Genetic variation in CaTIFY4b contributes to drought adaptation in chickpea.

Chickpea production is vulnerable to drought stress. Identifying the genetic components underlying drought adaptation is crucial for enhancing chickpea productivity. Here, we present the fine mapping and characterization of 'QTL-hotspot', a genomic region controlling chickpea growth with positive consequences on crop production under drought. We report that a non-synonymous substitution in the transcription factor CaTIFY4b regulates seed weight and organ size in chickpea. Ectopic expression of CaTIFY4b in Medicago truncatula enhances root growth under water deficit. Our results suggest that allelic variation in 'QTL-hotspot' improves pre-anthesis water use, transpiration efficiency, root architecture and canopy development, enabling high-yield performance under terminal drought conditions. Gene expression analysis indicated that CaTIFY4b may regulate organ size under water deficit by modulating the expression of GRF-INTERACTING FACTOR1 (GIF1), a transcriptional co-activator of Growth-Regulating Factors. Taken together, our study offers new insights into the role of CaTIFY4b and on diverse physiological and molecular mechanisms underpinning chickpea growth and production under specific drought scenarios.

Functional Dissection of the Chickpea (Cicer arietinum L.) Stay-Green Phenotype Associated with Molecular Variation at an Ortholog of Mendel's I Gene for Cotyledon Color: Implications for Crop Production and Carotenoid Biofortification.

"Stay-green" crop phenotypes have been shown to impact drought tolerance and nutritional content of several crops. We aimed to genetically describe and functionally dissect the particular stay-green phenomenon found in chickpeas with a green cotyledon color of mature dry seed and investigate its potential use for improvement of chickpea environmental adaptations and nutritional value. We examined 40 stay-green accessions and a set of 29 BC2F4-5 stay-green introgression lines using a stay-green donor parent ICC 16340 and two Indian elite cultivars (KAK2, JGK1) as recurrent parents. Genetic studies of segregating populations indicated that the green cotyledon trait is controlled by a single recessive gene that is invariantly associated with the delayed degreening (extended chlorophyll retention). We found that the chickpea ortholog of Mendel's I locus of garden pea, encoding a SGR protein as very likely to underlie the persistently green cotyledon color phenotype of chickpea. Further sequence characterization of this chickpea ortholog CaStGR1 (CaStGR1, for carietinum stay-green gene 1) revealed the presence of five different molecular variants (alleles), each of which is likely a loss-of-function of the chickpea protein (CaStGR1) involved in chlorophyll catabolism. We tested the wild type and green cotyledon lines for components of adaptations to dry environments and traits linked to agronomic performance in different experimental systems and different levels of water availability. We found that the plant processes linked to disrupted CaStGR1 gene did not functionality affect transpiration efficiency or water usage. Photosynthetic pigments in grains, including provitaminogenic carotenoids important for human nutrition, were 2-3-fold higher in the stay-green type. Agronomic performance did not appear to be correlated with the presence/absence of the stay-green allele. We conclude that allelic variation in chickpea CaStGR1 does not compromise traits linked to environmental adaptation and agronomic performance, and is a promising genetic technology for biofortification of provitaminogenic carotenoids in chickpea.

Plant vigour QTLs co-map with an earlier reported QTL hotspot for drought tolerance while water saving QTLs map in other regions of the chickpea genome.

BACKGROUND: Terminal drought stress leads to substantial annual yield losses in chickpea (Cicer arietinum L.). Adaptation to water limitation is a matter of matching water supply to water demand by the crop. Therefore, harnessing the genetics of traits contributing to plant water use, i.e. transpiration rate and canopy development dynamics, is important to design crop ideotypes suited to a varying range of water limited environments. With an aim of identifying genomic regions for plant vigour (growth and canopy size) and canopy conductance traits, 232 recombinant inbred lines derived from a cross between ICC 4958 and ICC 1882, were phenotyped at vegetative stage under well-watered conditions using a high throughput phenotyping platform (LeasyScan). RESULTS: Twenty one major quantitative trait loci (M-QTLs) were identified for plant vigour and canopy conductance traits using an ultra-high density bin map. Plant vigour traits had 13 M-QTLs on CaLG04, with favourable alleles from high vigour parent ICC 4958. Most of them co-mapped with a previously fine mapped major drought tolerance "QTL-hotspot" region on CaLG04. One M-QTL was found for canopy conductance on CaLG03 with the ultra-high density bin map. Comparative analysis of the QTLs found across different density genetic maps revealed that QTL size reduced considerably and % of phenotypic variation increased as marker density increased. CONCLUSION: Earlier reported drought tolerance hotspot is a vigour locus. The fact that canopy conductance traits, i.e. the other important determinant of plant water use, mapped on CaLG03 provides an opportunity to manipulate these loci to tailor recombinants having low/high transpiration rate and plant vigour, fitted to specific drought stress scenarios in chickpea.

Quantitative trait loci (QTLs) for water use and crop production traits co-locate with major QTL for tolerance to water deficit in a fine-mapping population of pearl millet (Pennisetum glaucum L. R.Br.).

KEY MESSAGE: Four genetic regions associated with water use traits, measured at different levels of plant organization, and with agronomic traits were identified within a previously reported region for terminal water deficit adaptation on linkage group 2. Close linkages between these traits showed the value of phenotyping both for agronomic and secondary traits to better understand plant productive processes. Water saving traits are critical for water stress adaptation of pearl millet, whereas maximizing water use is key to the absence of stress. This research aimed at demonstrating the close relationship between traits measured at different levels of plant organization, some putatively involved in water stress adaptation, and those responsible for agronomic performance. A fine-mapping population of pearl millet, segregating for a previously identified quantitative trait locus (QTL) for adaptation to terminal drought stress on LG02, was phenotyped for traits at different levels of plant organization in different experimental environments (pot culture, high-throughput phenotyping platform, lysimeters, and field). The linkages among traits across the experimental systems were analysed using principal component analysis and QTL co-localization approach. Four regions within the LG02-QTL were found and revealed substantial co-mapping of water use and agronomic traits. These regions, identified across experimental systems, provided genetic evidence of the tight linkages between traits phenotyped at a lower level of plant organization and agronomic traits assessed in the field, therefore deepening our understanding of complex traits and then benefiting both geneticists and breeders. In short: (1) under no/mild stress conditions, increasing biomass and tiller production increased water use and eventually yield; (2) under severe stress conditions, water savings at vegetative stage, from lower plant vigour and fewer tillers in that population, led to more water available during grain filling, expression of stay-green phenotypes, and higher yield.

Pearl Millet Aquaporin Gene PgPIP2;6 Improves Abiotic Stress Tolerance in Transgenic Tobacco.

Pearl millet [Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T2 generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops.

Pearl millet (Pennisetum glaucum) contrasting for the transpiration response to vapour pressure deficit also differ in their dependence on the symplastic and apoplastic water transport pathways.

Genotypic differences in transpiration rate responses to high vapour pressure deficit (VPD) was earlier reported. Here we tested the hypothesis that this limitation could relate to different degrees of dependence on the apoplastic (spaces between cells), and symplastic water transport pathways (through cells via aquaporin-facilitated transport), which are known to have different hydraulic conductivities. The low transpiration rate (Tr) genotype PRLT 2/89/33 either restricted its transpiration under high VPD, or was more sensitive to VPD than H77/833-2, when grown hydroponically or in soil. The slope of the transpiration response to an ascending series of VPD was lower in whole plants than in de-rooted shoots. In addition, the transpiration response of detached leaves to moderately high VPD (2.67kPa), normalised against leaves exposed to constant VPD (1.27kPa), was similar in low and high Tr genotypes. This suggested that roots hydraulics were a substantial limitation to water flow in pearl millet, especially under high VPD. The dependence on the apoplastic and symplastic water transport pathways was investigated by assessing the transpiration response of plants treated with inhibitors specific to the AQP-mediated symplastic pathway (AgNO3 and H2O2) and to the apoplastic pathway (precipitates of Cu(Fe(CN)6) or Cu(CuFe(CN)6)). When CuSO4 alone was used, Cu ions caused an inhibition of transpiration in both genotypes and more so in H77/833-2. The transpiration of high Tr H77/833-2 was decreased more by AQP inhibitors under low VPD (1.8kPa) than in PRLT 2/89/33, whereas under high VPD (4.2kPa), the transpiration of PRLT 2/89/33 was decreased more by AQP inhibitors than in H77/833-2. The transpiration rate of detached leaves from H77/833-2 when treated with AgNO3 decreased more than in PRLT 2/89/33. Although the root hydraulic conductivity of both genotypes was similar, it decreased more upon the application of a symplastic inhibitor in H77/833-2. The transpiration of low Tr PRLT 2/89/33 was decreased more by apoplastic inhibitors under both low and high VPD. Then the hydraulic conductivity decreased more upon the application of an apoplastic inhibitor in PRLT 2/89/33. In conclusion, both pathways contributed to water transport, and their contribution varied with environmental conditions and genotypes. Roots were a main source of hydraulic limitation in these genotypes of pearl millet, although a leaf limitation was not excluded. The similarity between genotypes in root hydraulic conductivity under normal conditions also suggests changes in this conductivity upon changes in the evaporative demand. The low Tr genotype depended more on the apoplastic pathway for water transport, whereas the high Tr genotype depended on both pathway, may be by 'tuning-up' the symplastic pathway under high transpiration demand, very likely via the involvement of aquaporins.

Chickpea Genotypes Contrasting for Vigor and Canopy Conductance Also Differ in Their Dependence on Different Water Transport Pathways.

Lower plant transpiration rate (TR) under high vapor pressure deficit (VPD) conditions and early plant vigor are proposed as major traits influencing the rate of crop water use and possibly the fitness of chickpea lines to specific terminal drought conditions-this being the major constraint limiting chickpea productivity. The physiological mechanisms underlying difference in TR under high VPD and vigor are still unresolved, and so is the link between vigor and TR. Lower TR is hypothesized to relate to hydraulic conductance differences. Experiments were conducted in both soil (Vertisol) and hydroponic culture. The assessment of the TR response to increasing VPD showed that high vigor genotypes had TR restriction under high VPD, and this was confirmed in the early vigor parent and progeny genotype (ICC 4958 and RIL 211) having lower TR than the late vigor parent and progeny genotype (ICC 1882 and RIL 022). Inhibition of water transport pathways [apoplast and symplast (aquaporins)] in intact plants led to a lower transpiration inhibition in the early vigor/low TR genotypes than in the late vigor/high TR genotypes. De-rooted shoot treatment with an aquaporin inhibitor led to a lower transpiration inhibition in the early vigor/low TR genotypes than in the late vigor/high TR genotypes. Early vigor genotypes had lower root hydraulic conductivity than late vigor/high TR genotypes. Under inhibited conditions (apoplast, symplast), root hydraulic conductivity was reduced more in the late vigor/high TR genotypes than in the early vigor/low TR genotypes. We interpret that early vigor/low TR genotypes have a lower involvement of aquaporins in water transport pathways and may also have a smaller apoplastic pathway than high TR genotypes, which could explain the transpiration restriction under high VPD and would be helpful to conserve soil water under high evaporative demand. These findings open an opportunity for breeding to tailor genotypes with different "dosage" of these traits toward adaptation to varying drought-prone environments.

Molecular cloning and expression analysis of Aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits.

Pearl millet is a crop of the semi-arid tropics having high degree of genetic diversity and variable tolerance to drought stress. To investigate drought tolerance mechanism that possibly accounts for differences in drought tolerance, four recombinant inbred lines from a high resolution cross (HRC) were selected for variability in their transpiration rate (Tr) response to vapour pressure deficit (VPD) conditions. The differential Tr response of the genotypes to increased VPD conditions was used to classify the genotypes as sensitive or insensitive to high VPD. Aquaporin (AQP) genes PgPIP1;1, PgPIP1;2, PgPIP2;1, PgPIP2;3, PgPIP2;6, PgTIP1;1 and PgTIP2;2 were cloned. Phylogenetic analysis revealed that the cloned PgAQPs were evolutionarily closer to maize AQPs than to rice. PgAQP genes, including PgPIP1;1 and PgPIP2;6 in root tissue showed a significant expression pattern with higher expression in VPD-insensitive genotypes than VPD-sensitive genotypes under low VPD conditions (1.2kPa) i.e when there is no high evaporative demand from the atmosphere. PgAQP genes (PgPIP2;1 in leaf and root tissues; PgPIP1;2 and PgTIP2;2 in leaf and PgPIP2;6 in root) followed a diurnal rhythm in leaves and roots that have either higher or lower expression levels at different time intervals. Under high VPD conditions (4.21kPa), PgPIP2;3 showed higher transcript abundance in VPD-insensitive genotypes, and PgPIP2;1 in VPD-sensitive genotypes, while rest of the PgAQPs showed differential expression. Our current hypothesis is that these differences in the expression of AQP genes under different VPDs suggests a role of the AQPs in tuning the water transport pathways with variation between genotypes.

Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization.

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.