Maria Celeste Arrabaça

Photorespiration in C4 grasses remains slow under drought conditions

The CO(2)-concentrating mechanism present in C(4) plants decreases the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and, consequently, photorespiratory rates in air. Under drought conditions, the intercellular CO(2) concentration may decrease and cause photorespiration to increase. The C(4) grasses Paspalum dilatatum Poiret, Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel were grown in soil and drought was imposed by ceasing to provide water. Net CO(2) assimilation (A) and stomatal conductance to water vapour decreased with leaf dehydration. Decreased carbon and increased oxygen isotope composition were also observed under drought. The response of A to CO(2) suggested that the compensation point was zero in all species irrespective of the extent of drought stress. A slight decrease of A as O(2) concentration increased above 10% provided evidence for slow photorespiratory gas exchanges. Analysis of amino acids contained in the leaves, particularly the decrease of glycine after 30 s in darkness, supported the presence of slow photorespiration rates, but these were slightly faster in Cynodon dactylon than in Paspalum dilatatum and Zoysia japonica. Although the contents of glycine and serine increased with dehydration and mechanistic modelling of C(4) photosynthesis suggested slightly increased photorespiration rates in proportion to photosynthesis, the results provide evidence that photorespiration remained slow under drought conditions.

Contributions of soluble carbohydrates to the osmotic adjustment in the C4 grass Setaria sphacelata: A comparison between rapidly and slowly imposed water stress

Photosynthetic carbohydrate content in Setaria sphacelata var. splendida under rapidly and slowly induced water deficit and its contribution to osmotic adjustment were studied. In short-term stress experiments, a decrease in the total content of sucrose (Su) and starch (St) was observed in leaf discs submitted to stress. An increase in the ratio between free hexoses and sucrose was found in stressed leaves, but no significant differences were found in the amount of free hexoses nor in the ratio between soluble and insoluble sugars. In long-term stress experiments, a higher amount of soluble sugars and a lower amount of starch were found in stressed leaves, when compared to the control. The ratios of free hexoses to sucrose and of soluble to insoluble sugars were also higher in stressed leaves. The contribution of the accumulation of soluble sugars to osmotic adjustment was absent in rapidly stressed leaves and was of minor importance in slowly stressed leaves.

Photosynthetic responses of three C4 grasses of different metabolic subtypes to water deficit

C4 plants are considered to be less sensitive to drought than C3 plants because of their CO2 concentrating mechanism. The C4 grasses, Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) were compared in their response to water deficit imposed by the addition of polyethylene glycol to the nutrient solution in which they were grown. The effects of drought on leaf relative water content (RWC), net photosynthesis, stomatal conductance, carboxylating enzyme activities and chlorophyll a fluorescence were investigated. In C. dactylon the RWC was more sensitive, but the photosynthetic activity was less sensitive, to water deficit than in P. dilatatum and Z. japonica. The decrease of photosynthesis in P. dilatatum under water deficit was not closely related to the activities of the carboxylating enzymes or to chlorophyll a fluorescence. However, decreased activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase, in addition to decreased stomatal conductance, may have contributed to the decrease of photosynthesis with drought in C. dactylon and Z. japonica. The different responses to water deficit are discussed in relation to the natural habitats of C4 grasses.

Grasses of different C4 subtypes reveal leaf traits related to drought tolerance in their natural habitats: Changes in structure, water potential, and amino acid content

Three grasses (Poaceae) of different C(4) subtypes, Paspalum dilatatum (NADP-malic enzyme [ME]), Cynodon dactylon (NAD-ME) and Zoysia japonica (phosphoenolpyruvate carboxykinase), occur in natural habitats that differ in annual rainfall. Their leaf characteristics were studied to identify traits related to drought tolerance. Plants were grown in pots, and water deficit was gradually induced by withholding water. Leaves of Z. japonica had the greatest and P. dilatatum the lowest relative dry matter content. Transverse sections of leaves that developed during the water deficit showed little change compared to control leaves, consistent with low phenotypic plasticity. Anatomical features distinguished the three species, with xeromorphic characteristics most strongly represented in Z. japonica. The leaf relative water content (RWC) decreased with the soil water content similarly for the three grasses. However, at 80% RWC, the leaf water potential was -3.1 MPa for Z. japonica and only -1.3 MPa for P. dilatatum and C. dactylon. Soluble amino acids, especially proline, increased as RWC decreased in leaves of C. dactylon and Z. japonica. Phenylalanine, valine, leucine, and isoleucine increased more in Z. japonica than in the other two species. The results provide evidence that C. dactylon and, especially, Z. japonica have evolved leaf traits better suited to arid habitats.

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses.

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C(4) grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and beta-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using (1)H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C(4) grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes.

The C4 photosynthetic pathway involves the assimilation of CO2 by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C4 acids. The enzymes of the CO2 concentrating mechanism could be affected under water deficit and limit C4 photosynthesis. Three different C4 grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C4 acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C4 enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of l-malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO2 by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.

Dorsoventral variations in dark chilling effects on photosynthesis and stomatal function in Paspalum dilatatum leaves

The effects of dark chilling on the leaf-side-specific regulation of photosynthesis were characterized in the C4 grass Paspalum dilatatum. CO2- and light-response curves for photosynthesis and associated parameters were measured on whole leaves and on each leaf side independently under adaxial and abaxial illumination before and after plants were exposed to dark chilling for one or two consecutive nights. The stomata closed on the adaxial sides of the leaves under abaxial illumination and no CO2 uptake could be detected on this surface. However, high rates of whole leaf photosynthesis were still observed because CO2 assimilation rates were increased on the abaxial sides of the leaves under abaxial illumination. Under adaxial illumination both leaf surfaces contributed to the inhibition of whole leaf photosynthesis observed after one night of chilling. After two nights of chilling photosynthesis remained inhibited on the abaxial side of the leaf but the adaxial side had recovered, an effect related to increased maximal ribulose-1,5-bisphosphate carboxylation rates (Vcmax) and enhanced maximal electron transport rates (Jmax). Under abaxial illumination, whole leaf photosynthesis was decreased only after the second night of chilling. The chilling-dependent inhibition of photosynthesis was located largely on the abaxial side of the leaf and was related to decreased Vcmax and Jmax, but not to the maximal phosphoenolpyruvate carboxylase carboxylation rate (Vpmax). Each side of the leaf therefore exhibits a unique sensitivity to stress and recovery. Side-specific responses to stress are related to differences in the control of enzyme and photosynthetic electron transport activities.

Effects of rapidly imposed water deficit on photosynthetic parameters of three C4 grasses

Water deficit, when rapidly imposed on three C4 grasses of the different metabolic subtypes, Paspalum dilatatum Poiret (NADP-malic enzyme), Cynodon dactylon (L.) Pers (NAD-malic enzyme) and Zoysia japonica Steudel (phosphoenolpyruvate carboxykinase), caused decreases in photosynthetic rates, in the quantum yield of PS II and photochemical quenching, and in the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC). The results provide evidence for non-stomatal limitations of photosynthesis differing in nature between the three species.

Photosynthetic enzymes of the C4 grass Setaria sphacelata under water stress: a comparison between rapidly and slowly imposed water deficit

Two stress imposing systems were used: a rapid stress developed by allowing excised leaves to loose water by transpiration, and a slow stress developed by withholding watering of potted plants. Carboxylating enzymes reacted differently on both types of stress. Rapid stress increased ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activation, but both activities (initial and total) showed little variation with stress. Under slow stress the activation did not change, although both activities decreased much under stress. Phosphoenolpyruvate carboxylase (PEPC) showed a deep decrease of activity under rapid stress, nevertheless, a certain recovery was found under extreme stress. On the other hand, under slow stress the activity of PEPC showed a linear increase with decreasing relative water content. The ratio between physiological and maximal activity increased slightly under both types of stress. The activity of malic enzyme did not change under rapid stress, and decreased linearly under slow stress.

Variations in the dorso‐ventral organization of leaf structure and Kranz anatomy coordinate the control of photosynthesis and associated signalling at the whole leaf level in monocotyledonous species

Photosynthesis and associated signalling are influenced by the dorso-ventral properties of leaves. The degree of adaxial/abaxial symmetry in stomatal numbers, photosynthetic regulation with respect to light orientation and the total section areas of the bundle sheath (BS) cells and the surrounding mesophyll (M) cells on the adaxial and abaxial sides of the vascular bundles were compared in two C(4)[Zea mays (maize) and Paspalum dilatatum] and one C(3)[Triticum turgidum (Durum wheat)] monocotyledonous species. The C(3) leaves had a higher degree of dorso-ventral symmetry than the C(4) leaves. Photosynthetic regulation was the same on each side of the wheat leaves, as were stomatal numbers and the section area of the BS relative to that of the M cells (BS/M section area ratio). In contrast, photosynthetic regulation in maize and P. dilatatum leaves showed a marked surface-specific response to light orientation. Compared to the adaxial sides of the C(4) monocotyledonous leaves, the abaxial surfaces had more stomata and the BS/M section area ratio was significantly higher. Differences in dorso-ventral structure, particularly in Kranz anatomy, serve not only to maximize photosynthetic capacity with respect light orientation in C(4) monocotyledonous leaves but also allow adaxial and abaxial-specific signalling from the respective M cells.