Heterogeneity in Carbon Isotope Discrimination in Leaves, Stalks and Spikes of Ten Annual Wild Triticeae Species

Czech J. Genet. Plant Breed., 41, 2005 (Special Issue)

J. Z�� and V. H��

Research Institute of Crop Production, 161 06 Praha-Ruzyně, Czech Republic e-mail: [email protected], [email protected]

Abstract: The wild Triticeae species are confined mostly to arid regions and thus, they are suitable material for study of their adaptation to dry conditions. Species from semidesert localities of Mediterranean, Balkans and Near East (genera Eremopyrum, Heteranthelium, Taeniatherum, Dasypyrum, and Aegilops) and wild wheats (Triticum) from steppe localities were studied. The studied species can be divided into two groups on the basis of their carbon isotope discrimination (Δ). The first group, which had high Δ, consists of species (Ta. crinitum, A. tauschii, H. piliferum, E. orientale, A. markgrafii, A. comosa) collected in the driest localities such as semidesert sites. The second group from steppe localities, which consists of D. villosum, T. urartu, T. boeoticum, and T. monococcum, had relatively low Δ. We conclude that wild species originally grown in dry regions discriminate more carbon isotope as they are adapted to the dry condition by stomata opening and/or Rubisco activity. We examined from high to low carbon isotope discrimination values of the flag leaves, stalks, and spikes.

Keywords: carbon isotope discrimination; delta; wild Triticeae; heterogeneity; drought adaptation

Wild wheat relatives, members of the Triticeae ing method for evaluation transpiration efficiency tribe, could represent a valuable source of genetic (TE). Δ is a measure of the ratio of stable carbon variation for improvement of abiotic stress tolerance isotopes (13C/12C) in plant dry ma�er compared in cultivated wheat (Triticum aestivum L.). A better with the same ratio in the atmosphere. 13 knowledge of the adaptive strategies developed Discrimination against CO2 is closely negatively by these species is needed. Species of Aegilops correlated with the transpiration efficiency inte- (goatgrass) are well adapted to dry environments. grated over the life of the sampled plant material Aegilops tauschii is of particular interest, since gene (F�� & R�� 1984; C�� et al. 1990; transfer from this drought-adapted species to bread C�� et al. 1992). Carbon isotope discrimination wheat is relatively easy (V�� et al. 2004). has been proposed as a useful indicator of TE in Isotopic differences in diverse elements make it wheat (F�� & R�� 1984; E�� et al. possible to demonstrate that fractionation events 1991) and in numerous species including other occur in nature. Fractionation processes are ther- cereals (C�� et al. 1993; M�� et al. 1996). The modynamic or kinetic reactions that produce dif- negative correlation of Δ and water use efficiency ferent abundances in reactants and products. Two (WUE, measured either as net photosynthesis/ 13 important processes in which CO is discriminated transpiration or plant biomass produced/water 2 are diffusion and carboxylation. Diffusion fractiona- transpired) led to Δ being proposed as selection tion is dependent on molecular mass. Diffusion of criteria for WUE (F�� & R�� 1984). It heavy atoms or molecules is slower than diffusion of is determined by the intercellular partial pressure lighter ones. Carboxylation by RUBISCO discrimi- of CO2, which is largely regulated by variation in nates strongly against 13CO typically in C plants. stomatal aperture (F�� et al. 1989). Under 2 3 Carbon isotope discrimination (Δ) is a very promis- drought stress, Δ can be considered as a good

212 Proc. 5th International Triticeae Symposium, Prague, June 6–10, 2005 Czech J. Genet. Plant Breed., 41, 2005 (Special Issue) predictor of stomatal conductance (C�� et al. tope discrimination and the total carbon content 1990). Carbon isotope discrimination positively in plant parts. correlates with yield and growth cycle duration (A�� et al. 1997) and negatively correlates with MATERIAL AND METHODS leaf temperature (A�� 1993). The strong correlation between carbon isotope discrimination and Material. Mature plants of wild Eremopyrum, yield in durum wheat grown in the Mediterranean Heteranthelium, Taeniatherum, Dasypyrum, Triticum conditions has been confirmed (H�� et al. 2000). and Aegilops species were collected at the end of It is easier to measure carbon isotope discrimina- vegetation period from semidesert and steppe tion than transpiration efficiency. Measuring of localities of the Mediterranean, Balkans and Near carbon isotope ratio in dry matter of a plant organ East from steppe localities (Table 1). The localities provides a measure of the discrimination of carbon are characterized by a dry summer season with isotope of this organ and carbon isotope ratio of high temperature and high irradiance. matter coming from other plant organs. Methods. Collected plants were dried until they The main objectives of the present study were (i) reached a constant weight. Twenty flag leaf blades, to examine the variation in plant carbon isotope twenty 10 cm sections from the upper part of the discrimination as a tool which represents water stalk, and twenty spikes per genotype were ran- use efficiency among accessions of the wild Tri- domly sampled from fully mature plants. The leaf, ticeae collected in their native habitats where they stalk and spike samples were separately grounded are well acclimated (ii) to analyze relationships in to a fine powder in a metal ball mill. Carbon isotope carbon isotope discrimination of different wild ratios were measured to 0.10‰ by isotope ratio mass Triticeae organs in generative stage important for spectrometer (IRMS). The total carbon content and reproduction at the end of plant ontogenesis and carbon isotope composition of leaves stalks and (iii) to evaluate relationship between carbon iso- spikes were determined by an Elemental Analyzer

Table 1. Species used for study, their geographic site and ecological characteristic

Accession Abbrevia- Collector/ Latin Name Geographic Site Ecology No. tion donor

C2100705 Aegilops comosa SIBTH. et SM. A. com NW Korfu on Isle of saline grassland Gatersleben ssp. heldreichii (BOISS.) EIG Korfu, Near Potamos River Aegilops markgraﬁi (GREUTER) Hasan Dag, nr. Hamrun, bazalt, lava pall, C2100428 A. mar VH HAMMER var. markgraﬁi Nigde, Turkey 1460 m TA2457 Aegilops tauschii COSS. A. tau Turkey dry steppe Kansas Dasypyrum villosum (L.) Sopot, 7 km N of C2300014 D. vil dry steppe, 415 m VH CANDARGY Kumanovo, Macedonia Eremopyrum orientale (L.) JAUB. Dzrvez, 2 km E of Erevan semidesert steppe, C3400006 E. ori VH et SPACH to Garni, Armenia 1000 m C3700001 Heteranthelium piliferum H. pil Iran semidesert Logan (BANKS et SOLANDER) HOCHST. Triticum boeoticum BOISS. var. Izgrev – Pcela, Rodopi dry steppe, 680 m VH C0104087 T. boe1 symbolense (Flaksb.) A. Filat. Mts., Bulgaria C0106065 Triticum boeoticum BOISS. T. boe2 Bayindr, Konya, Turkey dry steppe, 1195 m VH Taeniatherum crinitum Ni�aular nr. Dzebraili, semidesert steppe, C3300006 Ta. cri VH (SCHREBER) NEVSKI Georgia 1000 m C0106421 Triticum monococcum L. var. T. mon Transylvania, Romania old land race A. Szabo clusianum LR. Galara Kiss Triticum urartu THUM. ex dry steppe to C0106076 T. ura Sanli Urfa, Turkey ICARDA GANDIL. semidesert, 620 m

VH = Vojtech Holubec

Proc. 5th International Triticeae Symposium, Prague, June 6–10, 2005 213 Czech J. Genet. Plant Breed., 41, 2005 (Special Issue)

(EuroVector 3028/HT) coupled to an Isotope Ratio Statistica 6.1 (StatSoft) software was used for Mass Spectrometer (IRMS Isoprime). International statistical evaluation of the data. standards were used so all delta values are inter- comparable. We use delta notation (δ) to express RESULTS AND DISCUSSION carbon isotope ratio conveniently: Taeniatherum crinitum exhibited the highest 13 13 12 13 12 C = ([( C/ C) /( C/ C) ] – 1) 1000 (1) overall carbon isotope discrimination and Triti- δ sample standard cum monococcum the lowest (Figure 1). In general, delta values are expressed in parts “per mil” Dasypyrum and all studied Triticum had the lowest 13 (‰) for plant material ∂ C is always a negative carbon isotope discrimination. Interestingly, Ta. number. crinitum had values of more than Δ 20‰, which The standard error of determination was 0.10‰ is characteristic for species with low water use on average. Fractionation is also known as carbon efficiency. The Triticum spp. on the other hand, isotope discrimination (Δ). The Δ value of the sam- had low carbon discrimination, corresponding to ples was obtained according to the formula: the high WUE. For most of the species studied, the carbon com- Δ ≈ (δ – δ )/(1 + δ ), (2) a p p position of the leaves was low in comparison to rest of the plant (Figure 2). Most species had a Δ ≈ δsource – δproduct (3) carbon composition of 48–50% in their stalks and (F�� et al. 1989), where a and p refer to air and spikes. Aegilops comosa and Heteranthelium piliferum plant carbon isotope composition, respectively. The were exceptional in this respect, having values of carbon isotope composition of air was –8.3‰, δ 13C only 37% and 30%, respectively. The low carbon

of C3 plants is reported approximately –25‰. Each composition of the leaves of H. piliferum contrasts sample was analyzed at least 2 times. Direct transpi- with the values obtained for its stalks and spikes ration efficiency was not measured in this study. (Figure 3)

21 C (‰) 13 �

19 Carbon Carbon Isotope Discrimination

m ii i m le a 1 m u 2 m h fi u a s m u rt m u tu c ra r t o u s a u c i s g e n m c r c c in u k if ie o ti llo u ti o r ta r il r c o i o c c s a p o s e v m e o m p m p o m u o n u o s m m lo b u ic b o r il p iu ru i r it m e g o l y g m y r m th e il e p e u p T u m a A g th o A ic sy ic u i e n it a it ic n a m r r it e A r re T D T r a T T ete E H Figure 1 Average carbon isotope discrimination of species collected in their original growing area typical with shortage of precipitation, from semidesert localities of the Mediterranean, Balkans and Near East (genera Eremo- pyrum, Heteranthelium, Taeniatherum, Dasypyrum, and Aegilops), wild wheats (Triticum) from steppe localities

214 Proc. 5th International Triticeae Symposium, Prague, June 6–10, 2005 Czech J. Genet. Plant Breed., 41, 2005 (Special Issue)

23 Figure 2. Relation of carbon isotope discrimination of leaf, stalk and C (‰) C

13 22

� spike of wild Triticeae species to 21 their total carbon composition in dry ma�er 20

19 leaf 18 stalk spikes 17

Carbon isotope discrimination discrimination isotope Carbon 16 28 32 36 40 44 48 52

Carbon composition in dry matter (%)

Carbon isotope discrimination proved to be a The importance of spike Δ increased and Δ of valuable tool for evaluating contribution of different stalk decreased for the higher average values of Δ. organs to grain filling and yield. We examined the The Δ of leaves increased and was higher for all ratio of carbon isotope discrimination of individual range of Δ average. Similarly T�� et al. (2002) organs (Figure 3) by plotting the individual organ found higher Δ in leaves than in kernels in field Δ against the average Δ of these organs. There is grown barley. The relative contributions of the a significant linear trend for all organs. There is Δ values of flag leaf, peduncle and spike to Δ grain low Δ of spike and higher for stalk and leaf at of durum wheat genotypes were 51, 29, and 21%, low values of Δ average. The difference in Δ of respectively (H�� et al. 2000). With the exception stalk and spike is smaller with higher Δ average. of Aegilops comosa, we obtained the highest carbon

A. mar leaf 22 stalk Ta. cri A. tau spike H. pil D. vil E. ori 21 T. ura T. boe2 T. boe1 A. com 20 T.mon

18 � leaf = 3,2588+0,905*avg �; r=0,795** � stalk = 3,1319+0,8289*avg �; r=0,737** 17 � spike = -6,3907+1,2661*avg �; r=0,935** Leaf, stalk, spike isotope carbon discrimination (‰)

16 18,0 18,6 19,2 19,8 20,4 Average carbon isotope discrimination of whole plant (‰)

Figure 3. Relation of carbon isotope discrimination of separate plant parts (leaf, stalk and spike) on the average carbon isotope discrimination of tested plant parts. Coeﬃcients of linear regression are signiﬁcant on P < 0.99 levels

Proc. 5th International Triticeae Symposium, Prague, June 6–10, 2005 215 Czech J. Genet. Plant Breed., 41, 2005 (Special Issue) isotope discrimination in the leaves of all species References (characterised by the highest WUE). The two accessions of one species T. boeticum seem to behave A�� E. (1993): Potential of 13C discrimination as a like the majority of other species regardless T. boe1 selection criterion in barley breeding. In: E�� originating from Bulgaria had higher Δ on average J., H�� A.E., F�� G.D. (eds): Stable Isotopes than T. boe2 originating from Turkey. in Agriculture. Academic Press, USA. 399–417. The wild wheat relative Aegilops geniculata Roth. A�� J.L., A�� T., Z�� Y., N�� M.M. (1997): had highly significant correlations among carbon Effect of leaf structure and water status on carbon isotope discrimination (Δ) and plant temperature isotope discrimination in field-grown durum wheat. depression, biomass and grain weight per plant Plant, Cell and Environment, 20: 1484–1494. (Z�� et al. 2001). Strong negative correla- C�� A.G., F�� G.D., R�� R.A. (1990): tions between transpiration efficiency and carbon Genotypic variation in carbon isotope discrimina- isotope discrimination in wheat (Triticum aestivum tion and transpiration efficiency in wheat: Leaf gas L.) suggest that selection of progeny with low Δ exchange and whole plant studies. Australian Journal may increase transpiration efficiency and aerial of Plant Physiology, 17: 9–22. biomass under water-limited conditions (R�� C�� A.G., R�� R.A., F�� G.D. (1992): et al. 2002). The results of QTL study of carbon The effect of variation in soil water availability,vapour isotope discrimination measured in mature grains pressure deficit and nitrogen nutrition on carbon from plants grown in the Mediterranean field con- isotope discrimination in wheat. Australian Journal ditions indicate that several chromosomal regions of Agricultural Research, 43: 935. are involved in Δ variation (T�� et al. 2002). C�� A.G., R�� R.A., F�� G.D. (1993): The studied species can be divided in to two Relation-ships between carbon isotope discrimina- groups on the basis of their carbon isotope dis- tion and water-use efficiency for dryland wheat. crimination (Δ). The first group, which had high Δ, Australian Journal of Agricultural Research, 44: consists of Ta. crinitum, H. piliferum, E. orientale, 1693–1711. A. tauschii, A. markgrafii, and A. comosa collected E�� B., H�� A.E., F�� G.D., N�� H.T., in the driest localities such as semidesert sites. The W�� J.G. (1991): Water-use efficiency and carbon second group, which came from steppe localities, isotope discrimination in wheat. Crop Science, 31: had relatively low Δ. It included D. villosum, T. rartu, 1282–1288. T. boeoticum and T. monococcum. The wild Triticeae F�� G.D., R�� R.A. (1984): Isotopic com- species from dry areas had similar carbon isotope position of plant carbon correlates with water use discrimination as the advanced wheat cultivars efficiency of wheat. Australian Journal of Plant Physi- grown under full water supply. The first group had ology, 11: 539–552. Δ on average close to the Δ average value (21.4‰) F�� G.D., E�� J.R., H�� K.T. (1989): of 14 winter wheat cultivars grown under full water Carbon isotope discrimination and photosynthesis. supply. The second group were closer to the aver- Annual Review Plant Physiology Plant Molecular age value of 19.8‰ of the same cultivars grown Biology, 40: 503–537. under water stress (Z�� unpublished). The H�� M., M�� P., M�� O., D�� A. (2000): indigenous wild species growing in dry regions had Carbon isotope discrimination and yield in durum higher discrimination values. These comparisons wheat grown in the high plains of Sétif (Algeria). suggest a better adaptation of wild Triticeae species Contribution of different organs to grain filling. to dry conditions. The wild Triticeae species had Options Méditerranéennes, 40: 145–147. stomata open and/or high activity of RUBISCO M�� A., S�� A.E., V�� K�� C. (1996): Carbon even at very dry conditions. The carbon isotope isotope discrimination and indirect selection for discrimination was higher in leaves than in stalks transpiration efficiency at flowering in lentil (Lens and lowest in spikes in most species. culinaris Medikus), spring wheat (Triticum aestivum L.), durum wheat (Triticum turgidum L.) and canola Acknowledgements. This work was supported by Project (Brassica napus L.). Euphytica, 87: 141–151. No. QD1352 and by the National Programme for Plant Ge- R�� G.J., C�� A.G., R�� R.A., F�� netic Resources Conservation of the Ministry of Agriculture G.D. (2002): Selection for reduced carbon isotope dis- of the Czech Republic. crimination increases aerial biomass and grain yield

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of rainfed bread wheat. Crop Science, 42: 739–745. Directions for a Diverse Planet: Proc. 4th Int. Crop T�� B., M�� O., S�� X., B�� C., W�� Science Congr., Brisbane, Australia, 26 Sept.–1 Oct. R., T�� D. (2002): QTLs for grain carbon isotope 2004. discrimination in ﬁeld-grown barley. Theoretical Z�� M.E., G�� M., H�� E., A�� B., and Applied Genetics, 106: 118–126. M�� P. (2001): Drought and heat responses V�� J., A�� A., K�� J., S�� K., D� P�� in the potential interest for wheat improvement. E. (2004): Collection and conservation of genetic Crop Science, 41: 1321–1329. resources for dryland farming systems. In: New

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