Expression profiling and sequence diversity of novel DREB genes from common bean (Phaseolus vulgaris L.) and their association with drought-related traits

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance

SUMOylation represses SnRK1 signaling in Arabidopsis

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin-like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1-dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1-2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.

Seasonal patterns of growth, dehydrins and water-soluble carbohydrates in genotypes of Dactylis glomerata varying in summer dormancy

Background and Aims Summer dormancy in perennial grasses has been studied inadequately, despite its potential to enhance plant survival and persistence in Mediterranean areas. The aim of the present work was to characterize summer dormancy and dehydration tolerance in two cultivars of Dactylis glomerata (dormant 'Kasbah', non-dormant 'Oasis') and their hybrid using physiological indicators associated with these traits. Methods Dehydration tolerance was assessed in a glasshouse experiment, while seasonal metabolic changes which produce putative protectants for drought, such as carbohydrates and dehydrins that might be associated with summer dormancy, were analysed in the field. Key Results The genotypes differed in their ability to survive increasing soil water deficit: lethal soil water potential (ψ(s)) was -3(.)4 MPa for 'Kasbah' (although non-dormant), -1(.)3 MPa for 'Oasis', and -1(.)6 MPa for their hybrid. In contrast, lethal water content of apices was similar for all genotypes (approx. 0(.)45 g H2O g d. wt(-1)), and hence the greater survival of 'Kasbah' can be ascribed to better drought avoidance rather than dehydration tolerance. In autumn-sown plants, 'Kasbah' had greatest dormancy, the hybrid was intermediate and 'Oasis' had none. The more dormant the genotype, the lower the metabolic activity during summer, and the earlier the activity declined in spring. Decreased monosaccharide content was an early indicator of dormancy induction. Accumulation of dehydrins did not correlate with stress tolerance, but dehydrin content was a function of the water status of the tissues, irrespective of the soil moisture. A protein of approx. 55 kDa occurred in leaf bases of the most dormant cultivar even in winter. Conclusions Drought avoidance and summer dormancy are correlated but can be independently expressed. These traits are heritable, allowing selection in breeding programmes.

Seed treatment with gibberellic acid and glycinebetaine improves seedling emergence and seedling vigour of rice under low temperature

Prevalence of low temperature at sowing results in poor rice seed germination, seedling establishment and vigour in several temperate rice growing countries around the world. Rice seed of four cultivars (Sasanishiki, H433, HSC-55 and Doongara) was soaked in various combinations of gibberellic acid(3) (GA(3)) and glycinebetaine (GB) in petri dishes placed in a low temperature glasshouse (18/13 degrees C; day/night) for 2 days. After the 2 days soak, 10 treated seed were transferred into plastic pots filled with soil and seedlings were grown in the same glasshouse, where seed was treated. Seedling emergence was least affected by low temperature in cold tolerant cultivar, HSC-55, while other three cultivars showed reduced seedling emergence. However, seedling emergence increased significantly in some cultivars in response to seed treatment with GA(3) and/or GB. Seedlings emerged faster even in the cold tolerant cultivar, HSC-55, as measured by reduced mean emergence time (MET), in response to GB. Seedling height and seedling dry matter also increased in response to both GA(3) and GB. Combined treatment of both GA(3) and GB was more beneficial in increasing seedling emergence and vigour than the treatment with only GA3 or GB. We demonstrated significant genotypic differences for seedling emergence and vigour and not all cultivars responded to the treatment with GA(3) and GB, under low temperature.

Ecophysiology and biomechanics of Equisetum giganteum in South America

Equisetum giganteum L., a giant horsetail, is one of the largest living members of an ancient group of non-flowering plants with a history extending back 377 million years. Its hollow upright stems grow to over 5 m in height. Equisetum giganteum occupies a wide range of habitats in southern South America. Colonies of this horsetail occupy large areas of the Atacama river valleys, including those with sufficiently high groundwater salinity to significantly reduce floristic diversity. The purpose of this research was to study the ecophysiological and biomechanical properties that allow E. giganteum to successfully colonize a range of habitats, varying in salinity and exposure. Stem ecophysiological behavior was measured via steady state porometry (stomatal conductance), thermocouple psychrometry (water potential), chlorophyll fluorescence, and ion specific electrodes (xylem fluid solutes). Stem biomechanical properties were measured via a 3-point bending apparatus and cross sectional imaging. Equisetum giganteum stems exhibit mechanical characteristics of semi-self-supporting plants, requiring mutual support or support of other vegetation when they grow tall. The mean elastic moduli (4.3 Chile, 4.0 Argentina) of E. giganteum in South America is by far the largest measured in any living horsetail. Stomatal behavior of E. giganteum is consistent with that of typical C3 vascular plants, although absolute values of maximum late morning stomatal conductance are very low in comparison to typical plants from mesic habitats. The internode stomata exhibit strong light response. However, the environmental sensitivity of stomatal conductance appeared less in young developing stems, possibly due to higher cuticular conductance. Exclusion of sodium (Na) and preferential accumulation of potassium (K) at the root level appears to be the key mechanism of salinity tolerance in E. giganteum. Overall stomatal conductance and chlorophyll fluorescence were little affected by salinity, ranging from very low levels up to half strength seawater. This suggests a high degree of salinity stress tolerance. The capacity of E. giganteum to adapt to a wide variety of environments in southern South America has allowed it to thrive despite tremendous environmental changes during their long tenure on Earth.

Ecophysiology and Biomechanics of Equisetum Giganteum in South America

Equisetum giganteum L., a giant horsetail, is one of the largest living members of an ancient group of non-flowering plants with a history extending back 377 million years. Its hollow upright stems grow to over 5 m in height. Equisetum giganteum occupies a wide range of habitats in southern South America. Colonies of this horsetail occupy large areas of the Atacama river valleys, including those with sufficiently high groundwater salinity to significantly reduce floristic diversity. The purpose of this research was to study the ecophysiological and biomechanical properties that allow E. giganteum to successfully colonize a range of habitats, varying in salinity and exposure. Stem ecophysiological behavior was measured via steady state porometry (stomatal conductance), thermocouple psychrometry (water potential), chlorophyll fluorescence, and ion specific electrodes (xylem fluid solutes). Stem biomechanical properties were measured via a 3-point bending apparatus and cross sectional imaging. Equisetum giganteum stems exhibit mechanical characteristics of semi-self-supporting plants, requiring mutual support or support of other vegetation when they grow tall. The mean elastic moduli (4.3 Chile, 4.0 Argentina) of E. giganteum in South America is by far the largest measured in any living horsetail. Stomatal behavior of E. giganteum is consistent with that of typical C3 vascular plants, although absolute values of maximum late morning stomatal conductance are very low in comparison to typical plants from mesic habitats. The internode stomata exhibit strong light response. However, the environmental sensitivity of stomatal conductance appeared less in young developing stems, possibly due to higher cuticular conductance. Exclusion of sodium (Na) and preferential accumulation of potassium (K) at the root level appears to be the key mechanism of salinity tolerance in E. giganteum. Overall stomatal conductance and chlorophyll fluorescence were little affected by salinity, ranging from very low levels up to half strength seawater. This suggests a high degree of salinity stress tolerance. The capacity of E. giganteum to adapt to a wide variety of environments in southern South America has allowed it to thrive despite tremendous environmental changes during their long tenure on Earth.

Asian green mussels (Perna viridis) transplanted between Jakarta Bay and Lada Bay, West Java, Indonesia (April 2012 - November 2013)

The Asian green mussel Perna viridis is tolerant to environmental stress, but its robustness varies between populations from habitats that differ in quality. So far, it is unclear whether local adaptations through stressinduced selection or phenotypic plasticity are responsible for these inter-population differences. We tested for the relevance of both mechanisms by comparing survival under hypoxia in mussels that were transplanted from an anthropogenically impacted (Jakarta Bay, Indonesia) to a natural habitat (Lada Bay, Indonesia) and vice versa. Mussels were retrieved 8 weeks after transplantation and exposed to hypoxia in the laboratory. Additional hypoxia tests were conducted with juvenile mussels collected directly from both sites. To elucidate possible relationships between habitat quality and mussel tolerance, we monitored concentrations of inorganic nutrients, temperature, dissolved oxygen, salinity, phytoplankton density and the mussels' body condition index (BCI) for 20 months before, during and after the experiments. Survival under hypoxia depended mainly on the quality of the habitat where the mussels lived before the hypoxia tests and only to a small degree on their site of origin. Furthermore, stress tolerance was only higher in Jakarta than in Lada Bay mussels when the BCIs were substantially higher, which in turn correlated with the phytoplankton densities. We explain why phenotypic plasticity and high BCIs are more likely the causes of populationspecific differences in hypoxia tolerance in P. viridis than stress-induced selection for robust genotypes. This is relevant to understanding the role of P. viridis as mariculture organism in eutrophic ecosystems and invasive species in the (sub)tropical world.

Taxonomic and Functional Diversity of Soil and Hypolithic Microbial Communities in Miers Valley, McMurdo Dry Valleys, Antarctica

The McMurdo Dry Valleys of Antarctica are an extreme polar desert. Mineral soils support subsurface microbial communities and translucent rocks support development of hypolithic communities on ventral surfaces in soil contact. Despite significant research attention, relatively little is known about taxonomic and functional diversity or their inter-relationships. Here we report a combined diversity and functional interrogation for soil and hypoliths of the Miers Valley in the McMurdo Dry Valleys of Antarctica. The study employed 16S rRNA fingerprinting and high throughput sequencing combined with the GeoChip functional microarray. The soil community was revealed as a highly diverse reservoir of bacterial diversity dominated by actinobacteria. Hypolithic communities were less diverse and dominated by cyanobacteria. Major differences in putative functionality were that soil communities displayed greater diversity in stress tolerance and recalcitrant substrate utilization pathways, whilst hypolithic communities supported greater diversity of nutrient limitation adaptation pathways. A relatively high level of functional redundancy in both soil and hypoliths may indicate adaptation of these communities to fluctuating environmental conditions.

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris)

Common bean (Phaseolus vulgaris L.) is a leguminous in high demand for human nutrition and a very important agricultural product. Production of common bean is constrained by environmental stresses such as drought. Although conventional plant selection has been used to increase production yield and stress tolerance, drought tolerance selection based on phenotype is complicated by associated physiological, anatomical, cellular, biochemical, and molecular changes. These changes are modulated by differential gene expression. A common method to identify genes associated with phenotypes of interest is the characterization of Single Nucleotide Polymorphims (SNPs) to link them to specific functions. In this work, we selected two drought-tolerant parental lines from Mesoamerica, Pinto Villa, and Pinto Saltillo. The parental lines were used to generate a population of 282 families (F3:5) and characterized by 169 SNPs. We associated the segregation of the molecular markers in our population with phenotypes including flowering time, physiological maturity, reproductive period, plant, seed and total biomass, reuse index, seed yield, weight of 100 seeds, and harvest index in three cultivation cycles. We observed 83 SNPs with significant association (p < 0.0003 after Bonferroni correction) with our quantified phenotypes. Phenotypes most associated were days to flowering and seed biomass with 58 and 44 associated SNPs, respectively. Thirty-seven out of the 83 SNPs were annotated to a gene with a potential function related to drought tolerance or relevant molecular/biochemical functions. Some SNPs such as SNP28 and SNP128 are related to starch biosynthesis, a common osmotic protector; and SNP18 is related to proline biosynthesis, another well-known osmotic protector.

GENETIC VARIATION, LOCAL ADAPTATION AND POPULATION STRUCTURE IN NORTH AMERICAN RED OAK SPECIES, QUERCUS RUBRA L. AND Q. ELLIPSOIDALIS E. J. HILL

Forest trees, like oaks, rely on high levels of genetic variation to adapt to varying environmental conditions. Thus, genetic variation and its distribution are important for the long-term survival and adaptability of oak populations. Climate change is projected to lead to increased drought and fire events as well as a northward migration of tree species, including oaks. Additionally, decline in oak regeneration has become increasingly concerning since it may lead to decreased gene flow and increased inbreeding levels. This will in turn lead to lowered levels of genetic diversity, negatively affecting the growth and survival of populations. At the same time, populations at the species’ distribution edge, like those in this study, could possess important stores of genetic diversity and adaptive potential, while also being vulnerable to climatic or anthropogenic changes. A survey of the level and distribution of genetic variation and identification of potentially adaptive genes is needed since adaptive genetic variation is essential for their long-term survival. Oaks possess a remarkable characteristic in that they maintain their species identity and specific environmental adaptations despite their propensity to hybridize. Thus, in the face of interspecific gene flow, some areas of the genome remain differentiated due to selection. This characteristic allows the study of local environmental adaptation through genetic variation analyses. Furthermore, using genic markers with known putative functions makes it possible to link those differentiated markers to potential adaptive traits (e.g., flowering time, drought stress tolerance). Demographic processes like gene flow and genetic drift also play an important role in how genes (including adaptive genes) are maintained or spread. These processes are influenced by disturbances, both natural and anthropogenic. An examination of how genetic variation is geographically distributed can display how these genetic processes and geographical disturbances influence genetic variation patterns. For example, the spatial clustering of closely related trees could promote inbreeding with associated negative effects (inbreeding depression), if gene flow is limited. In turn this can have negative consequences for a species’ ability to adapt to changing environmental conditions. In contrast, interspecific hybridization may also allow the transfer of genes between species that increase their adaptive potential in a changing environment. I have studied the ecologically divergent, interfertile red oaks, Quercus rubra and Q. ellipsoidalis, to identify genes with potential roles in adaptation to abiotic stress through traits such as drought tolerance and flowering time, and to assess the level and distribution of genetic variation. I found evidence for moderate gene flow between the two species and low interspecific genetic differences at most genetic markers (Lind and Gailing 2013). However, the screening of genic markers with potential roles in phenology and drought tolerance led to the identification of a CONSTANS-like (COL) gene, a candidate gene for flowering time and growth. This marker, located in the coding region of the gene, was highly differentiated between the two species in multiple geographical areas, despite interspecific gene flow, and may play a role in reproductive isolation and adaptive divergence between the two species (Lind-Riehl et al. 2014). Since climate change could result in a northward migration of trees species like oaks, this gene could be important in maintaining species identity despite increased contact zones between species (e.g., increased gene flow). Finally I examined differences in spatial genetic structure (SGS) and genetic variation between species and populations subjected to different management strategies and natural disturbances. Diverse management activities combined with various natural disturbances as well as species specific life history traits influenced SGS patterns and inbreeding levels (Lind-Riehl and Gailing submitted).

Saccharomyces cerevisiae in the production of whisk(e)y

Whisky is a major global distilled spirit beverage. Whiskies are produced from cereal starches that are saccharified, fermented and distilled prior to spirit maturation. The strain of Saccharomyces cerevisiae employed in whisky fermentations is crucially important not only in terms of ethanol yields, but also for production of minor yeast metabolites which collectively contribute to development of spirit flavour and aroma characteristics. Distillers must therefore pay very careful attention to the strain of yeast exploited to ensure consistency of fermentation performance and spirit congener profiles. In the Scotch whisky industry, initiatives to address sustainability issues facing the industry (for example, reduced energy and water usage) have resulted in a growing awareness regarding criteria for selecting new distilling yeasts with improved efficiency. For example, there is now a desire for Scotch whisky distilling yeasts to perform under more challenging conditions such as high gravity wort fermentations. This article highlights the important roles of S. cerevisiae strains in whisky production and describes key fermentation performance attributes sought in distiller's yeast, such as high alcohol yields, stress tolerance and desirable congener profiles. We hope that the information herein will be useful for whisky producers and yeast suppliers in selecting new distilling strains of S. cerevisiae, and for the scientific community to stimulate further research in this area.

Phosphorus nutrition and tolerance of cotton to water stress: II. Water relations, free and bound water and leaf expansion rate

In previous experiments, increased leaf-Phosphorus (P) content with increasing P supply enhanced the individual leaf expansion and water content of fresh cotton leaves in a severely drying soil. In this paper, we report on the bulk water content of leaves and its components, free and bound water, along with other measures of plant water status, in expanding cotton leaves of various ages in a drying soil with different P concentrations. The bound water in living tissue is more likely to play a major role in tolerance to abiotic stresses by maintaining the structural integrity and/or cell wall extensibility of the leaves, whilst an increased amount of free water might be able to enhance solute accumulation, leading to better osmotic adjustment and tolerance to water stress, and maintenance of the volumes of sub-cellular compartments for expansive leaf growth. There were strong correlations between leaf-P%, leaf water (total, free and bound water) and leaf expansion rate (LER) under water stress conditions in a severely drying soil. Increased soil-P enhanced the uptake of P from a drying soil, leading to increased supply of osmotically active inorganic solutes to the cells in growing leaves. This appears to have led to the accumulation of free water and more bound water, ultimately leading to increased leaf expansion rates as compared to plants in low P soil under similar water stress conditions. The greater amount of bound and free water in the high-P plants was not necessarily associated with changes in cell turgor, and appears to have maintained the cell-wall properties and extensibility under water stressed conditions in soils that are nutritionally P-deficient.

Phosphorus nutrition and tolerance of cotton to water stress I. Seed cotton yield and leaf morphology

Seed cotton yield and morphological changes in leaf growth were examined under drying soil with different phosphorus (P) concentrations in a tropical climate. Frequent soil drying is likely to induce a decrease in nutrients particularly P due to reduced diffusion and poor uptake, in addition to restrictions in available water, with strong interactive effects on plant growth and functioning. Increased soil P in field and in-ground soil core studies increased the seed cotton yield and related morphological growth parameters in a drying soil, with hot (daily maximum temperature >33°C) and dry conditions (relative humidity, 25% to 35%), particularly during peak boll formation and filling stage. The soil water content in the effective rooting zone (top 0.4 m) decreased to -1.5 MPa by day 5 of the soil drying cycle. However, the increased seed cotton yield for the high-P plants was closely related to increasing leaf area with increased P supply. Plant height, leaf fresh mass and leaf area per plant were positively related to the leaf P%, which increased with increasing P supply. Low P plants were lower in plant height, leaf area, and leaf tissue water in the drying soil. Individual leaf area and the water content of the fresh leaf (ratio of dry mass to fresh mass) were significantly dependent on leaf P%.