 7 1271ndash1282

Hoagland DR amp Arnon DI (1938) The Water Culture Methodfor Growing Plants Without Soil University of California Agri-cultural Experiment Station Circular 347 pp 1ndash39 Universityof California Berkley CA USA

Kuzma J amp Fall R (1993) Leaf isoprene emission rate is depen-dent on leaf development and the level of isoprene synthasePlant Physiology 101 435ndash440

Loreto F amp Sharkey TD (1993) On the relationship betweenisoprene emission and photosynthetic metabolites under differ-ent environmental conditions Planta 189 420ndash424

Loreto F amp Velikova V (2001) Isoprene produced by leaves pro-tects the photosynthetic apparatus against ozone damagequenches ozone products and reduces lipid peroxidation of cel-lular membranes Plant Physiology 127 1781ndash1787

Monson RK amp Holland EA (2001) Biospheric trace gas fluxesand their control over tropospheric chemistry Annual Reviewof Ecology and Systematics 32 547ndash576

Monson RK Harley PC Litvak ME Wildermuth M Guen-ther AB Zimmerman PR amp Fall R (1994) Environmentaland developmental controls over the seasonal pattern of iso-prene emission from aspen leaves Oecologia 99 260ndash270

Pentildeuelas J Llusiagrave J Asensio D amp Munneacute-Bosch S (2005) Link-ing isoprene with plant thermotolerance antioxidants andmonoterpene emissions Plant Cell and Environment 28 278ndash286

Schnitzler J-P Arenz R Steinbrecher R amp Lehning A (1996)Characterization of an isoprene synthase from leaves of Quercuspetraea (Mattuschka) Liebl Botanica Acta 109 216ndash221

Schnitzler J-P Lehning A amp Steinbrecher R (1997) Seasonalpattern of isoprene synthase activity in Quercus robur leaves



and its significance for modeling isoprene emission rates Botan-ica Acta 110 240ndash243

Schwender J Zeidler J Groner R Muller C Focke M BraunS Lichtenthaler FW amp Lichtenthaler HK (1997) Incorpora-tion of 1-deoxy-D-xylulose into isoprene and phytol by higherplants and algae FEBS Letters 414 129ndash134

Sharkey TD (2005) Effects of moderate heat stress on photosyn-thesis importance of thylakoid reactions rubisco deactivationreactive oxygen species and thermotolerance provided by iso-prene Plant Cell and Environment 28 269ndash277

Sharkey TD amp Loreto F (1993) Water stress temperature andlight effects on the capacity for isoprene emission and photosyn-thesis of kudzu leaves Oecologia 95 328ndash333

Sharkey TD amp Yeh S (2001) Isoprene emission from plantsAnnual Review of Plant Physiology and Plant Molecular Biology52 407ndash436

Sharkey TD Chen XY amp Yeh S (2001) Isoprene increasesthermotolerance of fosmidomycin-fed leaves Plant Physiology125 2001ndash2006

Sharkey TD Singsaas EL Lerdau MT amp Geron C (1999)Weather effects on isoprene emission capacity and applicationsin emissions algorithms Ecological Applications 9 1132ndash1137

Sharkey TD Yeh S Wiberley AE Falbel TG Gong D ampFernandez DE (2005) Evolution of the isoprene biosyntheticpathway in kudzu Plant Physiology 137 700ndash712

Silver GM amp Fall R (1991) Enzymatic synthesis of isoprene fromdimethylallyl diphosphate in aspen leaf extracts Plant Physiol-ogy 97 1588ndash1591

Singsaas EL Lerdau M Winter K amp Sharkey TD (1997) Iso-prene increases thermotolerance of isoprene-emitting speciesPlant Physiology 115 1413ndash1420

Thompson AM (1992) The oxidizing capacity of the Earthrsquosatmosphere probable past and future changes Science 2561157ndash1165

Velikova V amp Loreto F (2005) On the relationship between iso-prene emission and thermotolerance in Phragmites australisleaves exposed to high temperatures and during the recoveryfrom heat stress Plant Cell and Environment 28 318ndash327

Wildermuth M (1997) Subcellular localization and biochemicalregulation of foliar isoprene production PhD Thesis Universityof Colorado Boulder CO USA

Wildermuth M amp Fall R (1996) Light-dependent isoprene emis-sion Characterization of a thylakoid-bound isoprene synthasein Salix discolor chloroplasts Plant Physiology 112 171ndash182

Wildermuth M amp Fall R (1998) Biochemical characterization ofstromal and thylakoid-bound forms of isoprene synthase in wil-low leaves Plant Physiology 116 1111ndash1123

Wolfertz M Sharkey TD Boland W Kuumlhnemann F Yeh S ampWeise SE (2003) Biochemical regulation of isoprene emissionPlant Cell and Environment 26 1357ndash1364

Received 15 December 2004 received in revised form 11 February2005 accepted for publication 11 February 2005

Development of kudzu isoprene emission

899



28

898ndash905

correlated with isoprene emission rates throughout leafdevelopment

MATERIALS AND METHODS

Plant growth conditions

Kudzu plants (

Pueraria montana

var

lobata

(Willd) Mae-sen amp S Almeida) were grown from stem cuttings in 10-Lpots containing a vermiculitepeat moss-based growthmedium (Metro-Mix 360 The Scotts Company Marys-ville OH USA) The plants were grown in temperature-controlled greenhouses at the Biotron facility of theUniversity of Wisconsin-Madison Three plants weregrown at 2016

infin

C daynight temperature with noextended day-length whereas two were grown at 3020

infin

Cdaynight temperature and day-length extended to 16 hwith high pressure sodium vapour lamps Plants werewatered with varying strength Hoaglandrsquos solution (Hoag-land amp Arnon 1938) and fertilized with 14-14-14 N-P-KOsmocote according to the manufacturerrsquos instructions(The Scotts Company)

Leaf measurement and sample collection

Leaves near the tops of the plants were tagged when theywere 1ndash2 cm in length and were measured daily thereafteruntil they were fully expanded Leaf length was measuredfrom the junction of the terminal leaflet blade and petiolealong the midrib to the tip of the blade Samples werecollected from mid-July up to the end of August 2004 andleaf measurements and collections were carried outbetween 1030 and 1230 h All tissue samples were collectedclose to the tips of the lateral leaflet blades and were frozenimmediately in dry ice and then stored at

-

80

infin

C until useSamples were collected in leaf lengths of 3ndash4 cm 4ndash5 cm

5ndash6 cm 6ndash7 cm and 7ndash8 cm also 24 h past full expansion1 week past full expansion and 2 weeks past full expansionThese categories were selected because healthy kudzuleaves can have a final length ranging from 8 to 20 cm andrarely stop growing before reaching 8 cm (unpublishedobservation) Collected leaf punches were 933 mm

2

inarea

Gas exchange and isoprene emission measurement

Gas exchange measurements were conducted as describedby Wolfertz

et al

(2003) all measurements were made at30

infin

C and 1000

m

mol m

-

2

s

-

1

The air source for the Li-Cor6400 (Li-Cor Inc Lincoln NE USA) was a cylinder ofcompressed air Ten millilitres of the air exiting the leafcuvette were collected by syringe and analysed for isoprenecontent by gas chromatography as described by Loreto ampSharkey (1993) with the following modifications the col-umn was maintained at 52

infin

C and the liquid isoprene stan-dard was serially diluted to 128 nmol mol

-

1

in N

2

Leafisoprene emission rates were calculated as described bySingsaas

et al

(1997)

RNA extraction and quantitative polymerase chain reaction analysis

Total RNA was extracted from leaf samples as follows Aleaf punch was ground in liquid nitrogen with a mortar andpestle Extraction buffer [01

M

Tris-HCl (Fisher ScientificPittsburgh PA) pH 80 01

M

LiCl 02 m

M

ethylenedi-aminetetraacetic acid(EDTA) 1 wv sodium dodecyl shl-phate (SDS) 50 vv phenol] heated to 80

infin

C was added(500

m

L per 10 mg tissue) and then homogenized The mix-ture was transferred to a polypropylene tube and shaken vig-orously 100

m

L 24 1 chloroform isoamyl alcohol wereadded and the tube was shaken again and then centrifugedat 12 000

yen

g

at 4

infin

C for 5 min The upper phase solution wascollected and precipitated with an equal volume of 4

M

LiClat 4

infin

C for 2 h This solution was centrifuged at 12000

yen

g at4

infin

C for 10 min the supernatant was removed and the pelletwas resuspended in 700

m

L diethylpyrocarbonate (DEPC)-treated H

2

O by pipetting This was extracted with 25 24 1phenol chloroform isoamyl alcohol twice and with 24 1chloroform isoamyl alcohol once Three molar sodium ace-tate (110 volume) and isopropanol (1 volume) were addedto the resulting solution and RNA was precipitated over-night at

-

20

infin

C This solution was centrifuged at 12000

yen

g

for 10 min the supernatant was removed and the pellet waswashed once with 70 ethanol and once with 100 ethanolThe pellet was air-dried and redissolved in 30ndash50

m

L DEPC-treated H

2

O All chemicals were obtained from Sigma-Aldrich (St Louis MO USA) unless otherwise noted

RNA concentration was determined using a BeckmanDUcopy 640 spectrophotometer (Beckman Coulter Inc Ful-lerton CA USA) and 0125

m

g from each sample werereverse transcribed using oligo(dT)15 primer and M-MLVreverse transcriptase according to the manufacturerrsquosinstructions (Promega Corp Madison WI

lt

USA) Quan-titative polymerase chain reaction (PCR) was then carriedout on 1 or 2

m

L of the reverse transcriptase (RT) productusing an Mx3000P

TM

real-time PCR system with Brilliant

reg

SYBR

reg

green QPCR master mix (Stratagene La JollaCA USA) according to the manufacturerrsquos instructionsThe primers used were forward 5

cent

-TGGCGAGTTATTTGTGCT-3

cent

reverse 5

cent

-CCTCTAGCTTTGTTGCCTT-3

cent

these span the first intron of the gene which is 357 bp long(Sharkey

et al

2005) and therefore prevented amplifica-tion of any contaminating genomic DNA The thermal pro-file was 95

infin

C for 10 min 40 cycles of 95

infin

C for 30 s 51

infin

Cfor 1 min and 72

infin

C for 30 s 95

infin

C for 1 min 45 cycles of30 s each with temperature beginning at 51

infin

C and increas-ing 1

infin

C per cycle The target sequence had previously beenamplified from a kudzu RNA preparation and reverse tran-scription with concentration determined as described forRNA extracts Dilutions of this each containing a knownnumber of copies of the target amplicon were used toprepare a standard curve that was used to quantify thePCR product in the plant samples To prevent the forma-tion of primer dimers which would give inaccurate quanti-tation each plant samplersquos reaction mixture was spikedwith 1000 copies of the amplicon this number was then

900

A E Wiberley

et al



28

898ndash905




et al


yen

g


M


a


infin


yen


TM


TM


Escher-ichia coli


et al


Ecoli


et al


MAGE




yen


M

Tris-HCl pH 8020 m

M

MgCl

2


M


M


M


et al


M


4

)

2

SO

4


M

bicine

pH 80 50 m

M

MgCl

2

5 m

M

KCl 2 m

M


m

L assay buffer 1

m

L1

M

DTT 10

m


RESULTS





















DISCUSSION





















ACKNOWLEDGMENTS


REFERENCES










































900

A E Wiberley

et al



28

898ndash905




et al


yen

g


M


a


infin


yen


TM


TM


Escher-ichia coli


et al


Ecoli


et al


MAGE




yen


M

Tris-HCl pH 8020 m

M

MgCl

2


M


M


M


et al


M


4

)

2

SO

4


M

bicine

pH 80 50 m

M

MgCl

2

5 m

M

KCl 2 m

M


m

L assay buffer 1

m

L1

M

DTT 10

m


RESULTS





















DISCUSSION





















ACKNOWLEDGMENTS


REFERENCES



























































DISCUSSION





















ACKNOWLEDGMENTS


REFERENCES















































DISCUSSION





















ACKNOWLEDGMENTS


REFERENCES























































ACKNOWLEDGMENTS


REFERENCES















































ACKNOWLEDGMENTS


REFERENCES





























































Documents

2005 Plant Cell Enviromnent