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226 Chapter 6 The Application of Carbon Isotope Discrimination in Cereal Improve~ne~lt for Water-Limited Environments Anthony 6. Condor1 Graharn D. Farquhar Greg J. Rebetzke Ricliard A. Richards Recently, two new cotnil~crcialwheat varieties were released for broadacre dryland production in Australia, which were very different from any pl-evious varieties. The varieties Drysdale (released in 2002) and Rees (releaced in 2003) carry a broad cpectrum of disease-resistance genes and produce high-quality tlour, as might be expected for new wheat varieties ~ 0 1 ~ 1 peting in a demanding l-r~arket.But Drysdale and Rees are ui~iquebecause they are the first varieties, of ally crop species, specifically bred for performance in dry environments using carbon isotope cliscrii-r~inationas an indirect selection criterion. Drysdale and liees were bred by backcrossing the high transpiration efficiency trait, i.e., the highly efficient exchange of CO, for water through the stotnata, into the widely grown, milling-quality variety Hartog. The backcrossing and selection process was based on the use of carbon isotope discrimination as a surrogate measul-e of transpiration efficiency, and resulted in a population of elite lines, including the two new varieties, that outyield the recurrent parent Hartog by 5 to 10 percent on average. The yield advantage tends to be greater in drier environillents and less in wetter environments. These new wheats are the first of what we anticipate will be several varieties bred for high transpiration efficiency and yield using carbon isotope discrimination as a seco~ldarytrait. These landmark releases come 20 years after it was first proposed that the isotopic coinposition of plant carbon in C3 species should reflect differDr.ought Ad~lptationi r l C ~ r c n l s 0 2006 by The Haworth Prcss. Inc. All rights rcscrvcd. doi: 10.130015781-06 1 72 DIIO I!C;H 1'AL)AI'T.l l'l Oil L1. CEIIE14LS cnces in the transpiration efficiency of leaf gas exchange (Farquhar et a]., 1982) arld that carbon isotope analysis may therefore prove a useful tool in breeding for improved water-use efficiency and yield in dry environments (Farquhar and Richards, 1984). In this chapter, we discuss the philosophy and practice behind the developtnent and use of carbon isotope discrimination as a se~ondilrytrait for cereal improvement and draw out some of the major conclusions and challenges that have emerged as the concept has progressed froill biophysical theory to the reality of new wheat varieties in frzr~rlers'fields. CARBON ISOTOPE L)ISCRlrl.lIIYAII'ZO,V:A PHYSIOLOGICAI, IMAKKER FOR HIGH TKANSPIKATIQIV EFFICIENCY In this section we present an overview of what is meant by the term w r borl isotope (li.scrirrzirllrtio?~, the basis for the association between carbon isotope discrinlination and transpiration efficiency, and why carbon isotope discrimination rnight be considered a useful secondary trait for yield irnprovenlent of dryland crops. The treatment ic not exhaostive. For a rnore conlplete coverage of the concepts covered here. the reader is referred to the early publications by Farquhar et al. (1982) and Farquhar and Richards (1984), and to more recent reviews such as those by Farqohar et al. (1989). Hall et al. (1994), Condon and Ha11 (1997), Brugnoli and Farquhar (2000). Condon et al. (2002), and Condon et al. (2004). Cnrborz Isotopes irz tlze Biosphere Carbon accounts for approximately 40 percent of plant dry weight, and is assimilated into plants by the process of photosynthesis. During photosynthesis, C0, from the atmosphere diffuses into the leaf interior through the thousands of tiny stomata1 pores in the leaf epidermis. The CO, is then assimilated to generate the sirnple sugars (carbon skeletons) that are a substrate for downsti-eanl syilthesis of the nlultitude of orgarlic compourlds that are important for plant growth. The carbon in atnlospheric C 0 2 and throughout the biosphere occurs as two stable (i.e., nonradioactive) isotopic for~ns.The nlost coillmo~~ form is 12C, which accounts for about 98.9 percent of the C in at~llosphericCO,. The other stable isotope, l3C, makes up about 1.1 percent of atmospheric CO,. The proportion of 13C in the biosphere is sufficiently large that very slnall variations in the 13C112C ratio can be measured accurately. Early measurements of the l'CI12C ratio revealed that the C-isotope composition of plant dry matter was different from the composition of the atiuosphere on which plants Seed. Plants were found to contain fractionally less 13C and thus relatively more 12Cthan the atmosphere. It has also been ~ e considerable variation e x i ~ t sin the l C / ' 2 C ratio known fi)r sorne t i ~ i that of plant dry matter (Brugnoli and Farcluhar, 2000). This variation has several implications for crop productivity, hut before expanding on these. we firat need to explore more closely the processes that cause variation in the C-isotope con~positionof plant dry matter. Before doing so, the principles involved in carbon isotope analysis and how the data are expressed are briefly summarized. Isotope Antrlvsis nrzd Ter-~ninology The isotopic composition of plant carbon is most often measured using isotope-ratio Inass spectrometry (Preston. 1992). The technique first requires the production of a pure sample of CO? gas from the plant material. This is usually achieved by combusting a small sample of dried. finely ground plant inaterial at high temperature. The C 0 2 produced is then purified. for example by gas chromatography, and introduced into the mass spectrometer wl~erethe C 0 2 nioleculcs are ioni/ed at high voltage and focused into a fast-moving beam. The isotopes are separated on the basis of mass-charge ratio by passing the beam of ions through a strong magnetic field. It is technically difficult to accurately measure the absolute isotopic con~positionof plant rnaterial because 13C is present in such low amounts (approximately 1.1 percent of total C at natur:ll abundance). Nevertheless, differences in conlposition between samples can be ~neasuredwith useful precision. The isotopic composition ic therefore expressed by comparing the molar abundance ratio, 13C/12C, of the plant sample (R,,} to the value of the molar abundance ratio in a standard (R,). Carbon isotope compocition (6°C) is labor cnlcolated as (R[, - R,)IR, (= R,,IR, - 1). For historical reasons. the standard for carbon has been Pee Dee belemnite (PDB), a carbonaceous rock. although a synthetic replacement is now provided by the International Atomic Energy Agency (IAEA). Values of 61TC,,, are often expressed as the value times 10' or per mil so that they appear as whole numbera. The 6''Ck,DBof plant material has :I v:ilue that is small and negative, in the range of approxiruately 1 0 per mil to 3 0 per mil. This is because thc 17C/12Cratio ol' plant material is less than that of PDR. The atmosphere has a value of 6"Cp,, that is also slightly negative, about -8 per mil relative to PDB. Carbon isotope composition (6l3Cp,,) has proven to be a ~isefulempirical measure, but as ~1nderst:indingdeveloped of the processes causing varin- Ecl~lations6.1 and 6.2 represent an essentially instantaneous process of discrimination. It is known that the value of c,/c,, is not constallt and neither, therefore, is the amount of discrimination. Rather, discrimination responds to nunlerous short-tern~and long-tenn environmental infloences, fluctuating depending on the extent that the stornatal step dominates or the carboxylation step dominates. These processes are also subject to some level of genetic control. Measuring A'" in dry matter of C-, species provides an assirnilation-weighted average value of c, / c , over the life of the plant material being analyzed. Carboil isotope discrillrination by plants of C, species is small co~upared with that by plants of C , species (Figure 6.1) and reflects to a much larger extent the discrimillation that takes place at the stomata. A small amount of variation exists in C-isotope composition of C4 plants resulting fro111 discrilllinntion duriilg C-fixation. This variation may be of siynificallce for plant productivity and will be considel-ecl in more detail toward the end of the chapter. Because of the greater potential for variation in C3 plants, discrilnillation hy C3 species has attracted far more attention niid forins the basis for ilrost of what follows Variation in Cctrhon Isotope Cornpusifion Awzorrg Metabolites The values of A13C measured in plant dry matter principally reflect discrimination during photosynthesis. yet in all plants sonre potential for additional small changes in C-isotope composition exists as C flows through biochemical pathways downstraaln from photosynthesis to form val-ious metabolites. For example, lipids tend to he depleted in I3C compared with bulk plant dry matter, whereas the reverse is true for carbohydrates (Figure 6.2). The reasons for these differences in C-isotope cornposition among metabolites are primarily due to fragmentation fractionations (Tcherkez et al.. 2004) associated with transfer of C atorns during the biochemical processes that generate these various compounds. These processes will not he discussed in detail here, however, Brugnoli and Farclohar (2000) provide a useful summary. It is possible that further, additional fractionation of C isotopes may take place during processes associated with transport of metabolites and respiration since these processes also often entail some I-erlrrangement of organic molecules. Leaf sugars Starch Cellulose Amino acids Proteins Lignin Lipids 5 FIGURE 6.1. Relationships for C3 and C4 plant species between carbon isotope discrimination during photosynthesis and the ratio of intercellular and atmospheric concentrations of Con (cJc,). The boxes represent the range of typical data points obtained in leaf gas exchange studies in which carbon isotope discrimination was determined "online" by measuring the carbon isotope composition of the gas stream before and after it passed over the leaf. Source: Adapted from Brugnoli and Farquhar, 2000. 10 15 20 25 Carbon isotope discrimination (103 x A%) 30 FIGURE 6.2. Carbon isotope discrimination in various metabolites in C3 species. The boxes represent the range of variation found in the literature, with the vertical bars being the mean values for each compound. The dashed line represents bl3C for bulk dry matter. Source: Adapted from Brugnoli and Farquhar, 2000. .4pi)liiofb,rr (?/G~rbiitlLotope Discriminntion in (>,recrl lr?~pioi~~~nrcttt181 level transpiration efficiency, fl,is directly related to only one co111ponent. W , the transpiration efficiency of biomass production, bat as will be discussed in following sections, A f l also has the potential to influence each of the other three components in tile yield framework. VARIATION IN A13C OF C, CEREALS Genotypic Variatiolt irz A13C Substantial genotypic v:lriation in A13C has been obqerved in many C3 cereal species. Numerous studies have shown variation in A13C of at least 2 per mil, occasionally closer to 3 per mil, in bread wheat (Condon et al., 1987: Condon et al.. 1990; Ehdiiie et al., 1991: Sayre et al., 1995), durulll wheat (Araus et a]., 1998; Merah et al., 1999; Royo et td.. 2002 ), and barley (Hubick and Farquhar, 1989; Craufurd ri 81.. 1991; Acrvedo, 1993; Voltas et al., 1999), and in various species of rangeland (Johnson et a].. 1990; Johnson and Bassett, 1991) and t ~ ~grasses rf (Ebdon et a]., 1998). Considerable genotypic variation is known to exist in A13Cwithin rice (Dingkuhn et al., 1991; Condon et al., 1999), and no obvious reasons explain why similar variation in Al3C should not be present in other cereals such as oats, rye, and triticale. For healthy, unstressed plallta of C3 species, average values of c,/c, are usually close to 0.7 (Farquhar, Ehleringer, and Hubick. 1989). This is equivalent to an average value of Al3C of ca. 21 per mil (froin Equation 6.2. Given this average value oEA13C,what does a range of k1per n i l genotypic variation in AnC mean in terms of pote~rtialvariation in A/T! This call be calculated by malung further use of Equatio~l6.2 (which relates variation in A13C to variation in c,/c,) and Equation 6.6 (which relates variation in to variation in c,/r,,,). From these it can bc dctcl-~uincdthat. in a relative sense. A/T of a genotype with a A'" value of 20 per mil could be approximately 1.3 times greater than A f l of a genotype with a Al3C of 22 per mil. Thos large potential gains in Aflare associated with relatively low valoes of Al3C. However, various reasons explain why it is onlikely that all of there gains will be realized in crops grown in the field. These searons, discucsed in detail in following sections, relate to a series of con~plicationsthat arise as processes of exchange of CO? for water are scaled up froin the level of instantaneous fluxes at the stolnata to crop growth and water use in field canopies. Many of these complications relate to the physiological basis of variation in A I T and A/T. Crrus~sof Genotypic ifariatiolz in A13C in C, Cereals As indicated earlier, variation in (;/c, and thus Al3C can result fro~nvariation in stomatal conducmnce. in pliotosynthetic capacity, or a cornbillation of both. In bread wheat. stomatal cond~ictanccand photosynthetic capacity contribute approxirnntely equally to genotypic variation in A13C(Condon et al., 1 990; hlorgnn and LeCain. 199 1 ). The data prcbentcd in Table 6.1 summarize obbervations rnnde on 6 of 14 bread wheat genotypes studied by Condon et al. ( 1990). Among these genotypes. both stomatal conductance and photoryntlietic capacity varied by 1.3-fold, while A13C varied over a range of 1.8 per mil. For pairs of genotypes with the qame conductance (e.g.. cvv. Veery 3 and Sunstar, Hartog and Quarrion), AIJC was lower in the genotype with higher photosynthetic capacity. For pairs of genotypes with the same photosynthetic capacity (e.g., Veery 3 and Quarrion), Al3C was lower in the genotype with lower stornat;il conductance. It is safe to assume that genotypic variation in Al3C will be the same for other cereals such as durum wheat, barley, and rice since all of these species show a siniilar range of variation in A13C 3s found in bread wheat. It is also likely that considerable genotypic variirtion may exist in both condilctance and photosynthetic capacity that is not revealed by me:isorin,g A T . This is because of the overall tendency in plants of C j species for conTABLE 6.1. Variation in stornatal conductance, photosynthetic capacity, and carbon isotope discrimination among representative Australian semidwarf wheat genotypes. Genotype Veery 3 Sunstar .. Hartog K1056 Quarrion M3844 Stomata1 conductances Photosynthetic capacity (rno1.m-2. s-1) (rnrnol-m-2. s-?-Pa-') 0.55 1.66 (%,) 21.O 0.49 0.46 1.56 1.60 20.2 19.8 0.48 0.43 1.68 1.79 19.5 19.3 Source: Adapted from Condon et al., 1990 astornatal conductance and photosynthetic capacity (initial slope of the relationship between A and ci) were measured on flag leaves of well-watered and fertilized plants. 3 ~ 1 was 3 ~measured on sink tissue for C assimilated by the flag leaf, i.e., growing ears enclosed in the flag leaf sheath. o ductance and capacity to covary, such that c,/c,, is maintained close to a value of 0.7, at least in leaves of healthy. unsti-essed plants (Farquhar, Ehleringer, and Huhick, 1989). This tendency for conductance and capacity to covary is illustrated in Table 6.2, which suminal.izes data on AI3C and related photosynthetic parameters obtained on a diverse collection of 41 rice genotypes grown under periuanently flooded conditions in southeast Australia. Among all 41 genotypes a range in A l C of recently grown leaf material froin 19.2 pel- mil to 21.1 per mil existed. Although substantial variation in leaf conductance (measured in this study as leaf porosity using a visconsflow porometer) was seen, very little relationship was apparent between Al3C and conduct;~nce.In fact, for the bulk of the 41 genotypes. considerable genotypic variation in conductance existed that was reflected in nl~~lost no change in A')C. This was because conductal~ceand capacity (inensured as leaf N content) tended to covary. When genotypes were arbitrarily split into three groups on the basis of N content, which is a robust measure of photosynthetic capacity in rice (Peng et al., 1995). they showed no difference in average A13C of leaves. Rather, diflerences in average N content aillong the groups were rellected in parallel differences in leaf conductance, canopy temperature depression (CTD), and ' 8 0 conlposition (8lXO)(Table 6.2). Similar observations of covariation of stomatal conductance and photosynthetic capacity were made by Fischcr ct al. ( I 998) on a "historic series" of semidwarf bread wheats released by CIMMYT. In this study. the genotypic variation in stomata1 conductance exceeded the variation in photosynthetic capacity. with the result that more recent, high capacity, high cunductance genotypes had highel- values of Al3C than earlier, low conductance, TABLE 6.2. Summary of variation in photosynthetic traits among 41 diverse rice genotypes grown under continuous flooding in southeast Australia, 1999. Conductance -. (per second) 1.8 i 0.2 1.4 i 0.2 1.1i0.2 - N-content groupa HighN (n= 14) Intermediate N (n = 13) LowN ( n = 14) N content (%) 2 3.40 3.03-3.40 53.03 D13Cb (%) 20.3 +- 0.1 20.2 ? 0.1 20.3i0.1 CTD ("C) 5.6k 0.2 5.2 + 0.2 4.9iO.l 6'80 (%.) 25.02 0.2 25.8 i 0.1 26.0+0.1 Source: Condon et al 1999. ; a Mean values of photosynthetic traits (L standard error) are shown for three equally sized groups. separated on the basis of leaf N content. b ~ 1 3 C6180 , (oxygen isotope composition with respect to standard mean ocean water [SMOW]) and stornatal conductance were measured on upper canopy leaves Stornatal conductance was measured using a viscous-flow porometer (Rebetzke et al,, 2001), CTD (canopy temperature depression) using an infrared thermometer. r f r o t s or o r I r /I tr t 181 low capacity genotypes. The range in AfiC ainong the w11e:its studies by Fischer et 211. (1998) \\Jiis 1 per mil. The me;lrnremrnt of AJ3Cprovides an integrated estinlirte of genotypic variation in c,/c,, and. potentially. A n : Unfortunately, A13Cprovides no information on whether I-, /c,~is v:l~yingdue to v:lriation in stomatal conductance or due to variation in photosynthetic capacity, and this infornl:~tion may be inlpoi-tant for several reasons. One reason is that it is unlikely that the uimpliqtic I-elationuhipbetween A1'C and Afldescribed by Equation 6.6 will f~illyapply in sit~~ations in which variation in AliC reilects variation in stomata1 conductance. Thic is b e c a ~ ~ any s c genotypic difrerence in stomatal corlductance will usually also be reflected in a dilference in leaf teniperature. Genotypes \\~ithlorver condrlctnnce will have warmer leaves (e.g., ?&ble 6.2) :ind therefore larger vapor pressure gr:ldients driving Lvater from the leaves (Farquhar, Hubick, et nl.. 1989). A a result, transpiration per unit conductance will also be greater (Equation 6.4). Ultimately this means that the term ( f i t i - M.~,) in Equation 6.6 is not an independent rariable. and any gain in ,4A" will be less than expected from the lower value of A13C that rcwits from the lower conductance. For individual leaves, this situation will become wol-ue the less the air is ?tin-ed around the leaves. This is because the air in the unstirred boundary layers around the leaves of a low-conduct ence genotypc will be warmer and drier than thc air in the boundary layers :[round thc leaves of a higher-conductance genotype. Thc situation is predicted to becoine worse still when leaves form extensive, dense canopies, \och ar in a well-fertilized cereal crop. This is largely because even less stirring of air around l e a e s will occur in such a canopy, although ndditio~lal reasons for this exist that are explored more fully elsewhere (Cowan, 1988; Jones. 1993). Despite the complications of elevated leaf temperature, in most situations genotypes with low A13C resulting frorn lower stomatal conductance I\ i l l still tend to have higher Am, but the gain in Am will be diluted sotnewhat. 01. collrsc another conwquence of low AI3C as\ociated with low ston~:ltalconductiince exists. Lower A1'C and higher A/l.resulting from low 5to1n:lt:ll conduct;ince are likely to be associ:~tetlwith lower photobynthetic rate per unit leaf ilre:l illld possibly a slower r:itr of crop growtll. Low AI'C i ~ \ \ ~ ~ i : with l t ~ dIO\\J ~t0111:ltiil conductance inay therefore result in lower productivity if no \tleong limitation to growth exists froill lack of water. The colnplications associated with low stomatal conductance are not anticipated if low Al3C is associiltad with high photosynthetic capacity (greater draw-down of ci per unit conductance). In this case no change in leaf teinpeleature should occur, so the I-csponse of N T t o a change in AuC sho~ildfillly correspolld to thnt given in Equation 6.6. Also. the expectation would be for greater photosyntlletic rate per unit leaf area and, therefore, prob;lbly a faster rate of crop growth, although as discussed in a following section. the latter may not necessarily follow. The values of A'3C nieasurecl in dry matter sanipled from plants of C3cereals under stress are alillost invariably lower than At3Cvalues ineasured on unstressed plants. Abiotic stresses such as soil-water deficit (e.g., Farc~obnr and Richards, 1984; Ehdaie et al., 1991; Condon et al., 1992: Merah et al., 1999), soil colnpaction (Masle and Farquhar, 1988), soil aalilrity (Isla et al., 1998: Rivelli et al., 2002), and low humidity (Condon et al., 1992) all result in lower values of A13Cbecauhe they result in some degrcc of stoiuatal closure, causing c, /c,, to be lower. In some circurnst:lnces, abiotic stresses such as salinity may be severe enough to cause damage to the photosynthetic apparatus, especially if' they are accompanied by conditiolls of' high light (e.g., James et nl., 2002). In such cases, a tendency toward a slightly higher value of (.,/(.,,may exist due to lower photosynthetic capacity, but a co~~currel~t tendency toward lower c,/c, will probably exist due to stornatal closure. In practice it is difficult to resolve these opposing influences on c,/c, and A ' T , even using sophisticated gas-exchange measurements. It might be expected that N starvation would be reflected in h i ~ h e values r of h13C since N starvation should i-ecult in lower photosyothetic capacity, but experiments conducted with wheat and barley Sound virlually no difference in A13C between N-sufficient and N-starved plants (Condon et al., 1992; Kang ct al., 1 996; Robinson et al., 2000). This is probably because N starvation was applied early in development. with two outcomes. First, the plants adjusted leaf area, through snialler leaves and fewer tillers. so that leaf N content was maintained at relatively high lcvcls (certainly less dif'ferent than the difference in applied N). Second, it is likely thnt stomata1 conductance fell to balance the photosynthetic capacity of thc leaves and coilstrain c,/c, near 0.7. Relatively low stomatal conductance is a comnlon observation for N-starved cereals (Wong et al., 1979). Values of A1jC meauured on unstressed plants of C3 cereals are typically in the vicinity of 19 to 22 per mil. For p1;ints subjected to soil-water deficit. A13C values measured on organs that have grown when plants were under severe l r r c have been fi)und to be as low as 12 to 14 per mil. Many of the lowest valves of A13C have been measured on grain of' teinperate cereals grouln in field e~pcrirncntuin very low rainfall environments or in container expcrjmcntu in ~vhiclisoil waler deficit became gradually more scverc (c.g., Condon ct al.. 1992; Voltas et al., 1999: Rotwl-ightet al., 2001). It is not curpri~ingthat a gmd~taldecline in the values ofA1C measured usually occur5 on rccentl y formed dry matter of temperate cereals grown in rain fed enviroi~mentc.since it is t19traIfor soil water availability to decline and the vapor presture deficit of the air to increate as the growi~lpseason PI-ogrescec.Both of these stresces will resolt in stomatal c1osilt-e and lower values of Al3C (ConcIon et al., 1 992; Figure 6.3). The very low values of A13C nleasured in grain have prompted hypotheses on 1-ecyclingof respired C 0 2 within the grain-enclosing stnictures of the ear (Arauu et al., 1993; Gebbing and Sclinyder. 200 1 ) and suggestions that an enllnnced contribution to C02-fixation from PEP c:lrboxylase present i n br:lcts and ylurnes rnay exist (Bort et al.. 1995). Whereas any enhanced contribution fro111PEP carboxylase rem:iins moot. it is highly likely thnt some recycling of respired CO, does take place within the ear. The effect of this t o 0 17 16 A A ~ ~ C 0 Soil water Conductance v vA B v Days after sowing FIGURE 6.3. Season-long changes in water in the 1.8 m soil profile, stomatal conductance, and values of A13C measured in most-recently expanded organs of wheat grown under rainfed conditions at Moombooldool, SE Australia in 1985. Data points shown for soil water depletion, stomatal conductance, and Al3C are mean values for eight genotypes. Note that changes in stomatal conductance measured on source organs are detected as changes in 6136 in sink organs sampled several days later. Source: Adapted from Condon et al., 1992. recycling on the Al3C value of grain is difficult to resolve since the carbon in the grain may be derived from numerous sources, including current photosynthate from leaves and ear structures and assimilates stored earlier in the sterns and leaf sheaths. Eaclr of these C sources may have quite different Al3C sig~lilturesthat largely derive fro111the environmental conditions prevailing at the time the C was acquired. Grain AI3C values are not always very different iiorn leaf A13C ~ ~ I L I C S . Mean grain A'" values of approuim:ltely 19 per mil have been measured in field experiments on durum wheat given supplemental irrigation (Merah et al.. 2001; Royo et al., 2002). In a study of 11 advanced breedillg lines of rice grown under flooded padi conditions in southeast Australia (Condon et al., 1999). the rrlcan A13Cvalue of grain harvested at maturity (mid-April) was 19.5 per mil (genotype range 18.6 to 20.3 per mil). This was very close to the mean Al3C value of 19.8 per mil measured on recently formed leaves sampled in early January (genotype range 19.4 to 20.2 per mil). The slightly lower values for grain Al3C than leaf Al3C could easily be attributed to the high starch content of the grain as compared with the relatively l~iglllipid content of the leaves. Starch is known to have a lower A]" value than lipids (Figure 6.2). Indeed, given the difference in chenlical conlposition betweell leaves and grain. it is sulprising that the difference in AI3C was not greater. Gerlotype x Environrnest Zrrlei-actioizfor A1"C Genotype x environment (G x E) interi~ctionsfor AI3C are potentially large because erivironment influences can have a very large effect on A13C values measured in plant dry matter, especially under conditions of declining soil water and rising ewlporative demand (see previous section for details), and the A13C value of all genotypes may not respond in the same manner or at the same time to these environmental perturbations. For example, the value of A13C inay fall faster in genotypes that use water faster or that have shallow root systems 01- that have stomata that are more senqitive to increasing vapor pressure deficit of the air. Among the eight wheat genotypes grown in the study suiumarized in Figure 6.3, genotypic variation in the slope of the seasonal decline in A1-T was strongly correlated with the proportion of profile water con{omed at anthe sis by each genotype. The rate of decline of AI3C was faster for genotypes that had used I-elatively more of the available soil water at anthesis (Condon et al., 1992). Broad-sense heritability is :I me:Isure of the extent to which phenotypic variation in a trait can be attributed to genot>~pic differences, rather than the effects of environn~ent,G x E, and sampling. Thus it reflects the extent to which the range in genotypic v:llues is repeatable, and therefore it also re- flects the potential for progress in selection during breeding. Estimates of' broad-sense heritability for A1?Ccan be high. in the order of 90 percent on a genotype-mean basis and 80 percent on a single-plot basis (Condon and Richards. 1992), but st~hqtantiallylower values are not uncommon (Ehdaie and Waines, 1994; Araus et nl., 1998; Merah et al., 2001). Different estimates of broad-scnsc heritability for AI3C may reflect the different tissues sampled for AI3C measurements. For temperate cereals. values of heritability for Ai3Care often lowest for plant material sampled near anthesis, such as flag leaves. the rachis. and peduncle. This is when any genotypic diSSerences in soil-water depletion are likely to be greatest. Herit;lbilitics may be higher for grain, but this is not always the case across contrasting soil rnoisturc I-egimes (Condon and Richards. 1992). Heritabilities have been found to be highest for dry matter laid dow~lbefore or during early stem elongation, when p1:ints are essentially unstressed and repeatability greatest (Condon and Richards, 1992). Values of n;rrrouT-senseheritability lor AI3C of bread wheat reported by Rebetzke, Condon, et 31. (2002) were also high: 93 percent on a yenotypemean haqis and 63 percent on a single-plot basis. In the same 5tudy. the corresponding n:irro\v-sense heritabililies for grain yield were 55 and 14 percent, respectively. The high values of heritability for A13Cwere obtained by employing dry matter sanlpling strategies that minimized thc iinpact of potenti;~llylarge G x E for A13C. Measurements o S A I T were done on vegetative leaf material laid down early in the standard growing season when plants weir effectively unstressed. as recommended by Condon and Richards (1992). Genetic Corltml of A]-'C Narrow-sense heritability reflects that portion of the phenotj p'lc variance that is transmitted from parent to progeny. Tlr~iq,it reflect$ breeding value. High narrow-sense heritabilities reported by Rebet~ke.Condon, et 211. (2002) for A13C of field-grown plants were consistent with high narrowsen$e heritabilities for AfiC rneasurcd for progeny from a range of wheat crosses evaluated in the glasshouse and field (Rebet~keet al., 2006). Furthermore, these high narrow-sense heritabilities were consistent with high heritabilities for tranqpiration efficiency measured on bread (Malik et al., 1999) and durum \\/heat (Solomon and Labuschagne, 2004) plants evaluated under water-limited and well-watered conditions in the glasshotlse. Genetic stildicq in u7henthave shown a piepondel-ance of additive gene effects for A1.'C (Rebelrke et al., 2006) and tr;~nspirationefficiency (M:~lik et al., 1999; Solon~onand Labuschagne, 2004). Although s~nall,some evi- 5 2. s." 5 b .zy I>.z 3-G g ga o% - s;".i% q a -.< W Vj I 5996 g -2& g u cog 53.. PF-.'P y . ; g9g K s o - g < g- 3 g-g 5 9 a sug g- -2:.2 Sf g g4 g g c w yj-rl." 5 0 z zc cy - ' m 3 C. c.5-z. 2 - s - s. E g'aO6 O Z 5 " c 5 5 3 " 3 6 fqe 2 $ & < $ 3 7 2 z d ' g T .3J. e g g m - J. 0 -2 s e 5 c : C PI. P -dI Transpiration efficiency (g.kg-~) 3 t-4 3 E Z @S+ 5 V E w z. 6. 0 "s g 05. 2rrG 0 - , 3 < 2 3 CD 5. EE':g7 -. 55 -8ga E g,, 0 0 2 =st< 3C3 Z " 3 Qg.g$ < 3 q 0 d 5. gz;zp. - . c 5: sg$?a 5.5.g B, w , ZFs r z cn 2 cr <cpg - c. 5 CD gez- 5 . d 2 9 'f-5.3 4. g g <2. 3 v.g-5'55 5 S 3 ~ , 0 " D z V E slower water use and perhaps consel-vative growth of low-Al3C genotypes may be adv:intageous in sustainillg higher grain ilumber and grain yield. Relationships between yield and AfiC may bc particularly variable in Mediterranean-type environments if low Al3C is associated with slow early growth. This is because in rainfed Mediten-anean-type environments any difference in transpiration between high-A'" and lo\v-A13C gellotypes may not be reflected in a similar difference in total crop-water use to anthesis if soil evaporiition is greater from under the slower-growing canopies of low-Al3C genotypes (Condon et al., 1993; Condon et al.. 2002). Why Would Low-A13C Cereals Grow More Slowly? As stated in the previous section, an obvio~lsreason why low A13Ccould be associated with relatively slow crop-growth rate in cereals is if low A1)C in the absence of soil-watel- deficit is the result of loiv stornatal conductance. Genotypes with lower sto1nat:ll conductance will tend to have higher A/T and lower A"C, all else being equal, but higher A/T is likely to be essociated with lower pllotosy~ltheticrate per unit leaf area and consequelltly a slower rate of crop growth. Low conductance iuay not be the only reason why low A13Cis associated with a slow crop-growth rate. Variation in A13C in cereals can also result froin variation in photocy~ltheticcapacity (Condon et al., 1990; Morgan and LeCain, 1991 ). If low Al3C is the result of high photosynthetic capacity, the expectation might be a higher rate of photosyllthesis per unit leaf area and thus faster crop-growth rate, However, crop growth rate may actually be slower becaose, in cereals, substantial illcreases in photosynthetic capacity arc most madily achieved by concentl-ating N into sinallsr leaves that have greater macs per unit area and that intercept less light per unit N. This may slow the rate of crop growth ~lntilfull light interception is achieved. IS fill1 light interception is achieved only briefly, as often occurs in drier cropping environments, or not achieved at all, then high photosynthetic capacity may not result in greater growth. In fact, the reverse may occur: cereal genotypes with low photosynthetic capacity may actually achiew faster crop growth. The eight genotypes shown in the s t ~ ~ ddepicted y in Figure 6.5 (Condon et al., 1993) provide an example. Biomass production to anthcsis was positively correlated with A'" (Figure 6.5a), but the three genotypes with the highest Al3C values did not have greater sto~natalcoilductallce (Figure 6.5b). Instead, the inference, giver1 they had higher valnec of A13C (ca. 1 per mil higher) at sinlilar values of conductance, was that these three genotypes had lower photosynthetic capacity. This inference was confirmed in studies on glasshouse-grown plants (Condon et al., 1090). 18.5 19.0 19.5 20.0 Discrimination (1 20.5 x 21 0 A'~c) - 0.35 0 40 0.45 0.50 Conductance (mmol m-2.s-') FIGURE 6.5. (a) The relationship between aboveground biomass production to anthesis and leaf Al3C, and (b) the relationship between leaf ~ 1 %and stomatal conductance, for eight wheat genotypes grown at Moombooldool, SE Australia in 1985. Values of A13C are average values for leaves from well-watered plants sampled well before anthesis. Values of stomatal conductance are the average of data obtained at five sampling times before anthesis. Least-squares linear regressions are shown for statistically-significant relationships (P < 0.05). Open symbols indicate genotypes with low photosynthetic capacity. Sources:Adapted from Condon et al., 1990; Condon et al., 1993. The eight genotypes grown in this study were very diverse, and other factors may have been associated with low photosynthetic capacity and high AJrC that were also associated with higher anthesis biomass. For example, just as substantial genotypic variation in dry matter and N partitioning appears to have existed within the leaves, affecting photosynthetic capacity, genotypic variation in factors iilfluencing within-plant partitioning may ha\-c nlco existed that could also have influenced crop growth rate. Detailed {tlrdies on ntclch rnol-e closely relatcd genotypes have been initiated to resolve accociations between factors contributing to variation in Al3C and factor5 contributing to variation in crop growth rate, factors influericing bioTnase partitioning at leaf and plant levels, and the extent to which such associations are due to pleiotropic effects or genetic linkage. CASE STUDY: AYI3LIGA7'IC)~VO F CANBONISOTOl'E ANALYSIS IN VVHEA T BREEL)INrr FOR A UL"iTRALIA Wheat production in Australia is a ~najorcomponent of a highly inechanized, commodity-driven agricultural system. The vast bulk of the wheat o I P 0 t G 0 ,0 2 2 2 , Drysdale yield (9/, of check) Yield advantage (%) maintain stomata more open after anthesis despite increaving soil and atrnospheric water stress (Condon et al., 1993). Any of these chiiracteristics acsociated with high grain Al3C (fast crop growth rate, ability to remobilize stored reserves, earlier flowering, better water extraction, stoniatal insensitivity to water deficit) would be ~lsefiilfor cereals in Meditermnean-type environments. Carbon lsotape Discrimination in C , Cereals Can variation in Al3C to breed for iniproved performance of C4 species in water-limited agriculture be exploited'? This n ~ a ybe possible, but both theory and available data indicate tliat progrevs with C, species is likely to be even less straightforward than with C3 species. S o ~ n evariation iii A13C has been observed within C4 specie? (e.g., Hubick et al., 1990; Mortlock and Hammer, 1999). but the extent of this variation in C, species is inuch less than in C3 species. In addition. the relationship between transpiration efficiency and A13C is different in C, species. This is because of the substaiitial differences between C3 and C, species in the processes of C-assimilation. In C, species C is fixed into 4carbon compounds (malate or aspartate) in ~liesophyllcells by the enzyme PEP carboxylase. With respect to gaseous CO,, this enryrne actually disin contrast to tlie C, enLynie R~~bisco. criminates a little in favor of 1". which has a relatively large discritnination against "CO?. After the initial carboxylation step in C, pilotosynthesis, tlie 4-carbon co~npoondsai-e then transported to the bundle-sheath cells surrounding the vascular bundles. Here. after a decarboxylation step, C 0 2 enters the C, pathway and is fixed by Rubisco. Tlie bundle sheath is very close to being gas-tight, and this has two implications: The first is that it allows the C0, concentration to be held mnuch higher than in the outside air. so c;trhoxylntion by Rubisco is much more efficient than in C, plants. Second, little opportunity exists for CO, to escape from the bundle sheath. and so little oppol-tunity exists for any disc~-iminntionagainst ' C O O by , Rubisco to be expressed. Discrimination by Rubisco can only be expressed if C 0 2 can escape to the atiiiosphere. In reality the bundle sheath is iiot absolutely gas-tight, so some discrimination by iiubisco occurs. 'rhese con~plcxitiesof discl-imination during C, photosynthesis can be expressed in a simple mathematical description relating Al3C to c/c,,, as follows (Farquhar, 1983; Henderson et al.. 1992: Rrugnoli and Farquhar, 2000): In this equation, n is the Sriiction:itinn dr~ringdiffusion through the stomata (4.4 per mil) and b' = [h, i-sP - (b, - s)]. where b4 is the discrimination by PEP carhouyla\c (-5.7 per mil), h, is the cli\crirnination hy Rubivco (30 per mil), s is dibcrinlination during lcakage fi-om the bundle sheath (1.8 per mil) and the factor @ accounts for the proportion of CO, released by dccorhoxylation that leak5 out of the hundlc vhcath (Rrugnoli and Farquhar, 2000). It should be noted tliat the value of cD is not constant. It can also be noted that Equation 6.8 has the same for111as Equation 6.1, which dcscrihed ditcrimination during C, photosynthesis. with the greater con~plexitiesof C , photosynthesis captul-ed in the term b' in Equation 6.8, analogous to the terrrl h in Equation 6.1 . Compared to C3 species, for C4 species relatively little variation in A T associated with the prirnary pi-ocecces of C assimilation exists. This is because the value of the term ( b - o) is close to zero, whereas the term ( h -a). from Eq~iatioti6.1, has a value of ca. 24 per mil. lo fact. for C, photosynthesis, the dependence of A T on c,/c,, [nay be positive or negative depending on whether the value of 17 5 s greater or legs than then value of a, arid this will depend an the iirnount of leakage from the bundle sheath. (Using the par;lmeter values cited, h' = a at @ = 0.36, and no dependence of A13C on c,/c, would exist.) In practice. the value of sP has been usually found to be a little less than 0.3. Conseqt~ently.the relationship between A'" ;and c,/c, for C, species is usually negative, but with a small slope (Figure 6.1). For C3plants tlie relationship between A13Cand c,/cclis positive, with a large slope. Discriminatioii during C4 photosynthrsis may therefore vary with variation in c/c, or with variation in @ (or both c,/c, and @). In practice, it is diffjcult to distinguish between these two inlluences, and both of'tlienl will be subject to genetic and environmental effects. Tlie value of c,/c,, would be expected to respond to changes in stol~iatalconductancc, which is under genetic and environmental intluenccs as found for C3 (pccics. The value of 0 is subject to several poorly understood inlluences. Genetic variation in @ has been indicated fronl the results of comparisons of C4 species that differ in aspects of their biochemistry and anatomy (Ehleringer and Pearcy, 1983). In soiiie C, specie%,the value of @ has bcen observed to change in response to drought and salinity stresses. These changes have been attributed to changes in the activity of R~ibiscorelative to PEP carboxylase (Bowman et al.. 1989; Saliendra et al.. 1996). Hubick et al. (1 990) attsibuted variation in A13C among soghurn (Soqqiium birolor L.) genotypes to variation in either or both c,/c,, and @, whereas Henderson et al. ( 1992) found cD to be relatively constant at 0.2 and variation in A l T to be related to variation in c-, /C(,. Farqli har ( 1 983) concluded that radiation-LIS~ efficiency may be higher if @ was low. and that this may be reflected i n productivity. Likewise, MI' Plat leaf area (cm*) LG D E t: J; 0 c 2 5 0-E g s sg zg g 6. 00: * m u m E rCD R CD t, Z m i T g $ q g g 'A 3 oG' P g k5s 02: g ez 8 6% rAE c. g z g UQ CTC, 3 5. s. -3-2 CD UQ Z Ec2. 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