The use of chitosan oligosaccharide to improve artemisinin yield in well-watered and drought-stressed plants.

Introduction: Artemisinin is a secondary metabolite well-known for its use in the treatment of malaria. It also displays other antimicrobial activities which further increase its interest. At present, Artemisia annua is the sole commercial source of the substance, and its production is limited, leading to a global deficit in supply. Furthermore, the cultivation of A. annua is being threatened by climate change. Specifically, drought stress is a major concern for plant development and productivity, but, on the other hand, moderate stress levels can elicit the production of secondary metabolites, with a putative synergistic interaction with elicitors such as chitosan oligosaccharides (COS). Therefore, the development of strategies to increase yield has prompted much interest. With this aim, the effects on artemisinin production under drought stress and treatment with COS, as well as physiological changes in A. annua plants are presented in this study. Methods: Plants were separated into two groups, well-watered (WW) and drought-stressed (DS) plants, and in each group, four concentrations of COS were applied (0, 50,100 and 200 mg•L-1). Afterwards, water stress was imposed by withholding irrigation for 9 days. Results: Therefore, when A. annua was well watered, COS did not improve plant growth, and the upregulation of antioxidant enzymes hindered the production of artemisinin. On the other hand, during drought stress, COS treatment did not alleviate the decline in growth at any concentration tested. However, higher doses improved the water status since leaf water potential (YL) improved by 50.64% and relative water content (RWC) by 33.84% compared to DS plants without COS treatment. Moreover, the combination of COS and drought stress caused damage to the plant's antioxidant enzyme defence, particularly APX and GR, and reduced the amount of phenols and flavonoids. This resulted in increased ROS production and enhanced artemisinin content by 34.40% in DS plants treated with 200 mg•L-1 COS, compared to control plants. Conclusion: These findings underscore the critical role of ROS in artemisinin biosynthesis and suggest that COS treatment may boost artemisinin yield in crop production, even under drought conditions.

Dynamic Regulation of Grapevine's microRNAs in Response to Mycorrhizal Symbiosis and High Temperature.

MicroRNAs (miRNAs) are non-coding small RNAs that play crucial roles in plant development and stress responses and can regulate plant interactions with beneficial soil microorganisms such as arbuscular mycorrhizal fungi (AMF). To determine if root inoculation with distinct AMF species affected miRNA expression in grapevines subjected to high temperatures, RNA-seq was conducted in leaves of grapevines inoculated with either Rhizoglomus irregulare or Funneliformis mosseae and exposed to a high-temperature treatment (HTT) of 40 °C for 4 h per day for one week. Our results showed that mycorrhizal inoculation resulted in a better plant physiological response to HTT. Amongst the 195 identified miRNAs, 83 were considered isomiRs, suggesting that isomiRs can be biologically functional in plants. The number of differentially expressed miRNAs between temperatures was higher in mycorrhizal (28) than in non-inoculated plants (17). Several miR396 family members, which target homeobox-leucine zipper proteins, were only upregulated by HTT in mycorrhizal plants. Predicted targets of HTT-induced miRNAs in mycorrhizal plants queried to STRING DB formed networks for Cox complex, and growth and stress-related transcription factors such as SQUAMOSA promoter-binding-like-proteins, homeobox-leucine zipper proteins and auxin receptors. A further cluster related to DNA polymerase was found in R. irregulare inoculated plants. The results presented herein provide new insights into miRNA regulation in mycorrhizal grapevines under heat stress and can be the basis for functional studies of plant-AMF-stress interactions.

High-throughput phenotyping of physiological traits for wheat resilience to high temperature and drought stress.

Interannual and local fluctuations in wheat crop yield are mostly explained by abiotic constraints. Heatwaves and drought, which are among the top stressors, commonly co-occur, and their frequency is increasing with global climate change. High-throughput methods were optimized to phenotype wheat plants under controlled water deficit and high temperature, with the aim to identify phenotypic traits conferring adaptative stress responses. Wheat plants of 10 genotypes were grown in a fully automated plant facility under 25/18 °C day/night for 30 d, and then the temperature was increased for 7 d (38/31 °C day/night) while maintaining half of the plants well irrigated and half at 30% field capacity. Thermal and multispectral images and pot weights were registered twice daily. At the end of the experiment, key metabolites and enzyme activities from carbohydrate and antioxidant metabolism were quantified. Regression machine learning models were successfully established to predict plant biomass using image-extracted parameters. Evapotranspiration traits expressed significant genotype-environment interactions (G×E) when acclimatization to stress was continuously monitored. Consequently, transpiration efficiency was essential to maintain the balance between water-saving strategies and biomass production in wheat under water deficit and high temperature. Stress tolerance included changes in carbohydrate metabolism, particularly in the sucrolytic and glycolytic pathways, and in antioxidant metabolism. The observed genetic differences in sensitivity to high temperature and water deficit can be exploited in breeding programmes to improve wheat resilience to climate change.

SUMO E3 ligase SIZ1 connects sumoylation and reactive oxygen species homeostasis processes in Arabidopsis.

The ubiquitin-like modifying peptide SMALL UBIQUITIN-LIKE MODIFIER (SUMO) has become a known modulator of the plant response to multiple environmental stimuli. A common feature of many of these external stresses is the production of reactive oxygen species (ROS). Taking into account that SUMO conjugates rapidly accumulate in response to an external oxidative stimulus, it is likely that ROS and sumoylation converge at the molecular and regulatory levels. In this study, we explored the SUMO-ROS relationship, using as a model the Arabidopsis (Arabidopsis thaliana) null mutant of the major SUMO-conjugation enhancer, the E3 ligase SAP AND MIZ 1 (SIZ1). We showed that SIZ1 is involved in SUMO conjugate increase when primed with both exogenous and endogenous ROS. In siz1, seedlings were sensitive to oxidative stress imposition, and mutants accumulated different ROS throughout development. We demonstrated that the deregulation in hydrogen peroxide and superoxide homeostasis, but not of singlet O2 (1O2), was partially due to SA accumulation in siz1. Furthermore, transcriptomic analysis highlighted a transcriptional signature that implicated siz1 with 1O2 homeostasis. Subsequently, we observed that siz1 displayed chloroplast morphological defects and altered energy dissipation activity and established a link between the chlorophyll precursor protochlorophyllide and deregulation of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), which is known to drive overproduction of 1O2. Ultimately, network analysis uncovered known and additional associations between transcriptional control of PORA and SIZ1-dependent sumoylation. Our study connects sumoylation, and specifically SIZ1, to the control of chloroplast functions and places sumoylation as a molecular mechanism involved in ROS homeostatic and signaling events.

Potential Phenotyping Methodologies to Assess Inter- and Intravarietal Variability and to Select Grapevine Genotypes Tolerant to Abiotic Stress.

Plant phenotyping is an emerging science that combines multiple methodologies and protocols to measure plant traits (e.g., growth, morphology, architecture, function, and composition) at multiple scales of organization. Manual phenotyping remains as a major bottleneck to the advance of plant and crop breeding. Such constraint fostered the development of high throughput plant phenotyping (HTPP), which is largely based on imaging approaches and automatized data retrieval and processing. Field phenotyping still poses major challenges and the progress of HTPP for field conditions can be relevant to support selection and breeding of grapevine. The aim of this review is to discuss potential and current methods to improve field phenotyping of grapevine to support characterization of inter- and intravarietal diversity. Vitis vinifera has a large genetic diversity that needs characterization, and the availability of methods to support selection of plant material (polyclonal or clonal) able to withstand abiotic stress is paramount. Besides being time consuming, complex and expensive, field experiments are also affected by heterogeneous and uncontrolled climate and soil conditions, mostly due to the large areas of the trials and to the high number of traits to be observed in a number of individuals ranging from hundreds to thousands. Therefore, adequate field experimental design and data gathering methodologies are crucial to obtain reliable data. Some of the major challenges posed to grapevine selection programs for tolerance to water and heat stress are described herein. Useful traits for selection and related field phenotyping methodologies are described and their adequacy for large scale screening is discussed.

Efficient Regulation of CO2 Assimilation Enables Greater Resilience to High Temperature and Drought in Maize.

Increasing temperatures and extended drought episodes are among the major constraints affecting food production. Maize has a relatively high temperature optimum for photosynthesis compared to C3 crops, however, the response of this important C4 crop to the combination of heat and drought stress is poorly understood. Here, we hypothesized that resilience to high temperature combined with water deficit (WD) would require efficient regulation of the photosynthetic traits of maize, including the C4-CO2 concentrating mechanism (CCM). Two genotypes of maize with contrasting levels of drought and heat tolerance, B73 and P0023, were acclimatized at high temperature (38°C versus 25°C) under well-watered (WW) or WD conditions. The photosynthetic performance was evaluated by gas exchange and chlorophyll a fluorescence, and in vitro activities of key enzymes for carboxylation (phosphoenolpyruvate carboxylase), decarboxylation (NADP-malic enzyme), and carbon fixation (Rubisco). Both genotypes successfully acclimatized to the high temperature, although with different mechanisms: while B73 maintained the photosynthetic rates by increasing stomatal conductance (gs), P0023 maintained gs and showed limited transpiration. When WD was experienced in combination with high temperatures, limited transpiration allowed water-savings and acted as a drought stress avoidance mechanism. The photosynthetic efficiency in P0023 was sustained by higher phosphorylated PEPC and electron transport rate (ETR) near vascular tissues, supplying chemical energy for an effective CCM. These results suggest that the key traits for drought and heat tolerance in maize are limited transpiration rate, allied with a synchronized regulation of the carbon assimilation metabolism. These findings can be exploited in future breeding efforts aimed at improving maize resilience to climate change.

Photoprotection and optimization of sucrose usage contribute to faster recovery of photosynthesis after water deficit at high temperatures in wheat.

Plants are increasingly exposed to events of elevated temperature and water deficit, which threaten crop productivity. Understanding the ability to rapidly recover from abiotic stress, restoring carbon assimilation and biomass production, is important to unravel crop climate resilience. This study compared the photosynthetic performance of two Triticum aestivum L. cultivars, Sokoll and Paragon, adapted to the climate of Mexico and UK, respectively, exposed to 1-week water deficit and high temperatures, in isolation or combination. Measurements included photosynthetic assimilation rate, stomatal conductance, in vitro activities of Rubisco (EC 4.1.1.39) and invertase (INV, EC 3.2.1.26), antioxidant capacity and chlorophyll a fluorescence. In both genotypes, under elevated temperatures and water deficit (WD38°C), the photosynthetic limitations were mainly due to stomatal restrictions and to a decrease in the electron transport rate. Chlorophyll a fluorescence parameters clearly indicate differences between the two genotypes in the photoprotection when subjected to WD38°C and showed faster recovery of Paragon after stress relief. The activity of the cytosolic invertase (CytINV) under these stress conditions was strongly related to the fast photosynthesis recovery of Paragon. Taken together, the results suggest that optimal sucrose export/utilization and increased photoprotection of the electron transport machinery are important components to limit yield fluctuations due to water shortage and elevated temperatures.

Using Rapid Chlorophyll Fluorescence Transients to Classify Vitis Genotypes.

When a dark-adapted leaf is illuminated with saturating light, a fast polyphasic rise of fluorescence emission (Kautsky effect) is observed. The shape of the curve is dependent on the molecular organization of the photochemical apparatus, which in turn is a function of the interaction between genotype and environment. In this paper, we evaluate the potential of rapid fluorescence transients, aided by machine learning techniques, to classify plant genotypes. We present results of the application of several machine learning algorithms (k-nearest neighbors, decision trees, artificial neural networks, genetic programming) to rapid induction curves recorded in different species and cultivars of vine grown in the same environmental conditions. The phylogenetic relations between the selected Vitis species and Vitis vinifera cultivars were established with molecular markers. Both neural networks (71.8%) and genetic programming (75.3%) presented much higher global classification success rates than k-nearest neighbors (58.5%) or decision trees (51.6%), genetic programming performing slightly better than neural networks. However, compared with a random classifier (success rate = 14%), even the less successful algorithms were good at the task of classifying. The use of rapid fluorescence transients, handled by genetic programming, for rapid preliminary classification of Vitis genotypes is foreseen as feasible.

All for One: The Role of Colony Morphology in Bryophyte Desiccation Tolerance.

In the last decade, several works showed that even bryophytes from aquatic environments, if slowly dehydrated, can cope with desiccation in a response like the one from desert bryophytes. This led to the hypothesis that, if bryophytes from contrasting habitats can have similar responses, desiccation tolerance (DT) is partially inductive and not only constitutive as previously proposed and, therefore, colony morphology might be the key trait responsible for controlling dehydration rate essential for DT induction. Morphology and life form may be determinant traits in the adaptation of bryophytes to habitats with different water availabilities and corresponding predicted levels in the DT inducibility spectrum. Bryophytes from habitats with different water availabilities were dried as individual shoots and as a colony. The bryophyte Fontinalis antipyretica is fully aquatic presenting a streamer life form, while the three terrestrial species present turf life form with different sizes and degrees of space between individuals in the colony. Two species were collected under trees with moist soil presenting short turf (Tortella tortuosa) and long turf (Campylopus pyriformis) life forms. Another species was completely exposed to sun light with no surrounding trees and a tall turf life form (Pleurochaete squarrosa). We used chlorophyll a fluorescence parameter Fv/Fm (maximum potential quantum efficiency of Photosystem II) as a proxy to photosynthetic fitness throughout the contrasting dehydration rates (fast and slow). These bryophytes with different life forms were submitted to an X-ray computed microtomography (µ-XCT) to assess the three-dimensional inner structure and visualize locations for water storage. Shoots dried slow or fast according to the dehydration they were exposed to, as expected, but they presented similar dehydration rates across different species. However, the aquatic moss F. antipyretica, was unable to recover from fast drying, and after 24 h the recovery following slow drying was lower than the other species. The other three species presented full recovery after 24 h, either at the individual or colony level, and either from slow or fast drying. The only exception was the colonies of Campylopus pyriformis following fast drying that presented a slightly lower recovery, probably due to a looser colony structure.

Opportunities and Limitations of Crop Phenotyping in Southern European Countries.

The Mediterranean climate is characterized by hot dry summers and frequent droughts. Mediterranean crops are frequently subjected to high evapotranspiration demands, soil water deficits, high temperatures, and photo-oxidative stress. These conditions will become more severe due to global warming which poses major challenges to the sustainability of the agricultural sector in Mediterranean countries. Selection of crop varieties adapted to future climatic conditions and more tolerant to extreme climatic events is urgently required. Plant phenotyping is a crucial approach to address these challenges. High-throughput plant phenotyping (HTPP) helps to monitor the performance of improved genotypes and is one of the most effective strategies to improve the sustainability of agricultural production. In spite of the remarkable progress in basic knowledge and technology of plant phenotyping, there are still several practical, financial, and political constraints to implement HTPP approaches in field and controlled conditions across the Mediterranean. The European panorama of phenotyping is heterogeneous and integration of phenotyping data across different scales and translation of "phytotron research" to the field, and from model species to crops, remain major challenges. Moreover, solutions specifically tailored to Mediterranean agriculture (e.g., crops and environmental stresses) are in high demand, as the region is vulnerable to climate change and to desertification processes. The specific phenotyping requirements of Mediterranean crops have not yet been fully identified. The high cost of HTPP infrastructures is a major limiting factor, though the limited availability of skilled personnel may also impair its implementation in Mediterranean countries. We propose that the lack of suitable phenotyping infrastructures is hindering the development of new Mediterranean agricultural varieties and will negatively affect future competitiveness of the agricultural sector. We provide an overview of the heterogeneous panorama of phenotyping within Mediterranean countries, describing the state of the art of agricultural production, breeding initiatives, and phenotyping capabilities in five countries: Italy, Greece, Portugal, Spain, and Turkey. We characterize some of the main impediments for development of plant phenotyping in those countries and identify strategies to overcome barriers and maximize the benefits of phenotyping and modeling approaches to Mediterranean agriculture and related sustainability.

Inorganic carbon availability in benthic diatom communities: photosynthesis and migration.

Diatom-dominated microphytobenthos (MPB) is the main primary producer of many intertidal and shallow subtidal environments, being therefore of critical importance to estuarine and coastal food webs. Owing to tidal cycles, intertidal MPB diatoms are subjected to environmental conditions far more variable than the ones experienced by pelagic diatoms (e.g. light, temperature, salinity, desiccation and nutrient availability). Nevertheless, benthic diatoms evolved adaptation mechanisms to these harsh conditions, including the capacity to move within steep physical and chemical gradients, allowing them to perform photosynthesis efficiently. In this contribution, we will review present knowledge on the effects of dissolved inorganic carbon (DIC) availability on photosynthesis and productivity of diatom-dominated MPB. We present evidence of carbon limitation of photosynthesis in benthic diatom mats and highly productive MPB natural communities. Furthermore, we hypothesize that active vertical migration of epipelic motile diatoms could overcome local depletion of DIC in the photic layer, providing the cells alternately with light and inorganic carbon supply. The few available longer-term experiments on the effects of inorganic carbon enrichment on the productivity of diatom-dominated MPB have yielded inconsistent results. Therefore, further studies are needed to properly assess the response of MPB communities to increased CO2 and ocean acidification related to climate change.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Dehydration rate determines the degree of membrane damage and desiccation tolerance in bryophytes.

Desiccation tolerant (DT) organisms are able to withstand an extended loss of body water and rapidly resume metabolism upon rehydration. This ability, however, is strongly dependent on a slow dehydration rate. Fast dehydration affects membrane integrity leading to intracellular solute leakage upon rehydration and thereby impairs metabolism recovery. We test the hypothesis that the increased cell membrane damage and membrane permeability observed under fast dehydration, compared with slow dehydration, is related to an increase in lipid peroxidation. Our results reject this hypothesis because following rehydration lipid peroxidation remains unaltered, a fact that could be due to the high increase of NO upon rehydration. However, in fast-dried samples we found a strong signal of red autofluorescence upon rehydration, which correlates with an increase in ROS production and with membrane leakage, particularly the case of phenolics. This could be used as a bioindicator of oxidative stress and membrane damage.

Photosynthesis in estuarine intertidal microphytobenthos is limited by inorganic carbon availability.

The effects of dissolved inorganic carbon (DIC) availability on photosynthesis were studied in two estuarine intertidal microphytobenthos (MPB) communities and in the model diatom species Phaeodactylum tricornutum. Kinetics of DIC acquisition, measured with a liquid-phase oxygen electrode, showed higher K(1/2)(DIC) (0.31 mM) and Vm (7.78 nmol min(-1) µg (Chl a)(-1)) for MPB suspensions than for P. tricornutum (K(1/2)(DIC) = 0.23 mM; Vm = 4.64 nmol min(-1) µg (Chl a)(-1)), suggesting the predominance of species with lower affinity for DIC and higher photosynthetic capacity in the MPB. The net photosynthetic rate of the MPB suspensions reached saturation at a DIC concentration of 1-1.5 mM. This range was lower than the concentrations found in the interstitial water of the top 5-mm sediment layer, suggesting no limitation of photosynthesis by DIC in the MPB communities. Accordingly, carbon isotope discrimination revealed a moderate activity of CO2-concentrating mechanisms in the MPB. However, addition of NaHCO3 to intact MPB biofilms caused a significant increase in the relative maximum photosynthetic electron transport rate (rETR max) measured by imaging pulse-amplitude modulated chlorophyll a fluorescence. These results suggest local depletion of DIC at the photic layer of the sediment (the first few hundred µm), where MPB cells accumulate during diurnal low tides. This work provides the first direct experimental evidence of DIC limitation of photosynthesis in highly productive intertidal MPB communities.

Performance index: An expeditious tool to screen for improved drought resistance in the Lathyrus genus.

Some species of the Lathyrus genus are among the most promising crops for marginal lands, with high resilience to drought, flood, and fungal diseases, combined with high yields and seed nutritional value. However, lack of knowledge on the mechanisms underlying its outstanding performance and methodologies to identify elite genotypes has hampered its proper use in breeding. Chlorophyll a fast fluorescence transient (JIP test), was used to evaluate water deficit (WD) resistance in Lathyrus genus. Our results reveal unaltered photochemical values for all studied genotypes showing resistance to mild WD. Under severe WD, two Lathyrus sativus genotypes showed remarkable resilience maintaining the photochemical efficiency, contrary to other genotypes studied. Performance index (PIABS) is the best parameter to screen genotypes with improved performance and grain production under WD. Moreover, we found that JIP indices are good indicators of genotypic grain production under WD. Quantum yield of electron transport (ϕEo) and efficiency with which trapped excitons can move electrons further than QA (ψ0) revealed as important traits related to improved photosynthetic performance and should be exploited in future Lathyrus germplasm improvements. The JIP test herein described showed to be an expeditious tool to screen and to identify elite genotypes with improved drought resistance.

Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism.

All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyretica Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.

The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss.

BACKGROUND AND AIMS: The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage. METHODS: Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed. KEY RESULTS: The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the initial stages of rehydration, perhaps due to the burst of ROS production. CONCLUSIONS: A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration.

Response of the diatom Phaeodactylum tricornutum to photooxidative stress resulting from high light exposure.

The response of microalgae to photooxidative stress resulting from high light exposure is a well-studied phenomenon. However, direct analyses of photosystem II (PSII) D1 protein (the main target of photoinhibition) in diatoms are scarce. In this study, the response of the diatom model species Phaeodactylum tricornutum to short-term exposure to high light was examined and the levels of D1 protein determined immunochemically. Low light (LL) acclimated cells (40 µmol photons m(-2) s(-1)) subjected to high light (HL, 1,250 µmol photons m(-2) s(-1)) showed rapid induction of non-photochemical quenching (NPQ) and ca. 20-fold increase in diatoxanthin (DT) concentration. This resulted from the conversion of diadinoxanthin (DD) to DT through the activation of the DD-cycle. D1 protein levels under LL decreased about 30% after 1 h of the addition of lincomycin (LINC), a chloroplast protein synthesis inhibitor, showing significant D1 degradation and repair under low irradiance. Exposure to HL lead to a 3.2-fold increase in D1 degradation rate, whereas average D1 repair rate was 1.3-x higher under HL than LL, leading to decreased levels of D1 protein under HL. There were significant effects of both HL and LINC on P. tricornutum maximum quantum yield of PSII (F(v)/F(m)), showing a reduction of active PSII reaction centres. Partial recovery of F(v)/F(m) in the dark demonstrates the photosynthetic resilience of this diatom to changes in the light regime. P. tricornutum showed high allocation of total protein to D1 and an active D1-repair cycle to limit photoinhibition.

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.