Exploiting the potential of plants with crassulacean acid metabolism for bioenergy production on ...

18 Pages·2009·0.54 MB·English

JournalofExperimentalBotany,Vol.60,No.10,pp.2879–2896,2009
doi:10.1093/jxb/erp118 AdvanceAccesspublication23April,2009
REVIEWPAPER
Exploiting the potential of plants with crassulacean acid
metabolism for bioenergy production on marginal lands
Anne M. Borland1,*, Howard Griffiths2, James Hartwell3 and J.Andrew C.Smith4
1 InstituteforResearchontheEnvironmentandSustainability,NewcastleUniversity,NewcastleUponTyne,NE17RU,UK
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2 DepartmentofPlantSciences,UniversityofCambridge,CambridgeCB23EA,UK o
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3 SchoolofBiologicalSciences,UniversityofLiverpool,LiverpoolL697ZB,UK nlo
4 DepartmentofPlantSciences,UniversityofOxford,OxfordOX13RB,UK ad
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Received23December2008;Revised17March2009;Accepted18March2009 m
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Abstract ://a
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Crassulacean acid metabolism (CAM) is a photosynthetic adaptation that facilitates the uptake of CO at night and m
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thereby optimizes the water-use efficiency of carbon assimilation in plants growing in arid habitats. A number of ic.o
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CAMspecieshavebeenexploitedagronomicallyinmarginalhabitats,displayingannualabove-groundproductivities p
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comparable with those of the most water-use efficient C or C crops but with only 20% of the water required for o
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cultivation. Such attributes highlight the potential of CAM plants for carbon sequestration and as feed stocks for /jx
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bioenergy production on marginal and degraded lands. This review highlights the metabolic and morphological /a
features of CAM that contribute towards high biomass production in water-limited environments. The temporal rtic
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separation of carboxylation processes that underpins CAM provides flexibility for modulating carbon gain over the -a
b
day and night, and poses fundamental questions in terms of circadian control of metabolism, growth, and stra
productivity.Theadvantagesconferredbyahighwater-storagecapacitance,whichtranslateintoanabilitytobuffer c
t/6
fluctuations in environmental water availability, must be traded against diffusive (stomatal plus internal) constraints 0
/1
imposed by succulent CAM tissues on CO supply to the cellular sites of carbon assimilation. The practicalities for 0
2 /2
maximizing CAM biomass and carbon sequestration need to be informed by underlying molecular, physiological, 8
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and ecological processes. Recent progress in developing genetic models for CAM are outlined and discussed in /5
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light of the need to achieve a systems-level understanding that spans the molecular controls over the pathway 7
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throughtotheagronomicperformanceofCAMandprovisionofecosystemservicesonmarginallands. 1
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Keywords: Biomass,CAM,carbonsequestration,circadiancontrol,marginallands,productivity. u
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Introduction
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The photosynthetic specialization of crassulacean acid include Ananas comosus (pineapple), Opuntia ficus-indica, 2
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metabolism (CAM) permits the net uptake of CO2 at night Agave sisalana, and A. tequilana, all of which can achieve 19
and thereby dramatically improves the water-use efficiency near-maximalproductivityoverareasinwhichprecipitation
(WUE) of carbon assimilation in plants growing in arid is inadequate or evapotranspiration so great that rainfall is
habitats. CAM is estimated to be expressed in ;7% of insufficient for the cultivation of many C and C crops. Of
3 4
vascular plant species (Winter and Smith, 1996a), many of these examples, pineapple (A. comosus: Fig. 1A) is culti-
which dominate the plant biomass of arid, marginal regions vated over 60(cid:1) of latitude and produces up to 86 Mg fruit
of the world. Typically, the water use-efficiency of CAM ha(cid:1)1;the international trade value ofthe freshproduce, not
plants,expressedasCO fixedperunitwaterlost,maybe3- including processed fruit, was recorded as US$1.9 billion
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fold higher than that of C plants and at least 6-fold higher year(cid:1)1 in 2003 (FAOSTAT, 2005). The high yielding
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than for C species. Examples of cultivated CAM species potential of Opuntia achieved notoriety in the 1900s when
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*Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected]
ªTheAuthor[2009].PublishedbyOxfordUniversityPress[onbehalfoftheSocietyforExperimentalBiology].Allrightsreserved.
ForPermissions,pleasee-mail:[email protected]
2880 | Borland et al.
productivity for O. ficus-indica of 47–50 Mg ha(cid:1)1 year(cid:1)1
(Nobel, 1996; where 1 ha ¼ 10 000 m2). The current
worldwide cultivation of Agave is >500 000 ha (Nobel
et al., 2002), mostly for fibre (primarily sisal: Fig. 1B) and
fodder, but also for the production of alcohol, either in the
form of tequila (produced from the double distillation of
fermented sugars from the stems and attached leaf bases of
A. tequilana), or as mezcal (a singly distilled beverage
extracted from ;10 other species).
The potential for Agave as an economically viable source
of bioethanol with a zero-waste platform has recently been
highlighted in Mexico as well as for the eroded lands of the
Great Karoo in South East Africa where the climate and
soil are not suitable for the cultivation of other crops D
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(Boguslavsky et al., 2007; Burger, 2008). The high annual w
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productivity of A. tequilana (50 Mg dry biomass ha(cid:1)1 lo
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year(cid:1)1 on semi-arid land) and high total sugar content (27– de
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38%) in leaves/stems/fruits (cf. sugar cane 15–22%) have led fro
to reports that distilled ethanol yields of 14 000 l ha(cid:1)1 can m
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be obtained from some cultivars, with predictions of ttp
a further 33 650 l ethanol ha(cid:1)1 from cellulose digestion s://a
(Burger 2008). It is recognized that economic and environ- c
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mental sustainability in the cultivation of dedicated bioen- e
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ergy crops will require greater emphasis on the application ic
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and diversification of low-input agriculture on marginal u
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land. This article will highlight the key attributes of CAM .c
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that contribute towards high biomass and bioenergy pro- /jx
duction in marginal habitats. Areas of current and future b/a
Fig.1. (A)Plantationofpineapple[Ananascomosus(L.)Merr.]at research are outlined for elucidating the causes and rtic
Rollingstone,Queensland,Australia(19(cid:1)02#S,146(cid:1)23#E).In2003, consequences of CAM and for providing a knowledge base le-a
worldwideproductionofpineapplewas153106Mg,withan thatmightinformandimprovethepotentialofCAMplants bs
internationaltradevalueofthefreshproduceofUS$1.9billion for carbon sequestration and bioenergy production on trac
year(cid:1)1,or;US$3.0billionyear(cid:1)1includingtheprocessedfruit marginal and degraded lands. t/6
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(http://www.fao.org/es/esc/en/15/217/highlight_218.html,Current /10
SituationandMedium-TermOutlookforTropicalFruits).(B) /28
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Plantationofsisal(AgavesisalanaPerrine)atBerenty,southern Biochemistry and regulation of CAM 9/5
Madagascar(25(cid:1)01#S,46(cid:1)18#E).Madagascaristheworld’ssixth 77
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largestproducerofsisal.In2006,worldwideproductionoffibre CAM, like C , employs the enzyme phosphoenolpyruvate 2
4 1
fromsisalwas2463103Mg,withafurther223103Mgbeing carboxylase (PEPC) for the capture of atmospheric and by
producedfromhenequen(AgavefourcroydesLem.),representing respiratory CO2, thereby providing a means of ‘turbo gue
acombinedexportvalueof;US$200million(http://www.fao.org/ charging’ Rubisco-mediated C3 photosynthesis and reduc- st o
es/esc/en/15/320/highlight_323.html,StatisticalBulletinJune ing photorespiration for much of the photoperiod. How- n
2
2008).Productionofsisalfibrehasgraduallydeclinedoverthelast ever, whilst the C4 pathway functions through spatial 9 M
40yearstoaboutone-thirdofitslevelinthemid-1960s,primarily separation of PEPC and Rubisco between the mesophyll a
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becauseofreplacementbysyntheticproductsmadefrompoly- andbundlesheathcells,thecomplete CAMpathwayoccurs h
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propylene.Itremainstobeseenwhetherdemandforthenatural in each photosynthetic mesophyll cell and relies on strict 01
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fibrewillincreaseagaininthefutureaspartofthetrendtoseek temporal regulation of C and C carboxylation processes.
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alternativestopetroleum-derivedsyntheticproducts.Photographs: CAM plants open their stomata and perform PEPC-
JACSmith. mediated CO uptake in the dark to form malic acid, which
2
is subsequently broken down to release CO that is fixed by
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Rubisco during the following day behind closed stomata.
an O. stricta monoculture grew to occupy >25 million The closure of stomata in the light period and concomitant
hectares in central eastern Australia and produced a total almost complete cessation of transpiration from the shoot
biomass of ;1.5 billion Mg in ;80 years (Osmond et al., surface underpins the high WUE of CAM plants. In
2008). Today, Opuntia species are part of natural and addition, the temporal separation of metabolism in CAM
agronomic ecosystems in many parts of the world, with providesplasticityforoptimizingcarbongaininresponseto
commercial cultivation (primarily for fodder and forage) changing environmental conditions via adjustments in both
occupying over 1 million hectares and annual dry biomass the magnitude and relative proportions of direct C - and
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CAM plants and bioenergy | 2881
C -mediated CO uptake. Whilst the enzymatic machinery nase. Malate is transported into the vacuole through
4 2
required for these carboxylation and decarboxylation reac- a voltage-gated, inward-rectifying anion channel (Hafke
tions is present in all higher plants, evolution of the et al., 2003). The current best candidate for the molecular
pathwayrequiredachangeintheregulationofkeyenzymes identity of this channel protein is the CAM orthologue of
andtransportersinordertosustainthetemporalseparation the Arabidopsis thaliana protein ALMT9 (aluminium-
ofthetwocarboxylationprocessesthatarecentraltoCAM. activated malate transporter 9: Kovermann et al., 2007),
although the ALMT family of proteins has yet to be
CAM biochemistry characterized in a CAM species. Inside the vacuole, malate
accumulates as malic acid due to the high concentration of
The 24 h day/night cycle istheonly meaningful unit of time H+generatedbythevacuolarH+-ATPaseand/orH+-PPase
i
withinwhichtoconsiderthebiochemicalprocessesofCAM (Bartholomewetal.,1996;Tsiantisetal.,1996).Indeed,itis
which may be delineated into four phases (Osmond, 1978). the electrical component of the H+ electrochemical differ-
Starting from the end of the photoperiod, the CAM cycle ence established by these two H+ pumps that maintains the
proceedswithphaseIandthemetabolicstepsareillustrated inside-positive potential needed to drive the influx of D
in Fig. 2. In the cytosol, PEPC uses phosphoenolpyruvate malate2(cid:1) anions across the tonoplast through the vacuolar ow
(PEP) and HCO3– (HCO3– resulting from the action of malate channel(Hafkeetal.,2003).CO2uptakeandmalate nloa
carbonic anhydrase on CO2) to generate oxaloacetate, accumulation continue for most of the dark period, such de
d
which is rapidly converted to malate by malate dehydroge- that the concentration of vacuolar malic acid can reach fro
upwards of ;200 mM by dawn. PEPC is activated during m
h
the dark period due to phosphorylation by a dedicated ttp
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PEPC kinase (PPCK), which mediates a 10-fold increase in ://a
the K of PEPC for its feedback inhibitor, malate (Nimmo c
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et al., 1984, 1987 Carter et al., 1991). The post-translational e
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activation of PEPC in the dark period is hypothesized to ic
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draw down the internal partial pressure of CO inside the u
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leaf, and it isfurther hypothesized that this signalsstomatal .c
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opening in the dark, thus sustaining the supply of CO2 to /jx
PEPC. b/a
In the few hours prior to dawn, PPCK is degraded and rtic
PEPC is dephosphorylated, rendering the enzyme 10 times le-a
moresensitivetoinhibitionbymalate.Themalate-mediated bs
shutdown of PEPC at the start of the photoperiod may be tra
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considered a critical step for curtailing futile cycling at the t/6
0
start of the photoperiod in CAM plants (Borland et al., /1
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Fig.2. Thepathwayofcrassulaceanacidmetabolisminameso- 1999). The export of malate from the vacuole to the cytosol /28
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phyllcellofanNADP-ME(malicenzyme)speciessuchas (the site of PEPC activity) has long been considered 9
/5
Mesembryanthemumcrystallinum.Filledarrowsrepresentdark apassiveprocess,coupledinsomewaytothestoichiometric 77
reactionswhilstopenarrowsrepresentlightreactions.Thedashed efflux of 2 H+ per malate (Smith et al., 1996), but the 62
1
linerunningacrossthecentreseparatesdarkatthetopfromlight molecularidentityofthistransportsystemhasalsonotbeen by
atthebottom.Theblack-filledboxesontheleftofthediagram identified. An intriguing possibility is that the CAM gu
e
representtheleafepidermis,withthegaprepresentingastomatal orthologue of the tonoplast dicarboxylate transporter st o
pore.Theenzymesthatcatalysethereactionsareasfollows:(1) identified in A. thaliana (Emmerlich et al., 2003; Hurth n
2
nocturnalstarchbreakdownpossiblyviachloroplasticstarch etal.,2005)couldmediatemalateeffluxfromthevacuoleat 9
M
phosphorylaseandtheexportofG6Pviatheglucose6- dawn. Rubisco activation, mediated via Rubisco activase, is a
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phosphate:phosphatetranslocator(GPT)followedbyglycolytic believed to commence at the start of the photoperiod, and h
2
conversionofG6PtoPEPwhichisthenusedbyphosphoenolpyr- for a brief period CO2 may be fixed by both PEPC and 01
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uvatecarboxylase(PEPC)asasubstrateforCO fixation;(2) Rubisco, a period referred to as phase II of CAM (Fig. 3).
2
carbonicanhydrase;(3)PEPC;(4)malatedehydrogenase;(5) However, it is noteworthy that a study on Kalanchoe¨
voltage-gatedmalatechannel,possiblyatonoplastmembrane- daigremontiana found that the Rubisco activation state was
targetedaluminium-activatedmalatetransporter;(6)vacuolarH+ low in phase II and increased gradually throughout the
ATPase;(7)unknownproteinproposedtomediatemalateefflux, photoperiod, peaking some 3–4 h before the end of the
possiblythetonoplastdicarboxylatetransporter;(8)NADP-ME;(9) photoperiod (Griffiths et al., 2002). This correlated with
ribulosebisphosphatecarboxylaseoxygenase;(10)unknown a peak in transcript and protein abundance of Rubisco
pyruvatetransporterontheinnerenvelopemembraneofthe activase, which occurred around the middle of the light
chloroplast;(11)pyruvateorthophosphatedikinase;(12)phos- period, in contrast to the peak of Rubisco activase
phoenolpyruvate:phosphatetranslocator;(13)gluconeogenesis; transcript abundance that occurs shortly after dawn in C
3
(14)GPT;(15)starchsynthesisbeginningwithADP-glucose species (Griffiths et al., 2002). Additionally, in Kalanchoe¨,
pyrophosphorylase. Rubisco has kinetic properties which are intermediate
2882 | Borland et al.
between C and C , with a reduced specificity (compared pair 5 showed most net CO uptake at night, and this was
3 4 2
with C ) but lower K for CO (compared with C ), reflected in a diminished phase II and IV and extended
3 m 2 4
perhaps reflecting the daily switch in carboxylase domi- phase III (the latter probably due to the time taken to
nance that occurs over the CAM cycle (Griffiths et al., decarboxylate the larger pool of malate present in leaf pair
2008). 5 compared with leaf pair 4). These different gas exchange
The decarboxylation of malate (phase III of CAM) may patterns can also be attributed to the contrasting degree of
be catalysed by: mitochondrial NAD+-ME (malic enzyme), succulenceandcommitmenttoCAMseeninyoungandold
cytosolic NADP+-ME, chloroplastic NADP+-ME, or cyto- leaves of K. fedtshenkoi, which is equivalent to the
solic PEPCK, depending on the plant species (Dittrich difference between mature leaves of K. daigremontiana and
et al., 1973; Dittrich, 1976; Holtum et al., 2005; the K. pinnata (Griffiths et al., 2008; von Caemmerer and
decarboxylation of malate via ME is shown in Fig. 2). In Griffiths,2009).
some CAMspecies,there iscorrelativeevidence thatmalate Energetically, the CAM cycle incurs an additional cost
is decarboxylated by more than one of these enzymatic when compared with the standard C mode of carbon
3
routesduetohighactivitiesofmorethanonedecarboxylase fixation. This arises from two sources: first, the cost of D
o
(Dittrich et al., 1973). Decarboxylation by ME generates transporting malic acid into the vacuole at night, driven by w
n
pyruvate. In order for this pyruvate to be recycled through one or both of the tonoplast H+ pumps; and secondly, the lo
a
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gluconeogenesis to starch, it must be converted to PEP by cost of converting the C3 residue resulting from malate ed
pyruvate, orthophosphate dikinase (PPDK; Fig. 2). PPDK decarboxylation during the daytime back to the level of fro
has long been assumed to be a chloroplastic enzyme, and storage carbohydrateviagluconeogenesis,thisbeingneeded m
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this was shown to be the case in Mesembryanthemum to provide the substrate for PEPC during the following ttp
s
crystallinum (Kondo et al., 1998). In cytosolic NADP-ME night-time. In aggregate, these processes probably represent ://a
and mitochondrial NAD-ME CAM species, PPDK in the an additional metabolic cost of ;10% compared with the c
a
d
chloroplast requires a pyruvate transporter on the chloro- standard C pathway (Winter and Smith, 1996b), which is e
3 m
plast inner envelope membrane (Kore-eda et al., 1996). The relatively minor when considering that CAM plants typi- ic
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molecular identity of this chloroplast membrane pyruvate cally grow in high-light environments in which photon u
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transporter remains to be elucidated. However, in contrast supply is not normally limiting for growth. In compensa- .c
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to M. crystallinum, other CAM species, including K. tion, however, CAM plants benefit during phase III from /jx
daigremontiana and K. pinnata, have isoforms of PPDK the suppression of photorespiration, which in C3 plants can b/a
localized to both the cytosol and chloroplast (Kondo et al., increase the cost of net CO2 fixation by a minimum of 25%. rtic
2000, 2001). These observations are consistent with data Even under optimal temperature conditions, therefore, the le-a
rPePpDorKtedgefnoer wthaesCfo3usnpdectioeshAa.vethtawlioanpa,roimnowtehrischthaatsipnrgole- eCnAerMgetpiclacnotsstthoafnneint CCO2pfilaxnattsio(nNoisbesilg,n1i9fi9c6a;ntWlyinlotewreranidn bstra
3 c
duced two different transcripts, one ofwhich was translated Smith, 1996b). This advantage increases with higher ambi- t/6
0
into a chloroplastic PPDK, whilst the other transcript ent temperatures, at which photorespiration increases /1
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produced a cytosolic isoform of PPDK (Parsley and steeply in C3 plants, which is relevant when considering the /287
Hibberd, 2006). The regulation and localization of ME temperature regimes characteristic of semi-arid habitats at 9
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isoforms, PEPCK, and PPDK have received very limited tropical and subtropicallatitudes. 7
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attention in CAM species, and the function of the PPDK- 2
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regulatory protein (Chastain et al., 2008)—a homologue of b
which has been identified in M. crystallinum—remains to be Temporal control of CAM y gu
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established in terms ofCAM operation. The complete 24 h cycle of CAM occurs within individual s
t o
The high internal concentration of CO generated in the leaf mesophyll cells, and strict temporal regulation of the n
2 2
intercellular spaces by malate decarboxylation in phase III various metabolic and transport components of the path- 9
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while stomata are closed effectively saturates the carboxyl- way is required to avoid the futile cycling that would result a
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ase activity and suppresses the oxygenase function of from uncontrolled, simultaneous CO fixation and malate h
2 2
Rubisco, even though internal O concentrations are also decarboxylation. The coordination and optimization of 0
2 1
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elevated at this time. In well-watered CAM plants, stomata CO fixation and subsequent metabolism during CAM is
2
may open once the supply of malate is exhausted and controlled by the endogenous circadian clock (Hartwell,
internal CO concentrations drop. Direct fixation of atmo- 2005a). In particular, the leaves of CAM species perform
2
sphericCO byRubiscocanthenproceed fortheremainder a persistent circadian rhythm of CO fixation at constant,
2 2
of the light period, a period known as phase IV (Fig. 3; permissive temperatures in continuous light, normal air,
Osmond, 1978). The magnitude and duration of each phase andcontinuousdarkness underCO -freeair (Wilkins, 1992;
2
of the CAM cycle is highly plastic and varies (i) between Hartwell, 2005a, b). Effective circadian control is probably
species; (ii) in response to the environment; and (iii) with critical to the optimal functioning of the CAM pathway,
leaf development (Winter et al., 2008). As illustrated for and has been covered in detail elsewhere (Borland and
Kalanchoe¨fedtshenkoi (Fig. 3), the magnitude and duration Taybi, 2004; Hartwell, 2005a, b). However, it is important
of phases II– IV is inversely related to the magnitude and to highlight some key features here, as circadian control
duration of CO taken up at night (phase I). The older leaf may be a major yield constraint in highly productive CAM
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CAM plants and bioenergy | 2883
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Fig.3. Thephasesofthe24 hCAMcycle.ThephasesproposedbyOsmond(1978)aresuperimposedontogasexchangedatafrom: 9
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(A)leafpair4and(B)leafpair5ofKalanchoe¨ fedtschenkoi.PhaseI:stomataopen,primaryCO fixationbyPEPCinthedarkleadingto 7
2 7
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malicacidaccumulation.PhaseII:stomataopen,simultaneousCO2fixationbyPEPCandRubiscoduringtheearlyhoursofthelight 21
period.PhaseIII:stomataclosed,refixationofCO fromdecarboxylatedmalatebyRubisco.PhaseIV:stomatareopenunderwell- b
2 y
wateredconditions,directfixationofatmosphericCO byRubisco. g
2 u
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s
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species such as Agave and Opuntia, and thus have relevance evolution of CAM exploited an existing C oscillator for n
3 2
in the selection of cultivars as feed stocks for bioenergy the coordination and optimization of C and C carboxyl- 9
3 4 M
production. ation processes. Moreover, phase advancing the M. crystal- a
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The control of carbon flux through PEPC in CAM linum multigene loop oscillator using a temperature pulse in h
2
species isknown to be mediated viacircadian control of the the late dark period resulted in a highly correlated phase 0
1
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synthesis and degradation of the dedicated regulatory advance of circadian-regulated processes associated with
kinase PPCK (Nimmo et al., 1984; Carter et al., 1991; CAM, including PPCK gene transcript abundance and
Hartwell et al., 1996, 1999, 2002). The nature of the malate oscillations (Hartwell, 2005b; SE Boxall, JM Foster,
underlying circadian oscillator that provides the temporal and J Hartwell, unpublished data). However, the most
signals to optimize the CAM cycle as a whole, however, convincing data linking the multigene loop oscillator in the
remains rather more elusive. Orthologues for many of the nucleus to the circadian regulation of CAM comes from
A. thaliana central clock genes have been cloned and transgeniclinesofK.fedtschenkoioverexpressingthecentral
characterized from the facultative CAM plant, M. crystal- circadian clock gene, TIMING OF CAB EXPRESSION1
linum(Boxalletal.,2005).Thisworkhasdemonstratedthat (TOC1). In these TOC1-overexpressor lines, the circadian
CAM species possess a multigene loop oscillator very rhythm of CO fixation collapses rapidly to arrhythmia
2
similar to that in A. thaliana (Hotta et al., 2007; McClung, followingtheonsetofconstantconditions(Hartwell,2005b;
2008), supporting the hypothesis that the convergent C Dall’omo, SE Boxall, JM Foster and J Hartwell,
2884 | Borland et al.
unpublished data). This is consistent with the phenotype of of soluble, low molecular weight sugars in the vacuole or
output rhythms in lines of A. thaliana that strongly over- as insoluble, polymeric glucan (starch) in the chloroplast
express TOC1 (Mas et al., 2003), and thus suggests that the (Kenyon et al., 1985; Christopher and Holtum, 1996,
multigene loop oscillator does coordinate CAM. The K. 1998). The typically high content of non-structural carbo-
fedtschenkoi TOC1-overexpressor lines grow less rapidly hydrates in CAM leaves can be viewed as a desirable trait
and have smaller leaves, indicating that circadian control of for bioethanol production (Smith, 2008). Moreover, de-
CAM iscritical tothe growth and developmentofthe plant spite the requirement to bank reserves for dark CO
2
(C Dall’omo and J Hartwell, unpublished). The detailed uptake, in many CAM species (e.g. pineapple, Agave,
characterization of the phenotype of these K. fedtschenkoi Opuntia) the potential for high biomass productivity is not
TOC1-overexpressor lines will be the subject of a future compromised. CAM allows metabolic flexibility in the use
publication. of different carbohydrate sources for nocturnal substrate
In the light of the growth penalties associated with provision, as demonstrated by the restoration of nocturnal
incorrect circadian control of CAM in TOC1-overexpressor acidification by feeding glucose or sucrose to leaves of
lines, it is clear that detailed understanding of circadian a starch-deficient mutant of M. crystallinum (Cushman D
o
control will be a key element in dissecting yield constraints et al., 2008a). Clearly, the partitioning of carbohydrate w
n
in potential CAM biofuel species such as Agave and for dark carboxylation must be modulated in line with lo
a
d
Opuntia. The importance of circadian control to growth the requirements of other competing sinks that include e
d
and reproductive success has previously been demonstrated dark respiration, export, and growth (Borland and Dodd, fro
in A. thaliana, where complete arrhythmia of the central 2002). m
h
circadian oscillator was accompanied by an ;50% decrease The phasing of leaf expansion growth over the diel ttp
s
in net carbon gain from photosynthesis (Dodd et al., 2005). CAM cyclehasimportantimplicationsforthemechanisms ://a
Furthermore, altered circadian control has recently been that regulate assimilate partitioning over a 24 h period. c
a
d
shown to regulate growth vigour in allotetraploids and F Using high-resolution digital image sequence processing to e
1 m
hybrids of Arabidopsis (Ni et al., 2009). It is generally examine diel patterns of leaf growth, Gouws et al. (2005) ic
.o
considered that CAM evolved via genome duplication or showed that leaf growth accelerated at night and de- u
p
polyploidy, followed by evolution of the CAM-specific role celerated during the day in C -performing M. crystallinum. .c
3 o
m
fortheextraredundantcopiesofrelevantgenessuchasPPC In contrast, leaves of the CAM species Kalanchoe¨ /jx
and PPCK (Cushman and Bohnert, 1999). It is intriguing to beharensisandcladodesofOpuntiaengelmaniiandO.oricola b/a
speculate that the clock-dependent up-regulation of output showed accelerated growth in the middle part of the day rtic
genes may predispose a newly formed allopolyploid to the (phase III of CAM) and little or no growth at night le-a
evolution of the CAM pathway under certain environmen- (Gouws et al., 2005). It was suggested that the markedly bs
tal conditions. Incorrect timing of biological activity in arid different diel growth patterns in CAM species as compared tra
c
habitats tends to have more rapid negative consequences with C can be explained in terms of both the distinctive t/6
3 0
simply by virtue of the greater environmental extremes (e.g. turgor relations and carbon supply of CAM plants (Gouws /1
0
temperature) that occur in such habitats. Thus, it can be et al., 2005). It has been reported for a range of C3 species /287
argued that accurate circadian control tends to be even that leaf growth occurs predominantly at night, and 9
/5
more important in arid-zone species. It remains to be studies on Arabidopsis have indicated a close coupling 7
7
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established if the clock in CAM species has an even more between the rate of growth and the rate of nocturnal starch 2
1
critical role in reproductive success than that in C species. degradation (Walter and Schurr 2005; Smith and Stitt, b
3 y
Certainly, it seems likely that the clock is central to the 2007). In contrast, nocturnal stomatal opening and transpi- gu
e
photoperiodic induction of CAM and onset of flowering in ration in CAM plants can cause leaf turgor to be low for s
t o
Kalanchoe¨ blossfeldiana (Taybi et al., 2002), and may be much of the dark period, with maximum turgor not n
2
associated with the onset of the dry season (Kluge and occurring until stomata are closed in phase III (Smith and 9
M
Brulfert, 1996). Lu¨ttge, 1985). The uncoupling of leaf expansion growth a
rc
from nocturnal carbohydrate degradation could provide h
2
a means of reconciling potential conflicts of demand 0
1
9
Carbohydrate economy and between accumulation of carbohydrate reserves for CAM
(or bioethanol production) and partitioning of resources
growth processes
forgrowth.
Whilst the circadian clock plays a cardinal role in
establishing and maintaining the characteristic phases of
Starch degradation in CAM plants
CAM, the day/night turnover of carbohydrate is a key
component in determining the magnitude of CAM expres- The enzymatic route by which starch is degraded at night
sion. The nocturnal uptake of CO is sustained by in CAM plants could have important implications for
2
degradation of carbohydrate reserves to PEP as substrate understanding the mechanisms that uncouple nocturnal
for PEPC-mediated carboxylation (Fig. 2). Up to 20% of starch breakdown from the synthesis of sucrose for
leaf dry biomass can be allocated to carbohydrates for growth. In leaves of Arabidopsis, the hydrolytic pathway
CAM, and these reserves may be accumulated in the form has been shown to be of prime importance for the
CAM plants and bioenergy | 2885
nocturnal conversion of starch to sucrose, with maltose require genetic manipulation of key enzymes and trans-
being the major export product from chloroplasts degrad- portersimplicated in these pathways ina CAM species.
ing starch at night (Niittyla et al., 2004). In contrast, the
phosphorolytic pathway of starch degradation has been
Day/nightturnover and storageof sugars in CAMplants
proposed to supply carbon for metabolism inside the
chloroplast of A. thaliana, with starch phosphorylase For CAM species such as pineapple and agave in which
providing glucose-6 P as substrate for the oxidative soluble sugars are the major storage carbohydrate and the
pentose phosphate pathway, particularly under conditions source of PEP for nocturnal carboxylation, the vacuole is
of stress and when photorespiration is elevated (Zeeman likely to play a key role in regulating partitioning of sugars
et al., 2004; Weise et al., 2006). between growth and CAM. The photosynthetically active
The induction ofCAM in M. crystallinum by exposure to cells of CAM plants are typically dominated by a large
salinity is accompanied by increased activities of a range of central vacuole that occupies ;95% of the cell volume.
starch-degrading enzymes implicated in both the hydrolytic Isolated vacuoles of A. comosus (pineapple) contain mainly
and phosphorolytic pathways (Paul et al., 1993). However, glucose and fructose (Kenyon et al., 1985; Christopher and D
o
a change in transport activities across the chloroplast Holtum, 1998), whilst whole-leaf extracts contain substan- w
n
envelope has been reported with the switch to CAM in M. tial amounts of sucrose (Kenyon et al., 1985). Given the lo
a
d
crystallinum. Chloroplasts isolated from C3 M. crystallinum veryhighextractableactivitiesofacidinvertasereportedfor ed
exported mainly maltose, whilst chloroplasts isolated from pineapple leaves (Black et al., 1996), it has been proposed fro
plants in the CAM mode exported mainly glucose-6 P that sucrose is synthesized in the cytoplasm during the day, m
h
(Neuhaus and Schulte, 1996). Two isogenes encoding transported across the tonoplast into the vacuole, and ttp
s
glucose-6 P/Pi translocators have been isolated from M. hydrolysed in the vacuolar lumen by acid invertase (Smith ://a
crystallinum (McGPT1 and McGPT2); CAM induction is and Bryce, 1992). To avoid futile cycling, the hexoses thus c
a
d
accompaniedbyanincreaseintranscriptabundanceofboth produced would need to be stored in the vacuole and not e
m
isogenes, which also show robust circadian patterns of released to the cytoplasm until the following dark period, ic
.o
abundance, implying a key role for these transporters in when they would be metabolized via glycolysis to provide u
p
CAM (Kore-eda et al., 2005). The nocturnal export of the C3 substrate (PEP) for nocturnal malate synthesis. This .co
m
glucose-6 P from the chloroplast to the cytosol in CAM model is supported by the finding that the tonoplast of /jx
plants might be predicted to cause allosteric activation of pineapple possesses a sucrose transport system with kinetics b/a
PEPC (Osmond and Holtum, 1982), whilst glycolytic appropriate to catalyse sucrose fluxes of the required rtic
conversion of glucose-6 P in the cytosol would provide magnitude. McRae et al. (2002) demonstrated that sucrose le-a
ATP by substrate-level phosphorylation and thus help to uptake by isolated tonoplast-enriched vesicles prepared bs
energize the nocturnal accumulation of malate in the from pineapple leaves exhibits concentration-dependent tra
c
vacuole (Holtum et al., 2005). Further indications that the saturation kinetics, ATP independence and trans stimula- t/6
0
phosphorolytic route may predominate over the hydrolytic tion by internal sucrose, characteristics that are consistent /1
0
route for starch degradation in CAM plants is provided by with the operation of a carrier type of sucrose transporter. /28
7
the very high extractable activities of chloroplastic starch Recently, a specific hexose transport system has also been 9
/5
phosphorylasefromleavesofM.crystallinum,K.fedstchenkoi, observed in isolated pineapple tonoplast vesicles (D Haines 77
6
and A. comosus, with activities in the CAM species and JAM Holtum, personal communication). Thus, sugar 2
1
;10-fold higher than that in Arabidopsis (T Taybi and AM transporters located at the tonoplast could play a strategic b
y
Borland, unpublished observation). In contrast, glucano- role in controlling the supply and demand for carbon over gu
e
transferase (DPE2), which in Arabidopsis acts on cytosolic the day–night cycle in sugar-accumulating CAM plants s
t o
maltose to provide a substrate for sucrose synthesis (Smith such aspineapple(Antony and Borland,2008). n
2
et al., 2005), shows very low activity in CAM-performing The mechanisms that control import and export of 9
M
M. crystallinum (AM Borland, unpublished observation). photoassimilates from the vacuole during the day–night a
rc
Interrogation of an expressed sequence tag (EST) database cycle are largely unexplored for any plant species, and the h
2
for M. crystallinum containing 25 000 ESTs failed to first plant tonoplast sugar carriers have only recently been 0
1
9
identify any candidate chloroplastic maltose transporters (J identified at the molecular level. Currently, the only
Hartwell, unpublished observation), which might suggest vacuolar sucrose transporters to be identified at the
that maltose export from the chloroplast in M. crystallinum molecular level (HvSUT2 from barley and AtSUC4 from
(and indeed other CAM species) is quantitatively less Arabidopsis; Endler et al., 2006) are believed to facilitate
important than that in Arabidopsis. sucrose export from the acidic lumen of the vacuole
Lu¨ttge et al. (1981) first pointed out that use of the (Neuhaus, 2007). The first tonoplast monosaccharide trans-
phosphorolytic pathway for starch breakdown would re- porters were recently identified from Arabidopsis (AtTMT)
duce the energetic cost of nocturnal malic acid accumula- and are believed to operate via an H+-coupled antiport
tion in CAM plants compared with the hydrolytic pathway. mechanismthatwouldallow importofglucoseandfructose
A full understanding of the relevance of utilizing the in the vacuole by a seconday active transport mechanism,
phosphorolytic pathway for directing carbon skeletons possibly in response to stimuli (i.e. cold, drought, salinity),
towards the synthesis of PEP rather than sucrose will that promotes sugar accumulation in Arabidopsis (Wormit
2886 | Borland et al.
et al., 2006). The model proposed for vacuolar sugar for increasing leaf sugar content whilst maintaining
transport in the leaves of A. comosus (Smith and Bryce, photosynthetic carbon assimilation by avoiding feedback
1992; McRae et al., 2002) implies the existence of a tono- repression of primary carbon metabolism and providing
plast hexose transport system to permit efflux of glucose substrate for nocturnal carboxylation.
and fructose at night to provide substrates for dark CO
2
uptake. A putative hexose transporter (AcMST1) recently
identified from pineapple leaves that localized to the
Genetic models for CAM
tonoplastoftobacco epidermalcellsisapotential candidate
for the energy-independent export of hexoses from the An important step towards maximizing the yield potential
vacuole (Antony et al., 2008). However, the crucial kinetic of CAM species as feed stock for biofuel production will be
mechanism that restricts efflux of vacuolar hexose to the the development of a systems-level understanding of the
dark period (thus avoiding futile cycling during the light molecular and metabolic controls over the pathway. Trac-
period)isstillcompletelyunknown.Thereisnoevidencefor table model CAM species will be key to dissecting the
diurnal or circadian control of transcript abundance of this pathway at molecular and biochemical levels. Early D
o
pineapple tonoplast hexose transporter, nor was there any attempts to develop a molecular–genetic model for the w
n
difference in transcript abundance of AcMST1 between study of CAM centred on M. crystallinum (Bohnert and lo
a
d
pineapple cultivars that differed in the magnitude of CAM Cushman, 2000). CAM may be induced on a C back- e
3 d
(Antony et al., 2008). Recent examinations of the phospho- ground via the imposition of salinity or drought in M. fro
proteomeofthetonoplastfromriceandArabidopsissuggest crystallinum (Winter and Holtum 2007), and this metabolic m
h
that phosphorylation of sugar transporters could be an switch has proved a very attractive system for identifying ttp
s
important mechanism for controlling the day/night loading CAM-associated genes and proteins (Cushman and ://a
and unloading of sugars across the vacuolar membrane Bohnert, 1989, 1999; Cushman, 1992; Boxall et al., 2005). c
a
d
(Whitemanet al., 2008a,b). Other beneficial attributes of M. crystallinum include: e
m
In Agave, fructans stored in the stems and leaf bases are a relatively rapid life cycle (7–14 weeks depending on ic
.o
the major source of ethanol from this plant and as such growth conditions); a relatively small genome, perhaps the u
p
represent an important vacuolar sink for photoassimilate. smallest known amongst CAM species measured to date .co
m
In mature leaves of Agave deserti, fructan biosynthesis was (;390 Mbp; De Rocher et al., 1990); and the setting of /jx
restricted to the vascular tissue (Wang and Nobel, 1998). thousands of small seeds which are ideal for mutagenesis, b/a
Long-distance transport in the phloem of fructans with allowing traditional forward genetic screens to be under- rtic
a low degree of polymerization has been reported for taken. Mutant populations of M. crystallinum have been le-a
a number of plant species, but the mechanisms whereby developed and screened to identify CAM-deficient mutants bs
fructans are unloaded into sink tissues and subsequently (Cushman et al., 2008a). A database of M. crystallinum tra
c
accumulated in the vacuole are unknown. Generally, it is ESTs containing some 27 000 sequences (Kore-eda et al., t/6
0
considered that fructans are synthesized in the vacuole via 2004) remains the largest readily accessible gene index for /1
0
the enzyme fructosyl transferase using imported sucrose as a CAM species. This gene index has been used to generate /28
7
substrate (Valluru and Van den Ende, 2008). Fructans are a Nimblegen oligonucleotide microarray with representa- 9
/5
believedtoprotectmembranesinplantsunderabioticstress tion of 8455 unique genes, which was used to investigate 77
6
and may also participate in the scavenging of reactive both the induction and circadian regulation of CAM 2
1
oxygen species in the vacuole (Van den Ende and Valluru, (Cushman etal.,2008b). b
y
2009). Substantial variation in sugar and fructan content The further development of M. crystallinum as a model gu
e
has been reported for different cultivars of Agave (Vargas- CAM system is presently hampered by the lack of an s
t o
Ponce et al., 2007) which could provide opportunities for efficient and stable transformation system to facilitate in n
2
selecting varieties for enhanced bioethanol production and vivo testing of gene function using both overexpression, 9
M
stress tolerance. To maximize such potential will require and gene silencing/RNAi (RNA interference) approaches. a
rc
a better understanding of the biochemical and molecular In addition, the study of CAM in M. crystallinum is h
2
basis of carbohydrate partitioning in Agave alongside complicated by the requirement for drought or salt stress 0
1
9
a systems-level approach to identify control points for to induce CAM, and it is therefore very difficult to
sugar partitioning. Genes that encode vacuolar sugar separate CAM genes from those genes that are more
transporters could be appropriate candidates for increas- directly involved in resisting the osmotic stress imposed by
ing sugar content in plants grown for bioethanol pro- the drought or salt. Given such constraints, attention is now
duction. Indeed, metabolic control analysis of the kinetics shifting towards the Madagascan endemic K. fedtschenkoi as
of sucrose accumulation in the maturing culm tissue of the next major genetic model for CAM based on the
sugar cane identified the rate of sucrose uptake by the following rationale. (i) K. fedtschenkoi is an obligate CAM
vacuole as a key control element in the magnitude of speciesthatdisplaysacleardevelopmentalprogressionfrom
sucrose accumulation (Uys et al., 2007). Given that leaf C to CAM, even under well-watered conditions. The
3
succulence and vacuolar capacity are particularly high in complication of the drought or salt stress response is
CAM species, knowledge of the tonoplast proteome in therefore avoided, and it is thus regarded as a much
a sugar-accumulating CAM species could offer potential ‘cleaner’ model for the study of CAM. (ii) K. fedtschenkoi
CAM plants and bioenergy | 2887
has the key advantage of a simple and efficient stable Carbon uptake and sequestration—from leaf
transformation system, permitting transgenic approaches to to canopy
decipheringgenefunction.(iii)K.fedtschenkoihasareason-
ably small genome at ;858 Mbp (De Rocher et al., 1990), Carbon uptake by leaf or stem succulent CAM plants
which places it well within the capabilities of the latest represents a classic example of conflict resolution in
DNA sequencing technologies. (iv) K. fedtschenkoi is very physiological ecology, whereby the potential for carbon
easy to grow and develops rapidly from cuttings or leaf gain across a 24 h cycle is traded against diffusive con-
plantlets, reaching a size suitable for CAM experiments straints imposed on CO2 supply by succulent tissues (Dodd
within2months.Theeaseofclonalpropagationmeansthat et al., 2002; Pierce et al., 2002; Griffiths et al., 2007). The
large quantities of developmentally coordinated plants can growth characteristics of CAM plants, as summarized in
be generated very quickly from individual transformants. Table 1, present a number of favourable attributes when
(v) K. fedtschenkoi has the further advantage that a wealth considering the cultivation of dedicated bioenergy crops in
ofbackgroundliteratureexistsprovidingdetailedcharacter- semi-arid regions of the world. However, the practicalities
ization of the physiology and biochemistry of the circadian for maximizing CAM biomass and carbon sequestration D
control of CAM (Wilkins, 1992; Hartwell et al., 2002; needtobeinformedbyunderlyingmolecular,physiological, ow
n
Hartwell, 2005b). and ecological processes. Understanding the integration of lo
a
The characteristics of K. fedtschenkoi described above molecular and cellular signals and their regulation by de
d
have led to a new project in the Hartwell laboratory environmental conditions will allow chemical and structural fro
(UniversityofLiverpool,UK)toperformin-depthsequenc- composition to be targeted towards a specific end-product, m
h
ingofthetranscriptomeofthisspecies.Theprojectwillalso provided that any potential market is sustainable and the ttp
e(BstAabCli)shlibararlyargaen-idnsepretrfobramcterpiaillotarBtiAficCialsecqhureonmcionsgomine l2o0c0a8l).cAomddmituionnitayllys,utphpeorertasrethuencienrittaiaintitviees(oCvoewr tlihnegmeatinatle.-, s://ac
a
readiness for whole genome sequencing. Digital transcrip- nance of potential productivity for the future, because de
m
tomics(the use ofthe number ofsequence reads pergene as achangingclimatewillleadtoalteredprecipitationpatterns ic
a quantitative digital read-out of transcript abundance in and tensions between food and fuel production, which are .ou
p
the original RNA sample) is being employed to identify current even in today’s marginal habitats. However, it .c
o
CAM-associated genes whose transcript abundance is up- should be noted that the exceptional degree of stress m/jx
regulated in CAM leaves relative to C3 leaves. Candidate tolerance of typical CAM plants towards restricted water b/a
CAM genes are subjected to more detailed real-time reverse availability, high temperatures, and high light intensities rtic
transcription-PCR (RT-PCR) analysis to test for circadian (Table 1) would be expected to make them relatively robust le-a
regulation in CAM leaves, and the most interesting tothe impactof futureclimatechange. bs
candidates will be overexpressed and silenced in transgenic tra
c
K.fedtschenkoi totesttheinvivo functionofeachcandidate t/6
Stomatal versus mesophyll (internal) constraints to 0
CAM gene. Thus, this project aims to identify all of the /1
carbon gain 0
genes that K. fedtschenkoi uses to perform CAM, and set /28
7
the stage for future proteomic and metabolomic analysis of At the scale of individual photosynthetic organs, stomata 9
/5
CAM in K. fedtschenkoi. The ultimate goal is to combine occur in relatively low densities and have low conductances 77
6
data concerning transcripts, proteins, and metabolites into to water vapour in CAM plants, reflecting the high water- 2
1
predictive models, which can be used to identify the key storage capacity, low external surface area:volume ratio, b
y
controlpoints foroptimalperformance andyieldassociated and high WUEs, for leaves, cladodes, and stems of CAM gu
e
with CAM. plants (Osmond, 1978; Nobel, 1988). The compromise s
t o
The phylogenetic position of K. fedtschenkoi within the between maximizing day- and night-time uptake is exempli- n
2
family Crassulaceae and order Saxifragales means that this fiedincomparisonsoftwoKalanchoe¨species(K.daigremontiana 9
M
speciesshareditslastcommonancestor with M.crystallinum and K. pinnata) which contrast in the degree of leaf a
rc
(Aizoaceae, Caryophyllales) some ;80–90 milllion years succulence (Griffiths et al., 2008; von Caemmerer and h
2
ago, and thus evolved the CAM pathway completely Griffiths, 2009). Stomatal densities were lower in the more 0
1
9
independently. Ongoing genomics projects with M. crystal- succulent species (K. daigremontiana), which was more
linum and K. fedtschenkoi will thus permit comparative committed to the conventional CAM cycle, with higher
analysis of the functional genomics of CAM in divergent rates of acid accumulation, CO uptake, and higher
2
speciesthat evolvedtheCAMpathwayindependently.Such stomatal conductance at night. In contrast, the less succu-
an approach may reveal differences in the regulatory and lent K. pinnata showed the more C -like expression of the
3
enzymatic steps that have been co-opted into a CAM- CAMphases byday,withahigher proportionofintegrated
specific role during the evolution of this pathway in 24 h net CO uptake mediated directly by Rubisco during
2
different taxonomic groups. This is important in light of phases II and IV. Overall, the ratios of internal:external
the fact that M. crystallinum and K. fedtschenkoi are not CO concentration weresimilar atnight for thetwo species,
2
closely related to the high-yielding CAM species of cacti and actually higher during phase IV for K. daigremontiana.
andagavesthatappeartobesuitedtobioenergyproduction However, the sensitivity of stomata to transient changes in
on marginallands. external CO concentration during the day–night cycle
2
2888 | Borland et al.
Table1. GrowthcharacteristicsofCAMplantsfavourableforcultivationasabioenergycropinsemi-aridregions
ExamplestakenfromNobel(1988,1994),Day(1993),andWinterandSmith(1996c).
Trait Example Comment
Highwater-useefficiency 5–16mmolCO permolH Oonanannualbasis Typically4-to10-foldhigherthanC plants
2 2 3
Highdroughttolerance Cangrowinareaswithaslittleas25mmyear(cid:1)1 Tissuescantolerateupto90%lossofwatercontent(cacti)
precipitation
Toleranceofhightemperatures Upto70(cid:1)C,basedon50%lossofcellviabilityafter Typicallyupperlimitof50–55(cid:1)CinC plants
3
1h;cansurviveexposureto74(cid:1)C
ToleranceofhighPPFD Cantolerate>1000lmolm(cid:1)2s(cid:1)1 GenerallymoretolerantofhighPPFDthanagronomically
(or>40molm(cid:1)2d(cid:1)1)withoutphotoinhibition importantC plants
3
ToleranceofUV-Bradiation Only1%incidentUV-Btransmittedthrough Generallythickepidermisandhighfoliarconcentrationsof
epidermisofYuccafilamentosa(Agavaceae) phenolicsinCAMplants
Entireshootsurfacetypically Wholeshootphotosyntheticinbothleaf-and ManyC3speciesdeciduous(sheddingphotosynthetic D
o
photosynthetic stem-succulentspecies;limitedbarkformation organsforpartofyear)orwoody(limitedstemphotosynthesis) w
n
evenonstemsofarborescentcacti lo
a
Highshoot:rootratioand Shoot:rootratioashighas10:1;above-ground d
e
d
hHaigrvhersetsiinsdtaenxcetoherbivores Ebifofemcativsesprehaydsiilcyahladrevfeesntceeds(stemsucculents)and from
chemicaldefences(leafsucculents) h
Highcontentofnon-structural Especiallymonocotyledons(;20%dryweigth); ttps
carbohydrate readyconversionofsolublesugarstobioethanol ://a
c
Lowlignincontent Weaksecondarythickeningandlackoftruewoodformation a
d
e
m
ic
.o
suggested that internal CO concentration is not the only ofcarbongainactuallyundertakenfortheshortperiodthat u
2 p
effector regulating the CAM diel stomatal cycle (von CAM may be active during extreme conditions (Pierce .c
o
m
Caemmerer and Griffiths 2009). et al., 2002; Winter and Holtum, 2002; Silvera et al., 2005). /jx
These data help to explain the compromise between the Additionally,theisotopiccompositionofCAMplantstends b/a
constraints imposed by succulent tissues, and the potential to be fairly conservative across a wide range of habitats rtic
for internal metabolism facilitated via high PEPC and (Griffiths, 1992), and may not always be indicative of le-a
Rubisco carboxylation capacities as a means of overcoming precipitation gradients (Amundson et al., 1994). This may bs
external diffusion limitation (Griffiths et al., 2002). How- limit the use of carbon isotopes as a tool in selecting tra
c
ever, with the succulent photosynthetic tissues of typical improved productivity and water use of bioenergy crop t/6
0
CAM plants containing only ;5% airspace (Smith and cultivars, which are mostly as yet unimproved. However, /1
0
Heuer, 1981; Maxwell et al., 1997), there are significant when combined with oxygen isotopes, which provide /28
7
constraints imposed on the internal diffusive supply of CO a marker for precipitation inputs and water sources within 9
2 /5
(outside the ‘regenerative’ phase III, when internal CO the soil profile, carbon isotope composition may be more 7
2 7
6
concentrations are likely to saturate Rubisco). Thus, at revealing. It has recently been shown that oxygen isotopes 2
1
night,thecarbonisotopesignalsassociatedwithPEPCwere can resolve water vapour exchanges by epiphytic CAM b
y
stronglysuggestiveofdiffusionlimitationinK.daigremontiana plants (Helliker and Griffiths, 2007; Reyes-Garcia et al., gu
e
(Griffiths et al., 2007). Additionally, mesophyll conductan- 2008) and act as a means of climate reconstruction from s
t o
ces derived during phase IV of gas exchange in the light saguaro spines (English etal.,2007). n
2
(and for a range of Clusia species) reflected the degree of 9
M
succulence, with internal CO supply at Rubisco potentially a
as low as110 lmol mol(cid:1)1 (G2riffiths etal., 1999). Tolerance of water deficits, water use and productivity rch
2
Given such diffusive constraints, the interpretation of Despite the metabolic limitations of high gas-phase resis- 0
1
9
carbon isotope composition of bulk organic material as tances associated with succulent tissues, high productivities
being representative of the balance between daytime (C ) can be achieved by CAM plants in habitats where pre-
3
and night-time (C ) carboxylation processes is complicated. cipitation is intermittent, but regular (i.e. seasonal) and
4
When carbon isotope composition is predicted by stomatal reasonablypredictableonanannualbasis(Ellenberg,1981).
conductance and carboxylase fractionations, lower discrim- With their relatively shallow root systems, CAM plants are
ination is likely to be associated with CO uptake during able to exploit efficiently the small amounts of water
2
phase IV in succulent tissues by day, and higher discrimina- available even from intermittent rainfall events delivering
tion may be predicted at night (Griffiths et al., 1990, 2007, <10 mm, which may be sufficient to moisten only the
2008; Roberts et al., 1997). The use of carbon isotope uppermost soil layers. Coupled with this efficient harvesting
composition to predict ‘furtive’ CAM species (or C –CAM of incident precipitation, leaf- and stem-succulent CAM
3
intermediates) in a given population may be confounded plantsalsohaveveryhighwater-storagecapacitanceintheir
both by internal constraints to diffusion and by the extent above-ground tissues, meaning that they are able to make