The Photosynthetic Role of Ears in C3 Cereals: Metabolism, Water ...

Critical Reviews in Plant Sciences, 26: 1–16, 2007 c Taylor & Francis Group, LLC Copyright ISSN: 0735-2689 print / 1549-7836 online DOI: 10.1080/07352680601147901

The Photosynthetic Role of Ears in C3 Cereals: Metabolism, Water Use Efficiency and Contribution to Grain Yield Eduardo A. Tambussi Instituto de Fisiolog´ıa Vegetal (INFIVE), Universidad Nacional de La Plata, cc 327, 1900, La Plata, Argentina

Jordi Bort Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Agda. Diagonal 645 (E-08028), Barcelona, Spain

Juan José Guiamet Instituto de Fisiolog´ıa Vegetal (INFIVE), Universidad Nacional de La Plata, cc 327, 1900, La Plata, Argentina

Salvador Nogués and José Luis Araus Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Agda. Diagonal 645 (E-08028), Barcelona, Spain Table of Contents I.

INTRODUCTION .............................................................................................................................................. 2

II.

CONTRIBUTION OF EAR PHOTOSYNTHESIS TO GRAIN FILLING ........................................................... 2 A. Photosynthetic Contribution of Ear Parts: Bracts and Awns .............................................................................. 4 B. Role of Awns in Grain Yield .......................................................................................................................... 4

III.

PERICARP PHOTOSYNTHESIS: NET FIXATION VERSUS RECYCLING .................................................... 5

IV.

REFIXATION OF RESPIRATORY CO2 ............................................................................................................ 6

V.

C3 VERSUS C4 METABOLISM ......................................................................................................................... 7

VI.

DISTRIBUTION PATTERN OF EAR AND FLAG LEAF ASSIMILATES ......................................................... 9

VII.

WATER USE EFFICIENCY OF THE EAR ........................................................................................................ 9 A. Instantaneous WUE ...................................................................................................................................... 9 B. Integrated WUE (Carbon Isotope Discrimination) ........................................................................................... 9

VIII. THE EAR UNDER WATER AND HEAT STRESS ............................................................................................10 A. Drought Tolerance or Drought Avoidance? ....................................................................................................10 B. Delayed Senescence Under Drought ..............................................................................................................11 C. Ear Photosynthesis Under High Temperatures: The Role of Awns ....................................................................11 IX.

INTERSPECIFIC AND GENOTYPIC VARIABILITY OF EAR PHOTOSYNTHESIS .....................................13

Address correspondence to Eduardo A. Tambussi, Instituto de Fisiolog´ıa Vegetal (INFIVE), Universidad Nacional de La Plata, cc 327, 1900, La Plata, Argentina. E-mail: [email protected]

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E. A. TAMBUSSI ET AL.

CONCLUSIONS AND UNRESOLVED QUESTIONS .......................................................................................13

ACKNOWLEDGEMENTS ...........................................................................................................................................13 REFERENCES ............................................................................................................................................................14

This review concerns ear photosynthesis and its contribution to grain filling in C3 cereals. Ear photosynthesis is quantitatively important to grain filling, particularly in dry areas where source (i.e., assimilate) limitations can occur. Compared to the flag leaf, ear photosynthesis exhibits higher water stress tolerance. Several factors could be involved in the ear’s “drought tolerance.” First, although degree of C4 metabolism in ear parts has been reported, current evidence supports only typical C3 metabolism. Second, recycling of respired CO2 (i.e., refixation) could have considerable impact on final crop yield by preventing loss of CO2 . Because refixation of CO2 is independent of atmospheric conditions, water use efficiency (measured as total ear photosynthesis divided by transpiration) could be higher in the ear than in the flag leaf. Moreover, ear parts (in particular awns) show higher relative water content and better osmotic adjustment under water stress compared to the flag leaf. This capacity, in addition to persistence of photosynthetic components under drought (delayed senescence), might help the ear to continue to fix CO2 late in the grain filling period. Keywords

I.

awn, barley, cereals, ear photosynthesis, grain yield, heat tolerance, refixation, water-stress, water use efficiency, wheat

INTRODUCTION In the past, flag leaf photosynthesis was considered to be the main source of assimilates for grain filling (e.g., Evans et al., 1975), even though the role of ear photosynthesis in C3 cereal productivity had been discussed for years (Thorne, 1966; Kriedemann, 1966). It is now generally accepted that ear photosynthesis makes an important contribution to final grain yield (Simmons, 1987; Araus et al., 1993a), especially under drought conditions where the ear may become the main photosynthetic contributor to grain filling (Bort et al., 1994; Sánchez-D´ıaz et al., 2002; Abbad et al., 2004). In fact, “high-spike photosynthesis” has been suggested to be an important trait in the conceptual model (i.e., ideotype) for drought tolerance in wheat (Reynolds et al., 2005). However, actual percentage contribution of the ear to grain yield is not clear and the mechanistic basis of ear photosynthetic performance is not completely understood. Importance of the ear as a source of assimilates seems to be related to its better photosynthetic performance under stress. Traits that have been proposed to account for better photosynthetic performance under water stress of the ear compared to the flag leaf can be summarized as the following: (a) Some degree of C4 metabolism (either constitutive or drought-induced) is thought to occur in the ear, (b) features of water relations dif-

fer (including xerophytic anatomy, osmotic adjustment, and a higher water use efficiency), (c) refixation of the CO2 respired by developing grains occurs and, (d) senescence of the ear is delayed compared to the lower and flag leaves (see references above). This review emphasizes the following points concerning ear photosynthesis of C3 cereals: (a) Ear photosynthesis makes a substantial contribution to grain yield in C3 cereals, in particular under water deficit conditions, (b) awns (see Figure 1) represent the main photosynthetic organ of the ear for assimilation of external CO2 , (c) recycling of respired CO2 (refixation) in green pericarp and inner bracts is a key process when considering total assimilation in the ear, (d) the ear has a higher water use efficiency than the flag leaf, (e) ear photosynthesis shows higher drought tolerance under water deficit conditions due to its capacity for osmotic adjustment, xeromorphic anatomy and delayed senescence, (f) the ear has a typical C3 metabolism and the apparent tolerance of the ear under water deficit is not explained by C4 photosynthesis as it was reported in the past, and (g) there is genotypic variability in ear photosynthesis and water use efficiency, creating an opportunity to use this trait as a target in breeding programs. Because of their economical importance, the discussion is focused mainly on three C3 cereals: bread (Triticum aestivum) and durum (T. turgidum var. durum) wheats, and barley (Hordeum vulgare).

II.

CONTRIBUTION OF EAR PHOTOSYNTHESIS TO GRAIN FILLING Grain filling in C3 cereals occurs by acquisition of assimilates from the following three sources: (a) flag leaf photosynthesis (including blade and sheath) (Evans et al., 1975); (b) translocation of C assimilated before anthesis, mainly stored in the internodes (Gebbing and Schnyder, 1999); and (c) ear photosynthesis (see references in Passioura, 1994). The proportion of grain weight that comes from these sources varies widely with the species, cultivars and environment, although the information available remains incomplete and fragmentary (Evans et al., 1975). For instance, Biscoe et al. (1975) estimated that the ear contribution to final grain yield in barley was low (ca. 13%). However, darkening experiments in barley showed that assimilates in grains come mainly from the ear (ca. 38%), especially in awned genotypes (Bort et al., 1994). Carr and Wardlaw (1965) reported that ear contribution to grain filling was ca. 50% in non-awned

PHOTOSYNTHETIC ROLE OF EARS

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FIG. 1. General morphology of the ear of C3 cereals. The inflorescence (ear or spike) of cereals consists of spikelets attached to a cauline structure, the rachis. Each spikelet consists of an axis (the rachilla) bearing two sterile glumes at the base, and an indefinite (e.g., in wheat) number of florets. Each floret is subtended by an external bract called the lemma. Internally, there is a second bract, the palea (commonly transparent). The lemmas can be prolonged or not (in the case of awned and awnless cultivars, respectively) in a filiform (i.e., thread-like) structure, the awn. Between the lemma and the palea are enclosed the reproductive structures (and consequently the grain). The last leaf developed in the tiller is the flag leaf (redrawn from Wilhelm and McMaster, 1996).

genotypes of wheat and was greater in awned cultivars. In durum wheat grown under Mediterranean conditions, darkening the flag leaf and ear reduced grain weight by 22% and 59% respectively, and genetic variability was observed (Araus et al., 1993a). “Darkening experiments” have been criticized because ear temperature can be altered (e.g., Kriedemann, 1966), thereby (hypothetically) affecting several physiological processes, such as respiration, assimilate translocation, ripening, etc. (Thorne, 1966). However, a temperature change during darkening experiments was not observed at night and mid-day in a greenhouse study (Guiamet, unpublished observations). Kriedemann developed the method of integrated short-term shading, in which groups within a population of ears are shaded for a few days at specific stages during their development and then harvested. The cumulative reduction in grain yield due to short-term shading is considered to be the contribution due to ear photosynthesis. Using this technique, Kriedemann observed an ear contribution of ca. 10%, this figure being higher when the ear was shaded from anthesis to maturity (i.e., continuous shading; Kriedemann, 1966). Under optimal agronomic conditions, it is generally considered that grain-filling of C3 cereals (in particular wheat) is sinkrather than source-limited (Slafer et al., 1999). Thus, photoassimilate availability does not seem to limit grain filling. Although some of the experimental approaches used to manipulate source-

sink ratios should be interpreted with caution (e.g., defoliation and de-graining experiments), the lack of source limitation has been reported in several studies (e.g., Kruk et al., 1997). Grain growth would not be limited by the availability of assimilate, but by capacity of grains to utilize assimilate (e.g Slafer and Savin, 1994). However, source limitation is present in water-stressed crops (Araus et al., 2002). Drought is the main abiotic stress limiting production of cereals and other major crops in many regions. For instance, the Mediterranean climate (where durum wheat and barley are widely cultivated) is characterized by a marked drought event during the summer period, i.e., during the grain filling of cereal crops (Acevedo et al., 1999 and references cited therein). In this context, the relative contribution of ear photosynthesis (compared with the flag leaf and other plant parts) may be more important in dry conditions. Abbad et al. (2004) reported that grain yield of durum wheat correlated strongly with the photosynthetic rate of the whole ear, both in well-watered and under water-stress conditions. Although correlations do not necessarily imply causal relationships, it is suggestive that grain yield correlated more closely with ear than flag leaf photosynthesis. Interestingly, the correlation coefficient between ear photosynthesis and grain yield improved as the stress level increased, which is consistent with the hypothesis of a shift from sink- to source-limitation of grain yield (Abbad et al., 2004). In summary, despite the variability between studies, growing conditions, species and cultivars, it is clear that ear supply of

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assimilates for grain filling is important. Even considering the minimal value reported (i.e., ca. 10%), ear contribution can be relevant, in particular when we consider final yield in economical terms. A.

Photosynthetic Contribution of Ear Parts: Bracts and Awns Before addressing the photosynthetic contribution of ear parts, we will briefly discuss how to standardize the assimilation rate of the ear. Measurements of ear photosynthesis are standardized on an area (e.g., Knoppik et al., 1986) or, more commonly, on organ basis (i.e., the entire spike is enclosed in a cuvette; e.g., Johnson et al., 1974; Araus et al., 1993a; SánchezD´ıaz et al., 2002; Abbad et al., 2004). Standardization on an organ basis is the more desirable method, since, when the total photosynthetic area (body of the ear plus awns) is estimated, a considerable error could be introduced by the irregular and complex nature of this organ. If post-anthesis water stress is applied, for instance, we can assume no differences in total ear area between irrigated and water-stressed plants; therefore, a comparison of photosynthesis between treatments on an organ basis is more realistic (Tambussi et al., 2005). However, standardization by chlorophyll has also been used (Lu and Lu, 2004). In this study, when net photosynthetic CO2 uptake was calculated per organ, assimilation of the ear represented 27% of that of the flag leaf. Since chlorophyll content is lower in ear parts than in leaves, the net photosynthetic assimilation of the ear expressed on a chlorophyll basis was 67% of that of the flag leaf (Lu and Lu, 2004). In summary, photosynthesis per organ seems to be better when the relative contribution of ear to the grain filling is compared with the flag leaf. However, photosynthesis per chlorophyll could be a better measurement when assimilation activity per unit of photosynthetic machinery is analyzed. Few studies show the relative photosynthetic contribution of various parts of the ear. However, it appears that, when present, awns are the main photosynthetic organs. In barley, awns represent ca. 90% of total ear photosynthesis (Ziegler-Jöns, 1989a), at least with regards to the fixation of external (i.e., atmospheric) CO2. In other cereals, like Triticum species, the contribution of awns is lower (see references in Ziegler-Jöns, 1989a) but important (Blum 1985). In durum wheat, presence of awns doubled total ear surface area and thereby light interception (Motzo and Giunta, 2002). Bremner and Rawson (1972), measuring 14 CO2 fixation, showed that lemmas (including awns) were the photosynthetically most active bracts in bread wheat. Relative contribution of bracts (glumes and lemmas, without considering awns) remains more difficult to assess, although it can be estimated by “detachment” experiments (e.g., ZieglerJöns, 1989a). However, results based on this type of experimental approach must be considered with caution, since compensation by other photosynthetic tissues can occur. In addition, light environment of some organs (e.g., lemmas) can be changed by removal of outer bracts. However, Bremner and Rawson (1972)

reported that glumes contribute ca. 20% of total 14 CO2 fixation, in agreement with results obtained by Ziegler-Jöns from detachment experiments (Ziegler-Jöns, 1989a; see above). Ziegler-Jöns (1989a) suggested that the uptake of CO2 by lemmas might be reduced as a result of the growing grains blocking stomata in their ventral (i.e., facing the grains) side. These stomata (ca. 50–60% in wheat lemmas; Ziegler-Jöns, 1989a) are excluded from gas exchange with the environment and can only recycle CO2 respired by the grains (see Refixation of Respiratory CO2 section). This blocked-stomata effect does not apparently result in a significant reduction of photosynthesis rates in intact ears (at least in wheat), because it is compensated for by better exposure to light of apical parts of bracts after the spikelet structures are pushed apart by growing grains (see below as well). In a similar way, we could speculate that stomata on the ventral side of glumes are blocked by the dorsal side of the lemma. Nevertheless, the lower uptake of atmospheric CO2 may be counterbalanced by the increased uptake of CO2 stemming from the respiration of growing kernels. As mentioned above, compared with flag leaf, total chlorophyll content on a dry weight basis is lower in ear parts (Lu and Lu, 2004). This is not surprising, given the abundance of schlerenchymatous tissue in these organs (e.g., Araus et al., 1993a; Li et al., 2006). Additionally, differences in light environment (the ear is positioned at the top of the canopy) could lead to changes in pigment composition (e.g., chlorophyll content, photoprotective carotenoids) relative to the flag leaf. In fact, a lower chlorophyll/carotenoid ratio is reported for glumes, lemmas and awns (Lu and Lu, 2004) and this could reflect a higher requirement for photoprotection in the ear. Measurements of quantum yield of photosynthesis in light response curves showed a higher photoinhibition susceptibility in more shaded ear parts, such as lemmas and paleas (Lu and Lu, 2004). B.

Role of Awns in Grain Yield Although the importance of awns in total ear photosynthesis is clear (see above), their role in final grain yield remains more controversial (e.g., see references in Fischer, 2001). There are reports noting that the presence of awns improved the weight of individual grains in wheat (Teare et al., 1972b; Olugbemi and Bush 1987; Chhabra and Sethi, 1989) and in isogenic lines of barley (Bort et al., 1994). Recently, Katsileros et al. (2002) reported an important decrease of kernel weight and grain yield in de-awned ears of durum wheat. Furthermore, in a study where awned and awnless near-isogenic lines of durum wheat were analyzed under Mediterranean conditions, awns positively affected grain yield, with an average increase between 10 and 16% depending on the year (Motzo and Giunta, 2002). Interestingly, under very severe drought, the authors observed early desiccation of awns, and therefore, no effect of awns on grain yield (Motzo and Giunta, 2002). Thus, although many authors have concluded that awns confer an advantage under water deficit conditions, the functionality of awns might be compromised by


the rapid desiccation by hot winds and severe drought (Motzo and Giunta, 2002, and references therein). In some cases, grain yield can improve to a lesser degree, or even decline, due to the presence of awns, particularly if kernel number decreases. There are reports on barley (Bort et al., 1994) and bread wheat (e.g., Teich, 1982), in which the presence of awns seemed to have a negative effect on number of kernels (i.e., number of viable florets). The causes of this phenomenon are unknown, although it has been postulated that awns could compete with florets for photoassimilates during ear development, thereby reducing grain number per ear (see references in Bort et al., 1994). In fact, an adverse influence of awns on wheat yield has been reported (McKenzie, 1972). However, Motzo and Giunta (2002) did not find any effect of awns on kernel numbers in durum wheat. These authors pointed out that the reported effects of awnedness on bread wheat fertility are few and inconsistent. Nevertheless, it is possible that under moderate drought the possible negative effects of awns on grain number could be more than compensated by their positive effects on grain weight. Additionally, in bread wheat plants, in which the flag leaf was affected by yellowing produced by rust (Puccinia triticina), the presence of awns partially compensated the loss of grain yield (Martin et al., 2003). Awnletted lines (i.e., ears with short awns) have lower grain yield (ca. 10%) compared to awned ones, particularly if they are susceptible to rust (Martin et al., 2003). Therefore, awns can contribute to grain filling even under optimal water regimes if the photosynthetic competence of leaves is affected by some other cause (e.g., pathogen attack). In summary, despite the controversial role of awns in final grain yield, there are several reports addressing crops grown in Mediterranean conditions (Motzo and Giunta, 2002) where a positive relationship was observed. Further research is needed to clarify under what particular conditions awnedness is a positive trait for improving grain yield. Conditions where the photosynthetic performance of leaves is compromised (e.g., moderate water stress, pathogen attack, etc.) are possibly those situations wherein awns play a major role. While awns probably represent the main ear part fixing atmospheric CO2 (see above), other parts (such as green pericarp) could prove to be more important in the re-assimilation of respired CO2 . We discuss this issue in the following sections. III.

PERICARP PHOTOSYNTHESIS: NET FIXATION VERSUS RECYCLING Photosynthesis of green pericarp of C3 cereals is influenced by the following three anatomical features: (a) Occasional presence of stomata in the external surface, (b) Green cells in the grain are surrounded by non-chlorenchymatous cells (known as a “transparent layer”; Cochrane and Duffus, 1979), (c) endosperm, a rapidly respiring tissue, is enclosed by the green layer mentioned above. Although grains could fix externally supplied CO2 , the transparent layer of barley pericarp restricts CO2 uptake, since

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fixation increases when it is removed (Watson and Duffus, 1988). Similar results were reported for wheat pericarp with respect to oxygen exchange (Caley et al., 1990). In short, the transparent layer has a low permeability to oxygen and carbon dioxide (Nutbean and Duffus, 1978; Caley et al., 1990). These authors speculated that the CO2 required by pericarp photosynthesis is supplied by grain respiration and that some of the oxygen for respiration is product of pericarp photosynthesis. In fact, Nutbean and Duffus (1978) reported that some of the oxygen generated by pericarp photosynthesis remains within the grain. A critical point for gas exchange with external air is the presence of stomata in the pericarp surface. However, there are contradictory data concerning this issue. Cochrane and Duffus (1979) reported the existence of stomata (although very few in number) in the pericarp of wheat and barley located in the “brush end” region of grain, near the flanks of the crease on the ventral side. Interestingly, these authors showed that parts of the grain with stomata are only loosely covered by lemmas when the pericarp is green. In contrast, we failed to find any stomata in the pericarp of durum wheat (Tambussi, personal observation), in agreement with a previous study (Kriedemann, 1966). Most likely, these discrepancies can be explained by specific and genotypic variability. Nonetheless, it seems clear that the number of stomata in the pericarp (if they are present) is very low (Cochrane and Duffus, 1979) and probably insufficient for gas exchange. Watson and Duffus (1988), using 14 C, found that intact grains of bread wheat and barley are capable of fixing externally supplied CO2 . This fixation is light dependent, although some (very low) fixation of external CO2 is also observed in dark. Surprisingly, CO2 fixation in the Chl-less mutant (named Albino lemma) of barley is significantly greater in light than in dark (Watson and Duffus, 1988). The authors explain this lightdependent CO2 -fixation in the albino mutant pericarp as result of light-induced opening of stomata. Presumably, CO2 uptake in the pericarp of the albino mutant (as in the normal genotype in dark) is catalyzed by phosphoenolpyruvate carboxylase (PEPc) (see also section C3 Versus C4 Metabolism in the ear). In short, although their data are inconclusive, Duffus’ group concluded that the major function of the pericarp is fixation of internally generated CO2 rather than photosynthetic uptake of atmospheric CO2 (Watson and Duffus, 1991). In a recent experiment, we found that the electron transport rate of green pericarp (estimated by modulated chlorophyll fluorescence) is quasi-insensitive to simultaneous changes in external CO2 and O2 concentrations (Figure 2), suggesting that pericarp photosynthesis operates on an internal source of CO2 . In summary, photosynthesis in the pericarp seems to be largely independent of atmospheric CO2 . An unanswered question is how much light reaches the pericarp surface. It must be remembered that the grain is surrounded by the lemma and palea, and is shaded by the glume. Thus, we can speculate that light could be a limiting factor for pericarp photosynthesis. Although the amount of light reaching the grain surface may increase as lemmas and glumes are pushed apart

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FIG. 2. Response of the relative quantum yield of PSII photochemistry (φPSII ) to changes in CO2 concentration at normal (ca. 21%) (A) or low (ca. 2%) oxygen concentrations (B) of glumes (open circles), lemmas (open squares) and the green pericarp (closed circles) of ears of durum wheat (Triticum turgidum L. var. durum) grown in a greenhouse. The φPSII was measured with a modulated chlorophyll fluorimeter MiniPAM (Waltz, Effeltrich, Germany). The conifer leaf cuvette of an infrared gas analyzer (LI-COR 6400) was modified to fit the optical fiber of the modulated fluorimeter. The optical fiber was obliquely oriented over the glume, lemma, or green pericarp. The measurements were made 20 days after anthesis at 800 μmol m−2 s−1 of PPFD. Each value represents the mean ± s.e. of four measurements.

by the growing grain (Kriedemann, 1966), this phenomenon occurs at the end of grain filling and probably has little impact on final kernel weight. In summary, an open question is whether the green pericarp can use the high CO2 levels surrounding the grain, keeping in mind that low PPFD levels could be a limiting factor. Further research is needed to elucidate this question. IV.

REFIXATION OF RESPIRATORY CO2 Recycling of respired CO2 (i.e., refixation) seems to be a common phenomenon in fruits of several species (for instance Wullschleger et al., 1991). Compared to other organs (e.g., flag

leaf), the ear has a high respiratory rate (Knoppik et al., 1986; Araus et al., 1993a), particularly during the mid grain-filling period (ca. 15 to 20 days after anthesis; Caley et al., 1990). The ear’s high respiration rate is explained by (a) presence of heterotrophic tissues in several organs, such as bracts, awns and cauline structures (i.e., rachis and rachilla of the spikelets), and (b) respiration of the growing grain (Knoppik et al., 1986). Therefore, CO2 refixation is not a trivial aspect of ear photosynthesis, since it can reduce respiratory loss and thus contribute to grain filling. Refixation in wheat ears was first shown by Kriedemann (1966). In this classic study, the author estimated the percentage of ear refixation, comparing CO2 emission in CO2 -free air versus CO2 emission in the dark (see explanation in Figure 3). Using this approach, the author calculated that ca. 60% of respired CO2 is refixed under light conditions. In addition, the relative contribution of net fixation (uptake of atmospheric CO2 ), refixation, and translocation (i.e., incorporation of assimilates from flag leaf, stem reserves, etc.) to grain yield was estimated to be 9, 21, and 70%, respectively. An awnless cultivar was used in this study (see photograph in Kriedemann, 1966). Because awns represent the main photosynthetic organ of the ear (at least with regard to fixation of external CO2 ), relative contribution of net assimilation could have been underestimated in this study. Direct evidence of refixation in bracts of the ear was first shown by Bort et al. (1996). In these experiments, ears of durum wheat and barley were fed with 14 C-sucrose, and evolution of 14 CO2 in darkness and light were compared (Figure 4). This study showed that refixation in the ear is notably higher than in the flag leaf. It seems reasonable to speculate that ear structures are better adapted than the flag leaf to recycle respired CO2 . Several traits of the ear could explain this capacity: (a) existence of photosynthetic tissues in the grain placed between endosperm and transparent layer (partially impermeable to CO2 ), as described above and (b) presence of green bracts enclosing the growing grain (lemma, palea and glume) (see more details in Figure 1). In other words, photosynthetic tissues of the ear can present a sequence of photosynthetic barriers, eventually recapturing (at least partially) the CO2 released by grain respiration. More recently, Gebbing and Schnyder (2001) assessed the fractional contribution of refixation to ear photosynthesis in bracts of bread wheat using 13 CO2 . In this study, the authors calculated that ca. 70% of the sucrose accumulated in “glumes,” including lemmas and paleas, came from fixation of respired CO2 (although the δ 13 C of the CO2 respired was not directly measured). It must be noted that in hexaploid wheat (as is the case with bread wheat used in this study), awns represent a lower proportion of total ear area (ca. 20% lower with respect to tetraploid wheat; Blum, 1985). In other words, the presence of a higher awn area in tetraploid wheat could have a significant impact on the relative contribution of net fixation and recycling in total ear photosynthesis. As mentioned above, a similar comment can be made with respect to Kriedemann’s study (1966), in which an awnless cultivar of wheat was used.


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FIG. 3. The historical study of ear photosynthesis carried out by Kriedemann (1966). This scheme shows the experimental treatments and the processes contributing to grain yield in each treatment. In the “dark” treatment, grain yield will depend exclusively on the import of assimilates. In L − CO2 , the ear is exposed to light in a CO2 -free atmosphere; therefore, no net photosynthesis results, only refixation of respired CO2 and translocation. In L + CO2 , the ear is exposed to light and atmospheric CO2 ; therefore, the three processes (i.e., ear net CO2 fixation, ear refixation of respired CO2 , and import of assimilates) will contribute to grain yield (redrawn from Kriedemann, 1966).

It is apparent that the ear does not refix all respiratory CO2 evolving in the light and that there may always be a loss of CO2 (Kriedemann, 1966; Bort et al., 1996). In addition, net fixation of external CO2 does occur and its importance should not be minimized. For instance, the awns and external surfaces of the glumes (where stomata are abundant; e.g., Tambussi et al., 2005) are putative sites for net fixation of atmospheric CO2 . By contrast, the internal surfaces of lemmas (facing the grain) and green pericarp probably have a role in refixation. Interestingly, we observed stomata on the internal side of the lemma of durum wheat (Tambussi et al., 2005), in contrast with Teare’s findings (1972a). Genotypic variability may explain this discrepancy. In summary, different studies support the view that refixation is a quantitatively relevant process in ears of cereals, such as barley and bread/durum wheat, and that it seems to be genotypically fixed (Bort et al., 1996). V. C3 VERSUS C4 METABOLISM In the 1970s to the 1980s, several studies either reported or suggested the possible existence of C4 metabolism in ears of C3 cereals (Nutbeam and Duffus, 1976; Wirth et al., 1977; Singal et al., 1986; Ziegler-Jöns, 1989b; Imaizumi et al., 1990). Evidence of C4 metabolism included the following: (a) Experiments

in which C4 metabolites (e.g., malate) were detected (Singal et al., 1986; Nutbean and Duffus, 1976), (b) Analysis of the activity of PEPc and enzymes of C4 metabolism (Wirth et al., 1977; Singal et al., 1986), (c) Gas exchange properties (Ziegler-Jöns, 1989b), and (d) Oxygen insensitivity of the photosynthetic rate (Imaizumi et al., 1990). Nutbeam and Duffus (1976) first reported C4 photosynthesis in the ear in experiments where the green pericarp of barley (without transparent layer) was incubated with 14 CO2 . In these experiments, 84% of the fixed CO2 was present as malate after 1 min. Similar results were reported by Singal et al. (1986) with bracts from the ear of wheat. However, these results must be regarded with caution. In these experiments, detached ears or isolated ear parts were incubated on a moist support (e.g., Singal et al., 1986), which may have increased the amount of inorganic 14 C present in the buffer as bicarbonate, the substrate of PEPc. Thus, the presence of high C4 acid levels could been an artefact (Bort et al., 1995). By contrast, research carried out by Araus’ group failed to detect C4 metabolism in the ear. Evidence that supports C3 -metabolism in ears of barley and durum wheat include the following: (a) A pulse with 14 CO2 showed insignificant labelling of C4 acids (Bort et al., 1995), (b) The linearity found in the O2 responses of the CO2 compensation

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FIG. 4. Refixation of respiratory CO2 by ears of barley (cvs. Roxana and Hatif; panel A and B respectively) and wheat (cv. Bidi 17; panel C). Ears were fed [14 C]sucrose during 30 min at 210 mmol mol−1 O2 and light (1100 μmol m−2 s−1 PPFD). The evolution of 14 CO2 was measured during the next 15 min exposure of the ears to one of four experimental conditions: L20, light at 20 mmol mol−1 O2 ; L210, light at 210 mmol mol−1 O2 ; D20, dark at 20 mmol mol−1 O2 ; and D210, dark at 210 mmol mol−1 O2 . Refixation (percentage of reduction in 14 CO2 evolution) was calculated comparing radioactivity trapped by each treatment to radioactivity trapped under D210. Values for each genotype are the means of three replicates (redrawn from Bort et al. 1996).

point is typical of C3 plants (Bort et al., 1995), (c) The absence of any effect on photosynthesis by DCDP (3,3-dichloro-2-hidroxyphosphinoylmethyl-2-propenoate), a specific inhibitor of PEPc (Araus et al., 1993b; Bort et al., 1996), (d) Rubisco distribution in glumes, lemmas and awns of durum wheat is typical of C3 plants (Tambussi et al., 2005), and (e) Photosynthetic electron transport rate of glumes and lemmas is markedly decreased by low levels of CO2 and O2 (Tambussi et al., 2005), a typical response of C3 metabolism (Lawson et al., 2002). In contrast, as mentioned above, green pericarp electron transport rate is insen-

sitive to external levels of CO2 and O2 . However, this apparent insensitivity is not surprising if we consider the existence of an internal source of CO2 in the grain (i.e., recycling of respired CO2 ). High PEPc activity in reproductive bracts has been reported in wheat ears (Wirth et al., 1977; Singal et al., 1986) and rice panicles (Imaizumi et al., 1990) and more recently, several nonleaf organs (glumes, lemmas, awns) of wheat (Xu et al., 2003). However, it must be noted that PEPc is present in all living plant cells (Chollet et al., 1996), and there is some agreement that PEPc in ears of C3 cereals is not directly involved in photosynthetic carbon fixation (Araus et al., 1993b). Immunolocalization studies in glumes of durum wheat showed that PEPc is localized mainly in “white granules” and vesiculations in the cytoplasm, which suggests a non-photosynthetic role for PEPc (Araus et al., 1993b). In fact, PEPc functions anaplerotically in C3 leaves (Chollet et al., 1996). Similarly, the enzyme pyruvate ortophosphate dikinase (PPDK), which mediates the conversion of pyruvate to PEP, has been found in the pericarp of C3 cereals (Meyer et al., 1982). The highest levels of PPDK occur later than the highest levels of chlorophyll and Rubisco, suggesting that its role is not photosynthetic (Aoyagi and Bassham, 1984). PPDK could function (together with PEPc) in the recapture of respiratory CO2 , and more likely, in amino acid inter-conversions during development of seed reserve material (Aoyagi and Bassham, 1984). Further evidence favoring C4 photosynthesis concerns the insensitivity of photosynthesis to O2 concentration in spikelets of rice panicles (Imaizumi et al., 1990), which suggests a lack of photorespiration, and thus, the possible existence of C4 metabolism. However, the authors pointed out that the occurrence of oxygen-insensitive photosynthesis does not necessarily exclude the existence of C3 metabolism. For instance, a decrease in low-oxygen stimulation of photosynthesis in C3 plants has been attributed to phosphate-limitation (Sharkey, 1985). Inorganic phosphate (Pi ), essential for photophosphorylation in the stroma, can be limiting if phosphorylated intermediates are not exported from chloroplasts. Thus, photosynthetic rate can be insensitive to a decrease in oxygen concentration under conditions where regeneration of RuBP is limiting (Nogués et al., 2005; Lawlor, 2002). In fact, O2 insensitivity is considered a symptom of Pi deficiency in vivo in C3 plants (Leegood, 1989). Thus, while the total lack of O2 sensitivity reported by Imaizumi et al. (1990) remains surprising, it is not conclusive evidence of C4 photosynthesis. Finally, even though low carbon isotope composition of bracts and awns (Araus et al., 1992a; Araus et al., 1992b; Gebbing and Schnyder, 2001) is also suggestive of some degree of C4 metabolism in the ear, these values (ranging between ca. −22 and −27%) fall within the normal range for C3 species (Pate, 2001). Although the existence of some degree of C4 -like metabolism cannot be totally ruled out, in addition to methodological considerations, differences in the plant material, species, cultivars,


and organs used in these studies could result in discrepancies. Therefore, several lines of evidence support the operation of C3 photosynthesis in ears. In some recent scientific literature, however, the ear parts are still cited as an intermediate C3 −C4 organ (e.g., Lu and Lu, 2004). One possibility not examined thus far is the existence of some degree of CAM metabolism in the ear, involving the fixation of external CO2 or (more likely) refixation of respired CO2 . In fact, re-assimilation of respired CO2 through CAM metabolism has been documented in organs of C3 species (named CAM cycling, e.g., Cushman, 2001). However, lack of stomatal opening at night and the CO2 exchange pattern do not provide evidence for CAM-like metabolism (Tambussi et al., 2005). Further research is necessary to evaluate whether some type of CAM metabolism is also present in ear parts. For instance, measurements of organic acids (e.g., malate) or vacuolar pH at night could prove interesting in evaluating this possibility. VI.

DISTRIBUTION PATTERN OF EAR AND FLAG LEAF ASSIMILATES The distribution pattern of assimilates produced by the ear or the flag leaf is to be different. In wheat, 14 C-labelling experiments showed that flag leaf exported photoassimilates mainly to central spikelets of the ear; by contrast, ear photoassimilates are exported more uniformly within the ear. Within each spikelet, the distribution between grains is much more uniform for ear rather than flag-derived assimilates (Rawson and Evans, 1970). At the plant scale level, ear and flag leaf assimilates seem to be distributed in different patterns. While ear assimilates are destined mainly to the grains, the flag leaf also exports assimilates to vegetative organs, such as stems and roots (Carr and Wardlaw, 1965). Thus, the distribution pattern of ear-derived photosynthates may confer an additional importance to this photosynthetic tissue (as source). In fact, it has been shown that the distribution of lemma assimilates (including the awn) is mainly toward the grain of the same floret (Bremner and Rawson, 1972). VII. WATER USE EFFICIENCY OF THE EAR High water use efficiency (WUE) of the ear of C3 cereals has been reported in some studies (Teare et al., 1972b; Araus et al., 1993a; Bort et al., 1994; Abbad et al., 2004). WUE of the ear has been estimated either by instantaneous measurement of the photosynthesis/transpiration ratio (i.e., gas exchange analysis) or by carbon isotope composition. A.

Instantaneous WUE Several analyses of gas exchange showed a higher instantaneous WUE in the ear compared to the flag leaf (Araus et al., 1993a; Bort et al., 1994). By contrast, Blum (1985) reported similar WUE in the flag leaf and whole ear of bread wheat. However, WUE was probably underestimated in this study since respired CO2 was not considered. Calculation of leaf WUE usually does

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not include respiration rate (e.g., Condon et al., 2002), but leaf respiration rate is negligible, typically around 5% (or lower) of the net photosynthetic rate in bread wheat and barley (e.g., Knoppik et al., 1986; Tambussi et al., 2004). In the ear, dark respiration rates are much higher (Knoppik et al., 1986), and if not taken into account, total ear photosynthesis (and thus, instantaneous WUE) can be severely underestimated (Araus et al., 1993a). Interestingly, WUE of the ear increases more than that of the flag leaf under severe water stress. Abbad et al. (2004) found that the ratio between ear WUE and flag leaf WUE increases substantially under water stress. Blum (1985) reported very high WUE in awns of detached ears of durum wheat, bread wheat and barley. We could not confirm this observation in durum wheat, as the awns showed a lower WUE than the flag leaf (Araus et al., 1993a). Awns of barley showed a higher WUE than the flag leaf (Bort et al., 1994), although the differences between both organs were lower than those reported by Blum (1985). The exceptionally high WUE of the awns (at least 5 times higher than that of the flag leaf) reported by Blum (1985) is surprising and might be explained by inter- and intra-specific variability. For instance, the awns of North African durum wheat genotypes had higher WUE than Middle Eastern varieties (Araus et al., 1992b). Again, in this study, instantaneous WUE of the awns was lower than the flag leaf. Consistent with the idea that awn WUE is lower than that of the flag leaf (at least in wheat), Teare et al. (1972b) reported a lower instantaneous WUE in awned vs. awnless cultivars of bread wheat. B.

Integrated WUE (Carbon Isotope Discrimination) Relationship between WUE and 13 C isotopic discrimination (13 C) is well known. In C3 species, 13 C is positively related to CO2 levels in intercellular spaces and (given a constant vapour pressure deficit), negatively related to WUE (Farquhar and Richards, 1984; Hubick and Farquhar, 1989). Ear photoassimilates show lower 13 C compared with the flag leaf (Araus et al., 1993a), suggesting a higher WUE (Hubick and Farquhar, 1989). The high-integrated WUE in ears might come from its capacity to recycle respired CO2 . In fact, triticale (x Triticosecale) has progressively higher 13 C isotopic composition (δ 13 C, i.e., a lower 13 C) from flag leaf to glumes and glumelles (i.e., towards the organs closest to respiring grains) (Araus et al., 1992b). Gebbing and Schnyder (2001) pointed out that the low 13 C in glumes (including lemmas) may be related to the following: (a) Some degree of C4 photosynthesis (as discussed above), (b) A closed system effect resulting from the arrangement of the glumes around the grains, and/or (c) Low CO2 conductance of the glumes. However, the interpretation of carbon isotopic composition in the ear remains uncertain for several reasons. First, although the δ 13 C of atmospheric CO2 is well characterized (ca. 8%; Pate, 2001), we do not know the δ 13 C of respired CO2 . Recently, it has been shown that discrimination occurs during respiration in leaves, leading to the production of CO2 enriched

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in 13 C. This enrichment in respired CO2 can be explained by the carbon source used for respiration, isotope effects of some respiratory enzymes, and/or non-statistical carbon isotope distribution in glucose (Tcherkez et al., 2003; Nogués et al., 2004). Therefore, the isotopic discrimination of ear bracts cannot be determined accurately if δ 13 C of respired CO2 is not measured. In addition, although the fractional contribution of net assimilation and refixation components has been estimated in one case (Gebbing and Schnyder, 2001), it is unknown whether this figures can be extrapolated to other species and cultivars.

VIII.

THE EAR UNDER WATER AND HEAT STRESS

A.

Drought Tolerance or Drought Avoidance? In comparison with the flag leaf, a better photosynthetic performance of the ear (evaluated by gas exchange) under water stress has been reported in several C3 cereals such as barley (e.g., Sánchez-Diaz et al., 2002), bread wheat (Xu et al., 1990) and durum wheat (Abbad et al., 2004; Tambussi et al., 2005). However, there are few studies analyzing the physiological traits underlying this phenomenon. The better photosynthetic performance of the ear under water stress conditions seems to be associated with its ability to maintain a higher relative water content (RWC). Xu and Ishii (1990) reported similar water relationships (water potential) for glumes and flag leaf of bread wheat (Triticum aestivum L.). In contrast, we found differences in RWC between the flag leaf and ear bracts (and mainly the awns) of water stressed plants of durum wheat (Tambussi et al., 2005; see Figure 5A). Likewise, Wardlaw (2002) reported that the glumes of bread wheat maintained higher RWC (with respect to the flag leaf) under progressive water stress. The higher RWC in ear parts under water stress could be explained by osmotic adjustment (OA) (i.e., accumulation of osmolyte compounds). The existence of OA in the whole spikelets of wheat was first shown by Morgan (1980) and confirmed by later studies in bread wheat (Blum et al., 1988). In durum wheat, OA is substantially higher in ear parts (glumes, lemmas, and awns) than the flag leaf (Tambussi et al., 2005). OA in plant cells results in a decrease of the cell osmotic potential, and consequently in the maintenance of water absorption and cell turgor pressure (Clarke, 1987, Kikuta and Richter, 1986). In fact, OA has been noted to be a crucial factor for drought tolerance in wheat (Morgan, 1984; Morgan and Condon, 1986; Sen Gupta and Berkowitz, 1987; Ludlow et al., 1990; Blum et al., 1999). In summary, OA in the ear could help to maintain a higher RWC with respect to the rest of the plant. In addition to osmotic adjustment, other factors could be involved in the drought response of ear photosynthesis. In wheat, a vertical heterogeneity in schlerophyllous characteristics (such as smaller intercellular spaces, smaller and packed cells, thicker cell walls and higher proportions of schlerenchymatic tissue) of photosynthetic organs has been reported (Araus et al., 1986; Tambussi et al., 2005), with the awns being the most schlerophyllous organ (e.g., Li et al., 2006). In this sense, we observed

FIG. 5. A. Relative water content (RWC) of leaves and ear parts of wellwatered (filled bars) and water-stressed (open bars) plants of durum wheat (Triticum turgidum L. var.durum) grown in a greenhouse. F, G, Ln, and A correspond to the flag leaf, glumes, lemma, and awns respectively. L1, L2, and L3 are the three leaves below the flag leaf, counting from the top. Each value represents the mean ± s.e. of four measurements. B. Relationship between the water content (as % of fresh weight) of the leaves and ear parts in well-watered plants versus the RWC of the same organs in water-stressed plants. Each point represents the mean ± s.e. of four measurements. Abbreviations as in A. For both panels, measurements were performed 20 days after anthesis (redrawn from Tambussi et al. 2005). With kind permission of Springer Science and Business Media.

a vertical gradient of water content in several organs of wellwatered plants, which was negatively related with the RWC of the same organs in water-stressed plants (Figure 5B; Tambussi et al., 2005). This could reflect a xeromorphic tendency in upper levels of the plants that might confer water stress tolerance. Consequently, the greater capacity of the ear to maintain a higher RWC with respect to the flag leaf could be also related to schlerophyllous traits of bracts and particularly awns. In summary, rather than drought tolerance, ears of wheat appear to employ an “avoidance” strategy against dehydration,


maintaining a higher water status in bracts and awns in waterstressed plants. Further research is necessary to elucidate the mechanistic basis of this phenomenon, in particular whether or not OA is causally related to ear performance under water stress and its impact on grain yield. B.

Delayed Senescence Under Drought The rapid degradation of photosynthetic components in leaves subjected to water stress is a well-documented phenomenon (e.g., Pic et al., 2002). By contrast, the photosynthetic apparatus of the ear can persist longer than that of the flag leaf during grain filling in bread wheat (Li et al., 2006), particularly under water stress conditions (Martinez et al., 2003). Key components of the photosynthetic machinery, such as chlorophyll, Rubisco and LHCII protein declined only slightly ahead of well-watered plants in bracts and awns of bread wheat plants subjected to water stress. In addition, photosynthetic competence (evaluated by chlorophyll fluorescence) was higher in the ear than the flag leaf under water stress in bread (Martinez et al., 2003) and durum wheat (Tambussi et al., 2005). The persistence of photosynthetic components (i.e., delayed senescence) in the ear could initially arise via two different processes. Since the ear represents the last organ developed in cereals, and considering the sequential bottom-to-top (acropetal) progress of senescence, it is not surprising that the ear persists longer than the leaves. On the other hand, superior water status (RWC) in ear parts could prevent drought-induced senescence observed in flag leaves. Martinez et al. (2003) ruled out this possibility, since similar time-courses of water potential in flag leaf and glumes had been observed (Barlow et al., 1980). However, it is important to note that RWC (rather than water potential per se) is a better indicator of tissue water status. Hence, observed differences in RWC between glumes and leaves under water stress (Tambussi et al., 2005; see above) could explain the lack of drought-induced senescence in the ear, which might help the ears to continue fixing CO2 late in the grain filling period. C.

Ear Photosynthesis Under High Temperatures: The Role of Awns There are studies where a role for awns in a “heat tolerance” of the ear has been pointed out (see references below). In this sense, the role of awns may involve two different aspects; the photosynthetic heat tolerance of awns per se and the effect of the presence of awns on spike temperature. Regarding the first point, Blum (1985) reported that the optimum temperature for photosynthetic rate in awns of (hexaploid and tetraploid) wheat is ca. 32◦ C (or higher), whereas, in leaves and glumes, the photosynthetic optimum occurs at lower temperatures (Blum et al., 1986). The causes underlying this heat tolerance of awns are unknown. Paradoxically, the extent of cellular damage (measured by electrolyte leakage; e.g., Prasad et al., 2006) under heat stress is higher in awns than in the flag leaf and glumes. These results suggest that membrane stability, a trait that when

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measured in leaves was associated with yield of wheat genotypes in a number of hot, irrigated environments (Reynolds et al., 1994), is not involved in thermotolerance of awn photosynthesis (Blum, 1985). Blum (1985) suggests that (unknown) “biochemical peculiarities” of the photosynthetic mechanism are perhaps involved in the heat tolerance of the awn. More recently, Xu et al. (2003) indicated that the thermal tolerance of awns is explained by high PEPc activity found in them and other non-leaf organs (e.g., glumes, lemmas). Compared with the flag leaf, high PEPc/Rubisco ratios were reported in ear organs and, as discussed earlier, a putative C4-like metabolism was proposed (Xu et al., 2003). When the ear is subjected to heat stress, PEPc activity is further increased. However, from a careful analysis of their results, we observed that the rise of PEPc activity during heat stress is associated with a decrease of Rubisco activity and is possibly a symptom of senescence (see Figure 1 in Xu et al., 2003). Consequently, the photosynthetic role of PEPc is improbable in this context (similar considerations were made about the enzyme PPDK; see section V). In short, the authors do not explain how a C4 metabolism could work when Rubisco activity is decreasing in senescing tissue under heat stress. An alternative explanation for the photosynthetic heat tolerance of ears might be related to refixation of CO2 . This is not, however, related to a higher PEPc. In fact it has for long been well known that plants exposed to high CO2 levels increase their temperature optimum for photosynthesis since photorespiration is prevented. However, in a study where several grain growth temperatures were tested, Olugbemi and Bush (1987) reported that the advantage of awned cultivars (compared with their awnless counterparts) is not related to thermal performance: awned cultivars showed higher grain weight per ear than did awnless cultivars, irrespective of the temperature during grain growth (Olugbemi and Bush, 1987). Thus, the nature of thermal tolerance of awns is not clear and further studies are needed. Regarding the second point (i.e., the effect of the presence of awns on spike temperature), in a study carried out in Mexico (under heat stress conditions) where several genotypes were analyzed, Ayeneh et al. (2002) found that the spike may be cooler than the surrounding air. The temperature of an organ depends on energy inputs (absorption) and outputs such as latent heat (evaporative cooling by transpiration) and sensible heat flux (e.g., convection). Temperature depression (i.e., temperature difference between the organ and the air) was lower in spike than in the flag leaf, suggesting that the energy inputs and/or outputs are different in both organs (i.e., higher energy absorption and/or a lower transpiration in the spike). In the same study, the authors reported a close correlation between the length of awns and the temperature depression in the spike. Counterintuitively, Panozzo et al. (1999) reported higher spike temperatures in an awned than in an awnless cultivar. Only on very hot days (when air temperature exceeded 40◦ C) did awned cultivar show a cooler spike than the awnless one. Thus, awns could increase the spike temperature (for instance, by increasing energy absorption) in some cases but cool the spike in very hot environments.

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FIG. 6. Schematic model of ear photosynthetic organs, summarizing some of the ideas discussed in this review. It must be noted that some statements are hypothetical (question marks). These are discussed extensively in the text. Photosynthetic tissues are colored black, the presence of stomata in glumes, lemmas, awns, and pericarp are noted by the symbol .


Light scattering, shading and evaporative cooling (by transpiration) from this organ could be implicated, but further research is needed to determine the relative contribution of each process. Unfortunately, studies where all the components of this thermal balance of the ear could be calculated are lacking. For instance, there are no reliable data on awn transpiration rate based on an area basis, considering the filiform shape of this organ. It seems clear that previous reports considering the awn to be a “nontranspirative” organ are erroneous (for instance, see references cited by Ayeneh et al., 2002). This is supported by the high stomatal density of awns (Tambussi et al., 2005), their potential to cool the ear (Ayeneh et al. 2002; Panozzo et al. 1999), and their photosynthetic contribution to ear photosynthesis (Blum, 1985) and the grain yield (e.g., Olugbemi and Bush, 1987). On other hand, genotypic variability in the relative contribution of different components of thermal balance, could explain some discrepancies between studies. IX.

INTERSPECIFIC AND GENOTYPIC VARIABILITY OF EAR PHOTOSYNTHESIS The relative importance of ear photosynthesis in different species of C3 cereals has received little attention, although some evidence indicates that it might be higher in barley than in wheat, in tetraploid than in hexaploid wheat species, and in six-rowed than in two-rowed barley (Blum, 1985 and references therein). This might be due to higher surface area of awns in the ears of barley, tetraploid wheat and six-rowed barley, respectively (Blum, 1985). Additionally, the lower area of the flag leaf in barley (e.g., Blum, 1985) could increase the relative contribution of ear photosynthesis in this species. Although there are few studies analyzing genotypic variability of ear photosynthesis and its impact on grain yield, some reports indicate that such variability does exist. Recently, Abbad et al. (2004), analyzing the behaviour of six cultivars of durum wheat from semi-arid regions in Morocco, found genotypic differences in ear photosynthetic rates, particularly under severe water stress. Moreover, considerable genotypic variability of ear A/g ratio (i.e., intrinsic WUE) was observed even under well-watered conditions (Abbad et al., 2004). Regarding the contribution of ear photosynthesis to final grain yield, Araus et al. (1993a) found considerable differences between five cultivars analysed under Mediterranean conditions. Interestingly, in a study carried out under dryland conditions in Jordan, Duwayri (1984) reported that grain yield of locally adapted cultivars was more affected by awn removal than that of cultivars developed in other areas. This supports the idea that awns contribute to grain yield under drought (as we discussed above), and suggests that empirical breeding could have improved this trait in local varieties native to dry areas. As far as we know, there are no studies analyzing genotypic variability and genotype x environment (GxE) interactions for ear refixation capacity. Refixation has been measured by nonstable (e.g., Bort et al., 1996) and, more recently, by stable isotope techniques (Gebbing and Schnyder, 2001). Analyzing culti-

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vars in great numbers using such methodologies is both complex and expensive, which may explain the lack of reports regarding the genotypic variability in ear refixation capacity. Spikes show genotypic variation for some morphological traits (e.g., wax and pubescence of bracts, awn color), but we do not know how such traits may interact with ear physiological parameters (e.g., photosynthesis, water relations). Research in this field could have considerable relevance, since some traits, in particular, those that are morphological in nature, could be easily evaluated in breeding programs. X.

CONCLUSIONS AND UNRESOLVED QUESTIONS Issues discussed here regarding ear photosynthesis can be summarised as follows: (a) Photosynthetic contribution of the ear to grain filling appears to be quite important, particularly when grain yield is source-limited (i.e., under drought); (b) Awns, when present and conspicuous, seem to be the main photosynthetic organ of the ear, at least with regard to net fixation of atmospheric CO2 ; (c) Refixation of respired CO2 is a well-documented process in ears of C3 cereals and represents a potentially important contribution to total photosynthesis; (d) Green pericarp and inner bracts (lemmas) are probably the main site for CO2 refixation; (e) Current evidence does not support the presence of C4 (or CAM) metabolism in ears of C3 cereals; (f) The ear exhibits better photosynthetic performance under water stress conditions, resulting from a higher RWC, and their capacity for osmotic adjustments compared to the flag leaf; (g) Delayed senescence (persistence of photosynthetic components) is a key process in maintaining ear photosynthesis, mainly under water stress. Some of these conclusions and open questions are summarized in Figure 6. Despite much research in this field, particularly during the ’80s and ’90s, several issues concerning photosynthesis in ears of cereals remain to be elucidated, including the following: (a) The extent of genotypic variability (and GxE interactions) in refixation of respired CO2 , net assimilation capacity and water use efficiency of ear, (b) Characterization of morphological and physiological traits affecting ear photosynthesis and its contribution to grain yield, and (c) The mechanistic basis underlying the performance of ear photosynthesis under water stress. Results from such studies can be useful for plant breeders, particularly when analytical approaches are used. Anatomical and physiological characterization of traits related to drought tolerance of ear photosynthesis can be used to obtain higher grain yield and water use efficiency from cereals, particularly in dry areas such as those typical of the Mediterranean climate. ACKNOWLEDGEMENTS This work was supported in part by the Spanish project (CICYT, AGL2002-04285-C03-03) and by the EC projects (TRITIMED, INCO-CT-2004-509136 and WASAMED, ICA3CT-2002-10013).

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