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.

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.

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.

Rubisco regulation: a role for inhibitors.

In photosynthesis Rubisco catalyses the assimilation of CO(2) by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.

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.

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.