Defective cytokinin signaling reprograms lipid and flavonoid gene-to-metabolite networks to mitigate high salinity in Arabidopsis.

Cytokinin (CK) in plants regulates both developmental processes and adaptation to environmental stresses. Arabidopsis histidine phosphotransfer ahp2,3,5 and type-B Arabidopsis response regulator arr1,10,12 triple mutants are almost completely defective in CK signaling, and the ahp2,3,5 mutant was reported to be salt tolerant. Here, we demonstrate that the arr1,10,12 mutant is also more tolerant to salt stress than wild-type (WT) plants. A comprehensive metabolite profiling coupled with transcriptome analysis of the ahp2,3,5 and arr1,10,12 mutants was conducted to elucidate the salt tolerance mechanisms mediated by CK signaling. Numerous primary (e.g., sugars, amino acids, and lipids) and secondary (e.g., flavonoids and sterols) metabolites accumulated in these mutants under nonsaline and saline conditions, suggesting that both prestress and poststress accumulations of stress-related metabolites contribute to improved salt tolerance in CK-signaling mutants. Specifically, the levels of sugars (e.g., trehalose and galactinol), amino acids (e.g., branched-chain amino acids and γ-aminobutyric acid), anthocyanins, sterols, and unsaturated triacylglycerols were higher in the mutant plants than in WT plants. Notably, the reprograming of flavonoid and lipid pools was highly coordinated and concomitant with the changes in transcriptional levels, indicating that these metabolic pathways are transcriptionally regulated by CK signaling. The discovery of the regulatory role of CK signaling on membrane lipid reprogramming provides a greater understanding of CK-mediated salt tolerance in plants. This knowledge will contribute to the development of salt-tolerant crops with the ability to withstand salinity as a key driver to ensure global food security in the era of climate crisis.

Differential metabolic rearrangements in the roots and leaves of Cicer arietinum caused by single or double nitrate and/or phosphate deficiencies.

Nitrate (NO3 - ) and phosphate (Pi) deficiencies are the major constraints for chickpea productivity, significantly impacting global food security. However, excessive fertilization is expensive and can also lead to environmental pollution. Therefore, there is an urgent need to develop chickpea cultivars that are able to grow on soils deficient in both NO3 - and Pi. This study focused on the identification of key NO3 - and/or Pi starvation-responsive metabolic pathways in the leaves and roots of chickpea grown under single and double nutrient deficiencies of NO3 - and Pi, in comparison with nutrient-sufficient conditions. A global metabolite analysis revealed organ-specific differences in the metabolic adaptation to nutrient deficiencies. Moreover, we found stronger adaptive responses in the roots and leaves to any single than combined nutrient-deficient stresses. For example, chickpea enhanced the allocation of carbon among nitrogen-rich amino acids (AAs) and increased the production of organic acids in roots under NO3 - deficiency, whereas this adaptive response was not found under double nutrient deficiency. Nitrogen remobilization through the transport of AAs from leaves to roots was greater under NO3 - deficiency than double nutrient deficiency conditions. Glucose-6-phosphate and fructose-6-phosphate accumulated in the roots under single nutrient deficiencies, but not under double nutrient deficiency, and higher glycolytic pathway activities were observed in both roots and leaves under single nutrient deficiency than double nutrient deficiency. Hence, the simultaneous deficiency generated a unique profile of metabolic changes that could not be simply described as the result of the combined deficiencies of the two nutrients.

The histidine phosphotransfer AHP4 plays a negative role in Arabidopsis plant response to drought.

Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to affect drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss-of-function approach we showed that AHP4 possesses an important role in the response of Arabidopsis to drought. This is evidenced by the higher survival rates of ahp4 than wild-type (WT) plants under drought conditions, which is accompanied by the downregulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4-mediated expression of several dehydration- and/or abscisic acid-responsive genes involved in modulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous abscisic acid at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. In addition, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species, reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to upregulation of genes that encode enzymes involved in reactive oxygen species scavenging and non-enzymatic antioxidant metabolism. Overall, our findings suggest that AHP4 plays a crucial role in plant drought adaptation.

Karrikin Receptor KAI2 Coordinates Salt Tolerance Mechanisms in Arabidopsis thaliana.

Plants activate a myriad of signaling cascades to tailor adaptive responses under environmental stresses, such as salinity. While the roles of exogenous karrikins (KARs) in salt stress mitigation are well comprehended, genetic evidence of KAR signaling during salinity responses in plants remains unresolved. Here, we explore the functions of the possible KAR receptor KARRIKIN-INSENSITIVE2 (KAI2) in Arabidopsis thaliana tolerance to salt stress by investigating comparative responses of wild-type (WT) and kai2-mutant plants under a gradient of NaCl. Defects in KAI2 functions resulted in delayed and inhibited cotyledon opening in kai2 seeds compared with WT seeds, suggesting that KAI2 played an important role in enhancing seed germination under salinity. Salt-stressed kai2 plants displayed more phenotypic aberrations, biomass reduction, water loss and oxidative damage than WT plants. kai2 shoots accumulated significantly more Na+ and thus had a lower K+/Na+ ratio, than WT, indicating severe ion toxicity in salt-stressed kai2 plants. Accordingly, kai2 plants displayed a lower expression of genes associated with Na+ homeostasis, such as SALT OVERLY SENSITIVE (SOS) 1, SOS2, HIGH-AFFINITY POTASSIUM TRANSPORTER 1;1 (HKT1;1) and CATION-HYDROGEN EXCHANGER 1 (NHX1) than WT plants. WT plants maintained a better glutathione level, glutathione-related redox status and antioxidant enzyme activities relative to kai2 plants, implying KAI2's function in oxidative stress mitigation in response to salinity. kai2 shoots had lower expression levels of genes involved in the biosynthesis of strigolactones (SLs), salicylic acid and jasmonic acid and the signaling of abscisic acid and SLs than those of WT plants, indicating interactive functions of KAI2 signaling with other hormone signaling in modulating plant responses to salinity. Collectively, these results underpin the likely roles of KAI2 in the alleviation of salinity effects in plants by regulating several physiological and biochemical mechanisms involved in ionic and osmotic balance, oxidative stress tolerance and hormonal crosstalk.

SUPPRESSOR of MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2) Negatively Regulate Drought Resistance in Arabidopsis thaliana.

Recent investigations in Arabidopsis thaliana suggest that SUPPRESSOR of MORE AXILLARY GROWTH 2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2) are negative regulators of karrikin (KAR) and strigolactone (SL) signaling during plant growth and development, but their functions in drought resistance and related mechanisms of action remain unclear. To understand the roles and mechanisms of SMAX1 and SMXL2 in drought resistance, we investigated the drought-resistance phenotypes and transcriptome profiles of smax1 smxl2 (s1,2) double-mutant plants in response to drought stress. The s1,2 mutant plants showed enhanced drought-resistance and lower leaf water loss when compared with wild-type (WT) plants. Transcriptome comparison of rosette leaves from the s1,2 mutant and the WT under normal and dehydration conditions suggested that the mechanism related to cuticle formation was involved in drought resistance. This possibility was supported by enhanced cuticle formation in the rosette leaves of the s1,2 mutant. We also found that the s1,2 mutant plants were more sensitive to abscisic acid in assays of stomatal closure, cotyledon opening, chlorophyll degradation and growth inhibition, and they showed a higher reactive oxygen species detoxification capacity than WT plants. In addition, the s1,2 mutant plants had longer root hairs and a higher root-to-shoot ratio than the WT plants, suggesting that the mutant had a greater capacity for water absorption than the WT. Taken together, our results indicate that SMAX1 and SMXL2 negatively regulate drought resistance, and disruption of these KAR- and SL-signaling-related genes may therefore provide a novel means for improving crop drought resistance.

KARRIKIN UPREGULATED F-BOX 1 negatively regulates drought tolerance in Arabidopsis.

The karrikin (KAR) receptor and several related signaling components have been identified by forward genetic screening, but only a few studies have reported on upstream and downstream KAR signaling components and their roles in drought tolerance. Here, we characterized the functions of KAR UPREGULATED F-BOX 1 (KUF1) in drought tolerance using a reverse genetics approach in Arabidopsis (Arabidopsis thaliana). We observed that kuf1 mutant plants were more tolerant to drought stress than wild-type (WT) plants. To clarify the mechanisms by which KUF1 negatively regulates drought tolerance, we performed physiological, transcriptome, and morphological analyses. We found that kuf1 plants limited leaf water loss by reducing stomatal aperture and cuticular permeability. In addition, kuf1 plants showed increased sensitivity of stomatal closure, seed germination, primary root growth, and leaf senescence to abscisic acid (ABA). Genome-wide transcriptome comparisons of kuf1 and WT rosette leaves before and after dehydration showed that the differences in various drought tolerance-related traits were accompanied by differences in the expression of genes associated with stomatal closure (e.g. OPEN STOMATA 1), lipid and fatty acid metabolism (e.g. WAX ESTER SYNTHASE), and ABA responsiveness (e.g. ABA-RESPONSIVE ELEMENT 3). The kuf1 mutant plants had higher root/shoot ratios and root hair densities than WT plants, suggesting that they could absorb more water than WT plants. Together, these results demonstrate that KUF1 negatively regulates drought tolerance by modulating various physiological traits, morphological adjustments, and ABA responses and that the genetic manipulation of KUF1 in crops is a potential means of enhancing their drought tolerance.

Comparative functional analyses of DWARF14 and KARRIKIN INSENSITIVE 2 in drought adaptation of Arabidopsis thaliana.

Functional analyses of various strigolactone-deficient mutants have demonstrated that strigolactones enhance drought resistance; however, the mechanistic involvement of the strigolactone receptor DWARF14 (D14) in this trait remains elusive. In this study, loss-of-function analysis of the D14 gene in Arabidopsis thaliana revealed that d14 mutant plants were more drought-susceptible than wild-type plants, which was associated with their larger stomatal aperture, slower abscisic acid (ABA)-mediated stomatal closure, lower anthocyanin content and delayed senescence under drought stress. Transcriptome analysis revealed a consistent alteration in the expression levels of many genes related to the observed physiological and biochemical changes in d14 plants when compared with the wild type under normal and dehydration conditions. A comparative drought resistance assay confirmed that D14 plays a less critical role in Arabidopsis drought resistance than its paralog karrikin receptor KARRIKIN INSENSITIVE 2 (KAI2). In-depth comparative analyses of the single mutants d14 and kai2 and the double mutant d14 kai2, in relation to various drought resistance-associated mechanisms, revealed that D14 and KAI2 exhibited a similar effect on stomatal closure. On the other hand, D14 had a lesser role in the maintenance of cell membrane integrity, leaf cuticle structure and ABA-induced leaf senescence, but a greater role in drought-induced anthocyanin biosynthesis, than KAI2. Interestingly, a possible additive relationship between D14 and KAI2 could be observed in regulating cell membrane integrity and leaf cuticle development. In addition, our findings also suggest the existence of a complex interaction between the D14 and ABA signaling pathways in the adaptation of Arabidopsis to drought.

Phosphate or nitrate imbalance induces stronger molecular responses than combined nutrient deprivation in roots and leaves of chickpea plants.

The negative effects of phosphate (Pi) and/or nitrate (NO3- ) fertilizers on the environment have raised an urgent need to develop crop varieties with higher Pi and/or nitrogen use efficiencies for cultivation in low-fertility soils. Achieving this goal depends upon research that focuses on the identification of genes involved in plant responses to Pi and/or NO3- starvation. Although plant responses to individual deficiency in either Pi (-Pi/+NO3- ) or NO3- (+Pi/-NO3- ) have been separately studied, our understanding of plant responses to combined Pi and NO3- deficiency (-Pi/-NO3- ) is still very limited. Using RNA-sequencing approach, transcriptome changes in the roots and leaves of chickpea cultivated under -Pi/+NO3- , +Pi/-NO3- or -Pi/-NO3- conditions were investigated in a comparative manner. -Pi/-NO3- treatment displayed lesser effect on expression changes of genes related to Pi or NO3- transport, signalling networks, lipid remodelling, nitrogen and Pi scavenging/remobilization/recycling, carbon metabolism and hormone metabolism than -Pi/+NO3- or +Pi/-NO3- treatments. Therefore, the plant response to -Pi/-NO3- is not simply an additive result of plant responses to -Pi/+NO3- and +Pi/-NO3- treatments. Our results indicate that nutrient imbalance is a stronger stimulus for molecular reprogramming than an overall deficiency.

[THE STATUS OF ADMISSION TO FACILITIES FOR CHILDREN WITH FOOD ALLERGIES].

BACKGROUND: To clarify the status of admission to facilities for food allergy (FA) children. METHODS: Guardians of FA children who underwent oral food challenges at Sagamihara National Hospital from September to December 2018 were enrolled. We surveyed the experience of refusal to enter facilities, the reason for refusal and so on using a self-administered questionnaire. RESULTS: We distributed a questionnaire to 205 guardians, of which 168 responded (response rate 82%). The median age (range) at the time of the survey was 4.5 (0 to 12) years old, 2 (1 to 11) food items had been removed at the time of admission, and 56 (33%) had a history of anaphylaxis before admission. Twenty-nine patients (17%) were prescribed an adrenaline auto injector. Twenty patients (12%) had been denied entry, the median number of refusals (range) was 1.5 (1 to 30). History of anaphylaxis before admission (odds ratio 2.80 [1.08-7.22]) and having 5 or more causative foods (odds ratio 3.44 [1.27-9.32]) were associated with admission refusal. 〔' Factors related to children with FA〕, 〔Factors related to facilities〕, and 〔Factors related to facility staff〕 were extracted as the reasons for refusal. CONCLUSIONS: In addition to the factors related to children with FA, the factors related to facilities and facility staff were related to admission refusal. Therefore, cooperation between medical care, local governments, and facility that comprehensively supports the living environment of children with FA is needed.

The karrikin receptor KAI2 promotes drought resistance in Arabidopsis thaliana.

Drought causes substantial reductions in crop yields worldwide. Therefore, we set out to identify new chemical and genetic factors that regulate drought resistance in Arabidopsis thaliana. Karrikins (KARs) are a class of butenolide compounds found in smoke that promote seed germination, and have been reported to improve seedling vigor under stressful growth conditions. Here, we discovered that mutations in KARRIKIN INSENSITIVE2 (KAI2), encoding the proposed karrikin receptor, result in hypersensitivity to water deprivation. We performed transcriptomic, physiological and biochemical analyses of kai2 plants to understand the basis for KAI2-regulated drought resistance. We found that kai2 mutants have increased rates of water loss and drought-induced cell membrane damage, enlarged stomatal apertures, and higher cuticular permeability. In addition, kai2 plants have reduced anthocyanin biosynthesis during drought, and are hyposensitive to abscisic acid (ABA) in stomatal closure and cotyledon opening assays. We identified genes that are likely associated with the observed physiological and biochemical changes through a genome-wide transcriptome analysis of kai2 under both well-watered and dehydration conditions. These data provide evidence for crosstalk between ABA- and KAI2-dependent signaling pathways in regulating plant responses to drought. A comparison of the strigolactone receptor mutant d14 (DWARF14) to kai2 indicated that strigolactones also contributes to plant drought adaptation, although not by affecting cuticle development. Our findings suggest that chemical or genetic manipulation of KAI2 and D14 signaling may provide novel ways to improve drought resistance.

Crosstalk between the cytokinin and MAX2 signaling pathways in growth and callus formation of Arabidopsis thaliana.

Cytokinin (CK) signaling has been shown to play important roles in callus formation and various developmental processes by analyzing different CK-responsive mutants, including the ahk2 ahk3 (AHK, Arabidopsis histidine kinase) double mutant. Recently, an F-box protein, called MAX2 (more axillary growth 2) was identified as a key component regulating many growth and developmental processes through the strigolactone and/or karrikin pathways. However, the function of MAX2 signaling in callus formation, seed size and yield, as well as the effects of its crosstalk with CK signaling on plant growth and development remain elusive. Here, we constructed the triple mutant ahk2 ahk3max2 and analyzed the callus formation and various phenotypic traits of all three max2, ahk2 ahk3 and ahk2 ahk3 max2 mutants along with wild-type (WT) during plant growth and development. We showed that MAX2 acted as a negative regulator of seed size, but positive regulator of callus formation and seed yield albeit at lower degree, as the CK receptor kinases. Importantly, our comparative analyses revealed interactive effects of CK and MAX2 pathways on primary root growth, hypocotyl elongation and shoot branching. However, these two pathways might independently regulate root hair growth, leaf development, leaf senescence, plant height, seed size, seed yield and callus formation. Our findings provide not only evidence for the involvement of MAX2 in regulating callus formation, seed size and seed yield, but also a better understanding of the relationship between CK and MAX2 signaling pathways in many key developmental processes across a plant's life.

Adaptation of the symbiotic Mesorhizobium-chickpea relationship to phosphate deficiency relies on reprogramming of whole-plant metabolism.

Low inorganic phosphate (Pi) availability is a major constraint for efficient nitrogen fixation in legumes, including chickpea. To elucidate the mechanisms involved in nodule acclimation to low Pi availability, two Mesorhizobium-chickpea associations exhibiting differential symbiotic performances, Mesorhizobium ciceri CP-31 (McCP-31)-chickpea and Mesorhizobium mediterranum SWRI9 (MmSWRI9)-chickpea, were comprehensively studied under both control and low Pi conditions. MmSWRI9-chickpea showed a lower symbiotic efficiency under low Pi availability than McCP-31-chickpea as evidenced by reduced growth parameters and down-regulation of nifD and nifK These differences can be attributed to decline in Pi level in MmSWRI9-induced nodules under low Pi stress, which coincided with up-regulation of several key Pi starvation-responsive genes, and accumulation of asparagine in nodules and the levels of identified amino acids in Pi-deficient leaves of MmSWRI9-inoculated plants exceeding the shoot nitrogen requirement during Pi starvation, indicative of nitrogen feedback inhibition. Conversely, Pi levels increased in nodules of Pi-stressed McCP-31-inoculated plants, because these plants evolved various metabolic and biochemical strategies to maintain nodular Pi homeostasis under Pi deficiency. These adaptations involve the activation of alternative pathways of carbon metabolism, enhanced production and exudation of organic acids from roots into the rhizosphere, and the ability to protect nodule metabolism against Pi deficiency-induced oxidative stress. Collectively, the adaptation of symbiotic efficiency under Pi deficiency resulted from highly coordinated processes with an extensive reprogramming of whole-plant metabolism. The findings of this study will enable us to design effective breeding and genetic engineering strategies to enhance symbiotic efficiency in legume crops.

Arabidopsis type B cytokinin response regulators ARR1, ARR10, and ARR12 negatively regulate plant responses to drought.

In this study, we used a loss-of-function approach to elucidate the functions of three Arabidopsis type B response regulators (ARRs)--namely ARR1, ARR10, and ARR12--in regulating the Arabidopsis plant responses to drought. The arr1,10,12 triple mutant showed a significant increase in drought tolerance versus WT plants, as indicated by its higher relative water content and survival rate on drying soil. This enhanced drought tolerance of arr1,10,12 plants can be attributed to enhanced cell membrane integrity, increased anthocyanin biosynthesis, abscisic acid (ABA) hypersensitivity, and reduced stomatal aperture, but not to altered stomatal density. Further drought-tolerance tests of lower-order double and single mutants indicated that ARR1, ARR10, and ARR12 negatively and redundantly control plant responses to drought, with ARR1 appearing to bear the most critical function among the three proteins. In agreement with these findings, a comparative genome-wide analysis of the leaves of arr1,10,12 and WT plants under both normal and dehydration conditions suggested a cytokinin (CK) signaling-mediated network controlling plant adaptation to drought via many dehydration/drought- and/or ABA-responsive genes that can provide osmotic adjustment and protection to cellular and membrane structures. Expression of all three ARR genes was repressed by dehydration and ABA treatments, inferring that plants down-regulate these genes as an adaptive mechanism to survive drought. Collectively, our results demonstrate that repression of CK response, and thus CK signaling, is one of the strategies plants use to cope with water deficit, providing novel insight for the design of drought-tolerant plants by genetic engineering.

The Soybean GmNAC019 Transcription Factor Mediates Drought Tolerance in Arabidopsis in an Abscisic Acid-Dependent Manner.

Being master regulators of gene expression, transcription factors (TFs) play important roles in determining plant growth, development and reproduction. To date, many TFs have been shown to positively mediate plant responses to environmental stresses. In the current study, the biological functions of a stress-responsive NAC [NAM (No Apical Meristem), ATAF1/2 (Arabidopsis Transcription Activation Factor1/2), CUC2 (Cup-shaped Cotyledon2)]-TF encoding gene isolated from soybean (GmNAC019) in relation to plant drought tolerance and abscisic acid (ABA) responses were investigated. By using a heterologous transgenic system, we revealed that transgenic Arabidopsis plants constitutively expressing the GmNAC019 gene exhibited higher survival rates in a soil-drying assay, which was associated with lower water loss rate in detached leaves, lower cellular hydrogen peroxide content and stronger antioxidant defense under water-stressed conditions. Additionally, the exogenous treatment of transgenic plants with ABA showed their hypersensitivity to this phytohormone, exhibiting lower rates of seed germination and green cotyledons. Taken together, these findings demonstrated that GmNAC019 functions as a positive regulator of ABA-mediated plant response to drought, and thus, it has potential utility for improving plant tolerance through molecular biotechnology.

Comparative transcriptome analysis of nodules of two Mesorhizobium-chickpea associations with differential symbiotic efficiency under phosphate deficiency.

Phosphate (Pi) deficiency is known to be a major limitation for symbiotic nitrogen fixation (SNF), and hence legume crop productivity globally. However, very little information is available on the adaptive mechanisms, particularly in the important legume crop chickpea (Cicer arietinum L.), which enable nodules to respond to low-Pi availability. Thus, to elucidate these mechanisms in chickpea nodules at molecular level, we used an RNA sequencing approach to investigate transcriptomes of the nodules in Mesorhizobium mediterraneum SWRI9-(MmSWRI9)-chickpea and M. ciceri CP-31-(McCP-31)-chickpea associations under Pi-sufficient and Pi-deficient conditions, of which the McCP-31-chickpea association has a better SNF capacity than the MmSWRI9-chickpea association during Pi starvation. Our investigation revealed that more genes showed altered expression patterns in MmSWRI9-induced nodules than in McCP-31-induced nodules (540 vs. 225) under Pi deficiency, suggesting that the Pi-starvation-more-sensitive MmSWRI9-induced nodules required expression change in a larger number of genes to cope with low-Pi stress than the Pi-starvation-less-sensitive McCP-31-induced nodules. The functional classification of differentially expressed genes (DEGs) was examined to gain an understanding of how chickpea nodules respond to Pi starvation, caused by soil Pi deficiency. As a result, more DEGs involved in nodulation, detoxification, nutrient/ion transport, transcriptional factors, key metabolic pathways, Pi remobilization and signalling were found in Pi-starved MmSWRI9-induced nodules than in Pi-starved McCP-31-induced nodules. Our findings have enabled the identification of molecular processes that play important roles in the acclimation of nodules to Pi deficiency, ultimately leading to the development of Pi-efficient chickpea symbiotic associations suitable for Pi-deficient soils.

Identification, Structural Characterization and Gene Expression Analysis of Members of the Nuclear Factor-Y Family in Chickpea (Cicer arietinum L.) under Dehydration and Abscisic Acid Treatments.

In plants, the Nuclear Factor-Y (NF-Y) transcription factors (TFs), which include three distinct types of NF-YA, NF-YB, and NF-YC TFs, have been identified to play key roles in the regulation of various plant growth and developmental processes under both normal and environmental stress conditions. In this work, a total of 40 CaNF-Y-encoding genes, including eight CaNF-YAs, 21 CaNF-YBs, and 11 CaNF-YCs, were identified in chickpea, and their major gene and protein characteristics were subsequently obtained using various web-based tools. Of our interest, a phylogenetically-based analysis predicted 18 CaNF-Ys (eight CaNF-YAs, seven CaNF-YBs, and three CaNF-YCs) that potentially play roles in chickpea responses to dehydration according to their close relationship with the well-characterized GmNF-Ys in soybean. These results were in good agreement with the enrichment of drought-responsive cis-regulatory motifs and expression patterns obtained from in silico analyses using publically available transcriptome data. Most of the phylogenetically predicted drought-responsive CaNF-Y genes (15 of 18) were quantitatively validated to significantly respond to dehydration treatment in leaves and/or roots, further supporting the results of in silico analyses. Among these CaNF-Y genes, the transcript levels of CaNF-YA01 and CaNF-YC10 were the most highly accumulated in leaves (by approximately eight-fold) and roots (by approximately 18-fold), respectively, by dehydration. Furthermore, 12 of the 18 CaNF-Y genes were found to be responsive to the most well-known stress hormone, namely abscisic acid (ABA), in leaves and/or roots, suggesting that these genes may act in chickpea response to dehydration in ABA-dependent manner. Taken together, our study has provided a comprehensive and fundamental information for further functional analyses of selected CaNF-Y candidate genes, ultimately leading to the improvement of chickpea growth under water-limited conditions.

Positive regulatory role of strigolactone in plant responses to drought and salt stress.

This report provides direct evidence that strigolactone (SL) positively regulates drought and high salinity responses in Arabidopsis. Both SL-deficient and SL-response [more axillary growth (max)] mutants exhibited hypersensitivity to drought and salt stress, which was associated with shoot- rather than root-related traits. Exogenous SL treatment rescued the drought-sensitive phenotype of the SL-deficient mutants but not of the SL-response mutant, and enhanced drought tolerance of WT plants, confirming the role of SL as a positive regulator in stress response. In agreement with the drought-sensitive phenotype, max mutants exhibited increased leaf stomatal density relative to WT and slower abscisic acid (ABA)-induced stomatal closure. Compared with WT, the max mutants exhibited increased leaf water loss rate during dehydration and decreased ABA responsiveness during germination and postgermination. Collectively, these results indicate that cross-talk between SL and ABA plays an important role in integrating stress signals to regulate stomatal development and function. Additionally, a comparative microarray analysis of the leaves of the SL-response max2 mutant and WT plants under normal and dehydrative conditions revealed an SL-mediated network controlling plant responses to stress via many stress- and/or ABA-responsive and cytokinin metabolism-related genes. Our results demonstrate that plants integrate multiple hormone-response pathways for adaptation to environmental stress. Based on our results, genetic modulation of SL content/response could be applied as a potential approach to reduce the negative impact of abiotic stress on crop productivity.

OaMAX2 of Orobanche aegyptiaca and Arabidopsis AtMAX2 share conserved functions in both development and drought responses.

Previous studies in Arabidopsis reported that the MAX2 (more axillary growth 2) gene is a component of the strigolactone (SL) signaling pathway, which regulates a wide range of biological processes, from plant growth and development to environmental stress responses. Orobanche aegyptiaca is a harmful parasitic plant for many economically important crops. Seed germination of O. aegyptiaca is very sensitive to SLs, suggesting that O. aegyptiaca may contain components of the SL signaling pathway. To investigate this hypothesis, we identified and cloned a MAX2 ortholog from O. aegyptiaca for complementation analyses using the Arabidopsis Atmax2 mutant. The so-called OaMAX2 gene could rescue phenotypes of the Atmax2 mutant in various tested developmental aspects, including seed germination, shoot branching, leaf senescence and growth and development of hypocotyl, root hair, primary root and lateral root. More importantly, OaMAX2 could enhance the drought tolerance of Atmax2 mutant, suggesting its ability to restore the drought-tolerant phenotype of mutant plants defected in AtMAX2 function. Thus, this study provides genetic evidence that the functions of the MAX2 orthologs, and perhaps the MAX2 signaling pathways, are conserved in parasitic and non-parasitic plants. Furthermore, the results of our study enable us to develop a strategy to fight against parasitic plants by suppressing the MAX signaling, which ultimately leads to enhanced productivity of crop plants.

Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response.

Cytokinin is an essential phytohormone controlling various biological processes, including environmental stress responses. In Arabidopsis, although the cytokinin (CK)-related phosphorelay--consisting of three histidine kinases, five histidine phosphotransfer proteins (AHPs), and a number of response regulators--has been known to be important for stress responses, the AHPs required for CK signaling during drought stress remain elusive. Here, we report that three Arabidopsis AHPs, namely AHP2, AHP3, and AHP5, control responses to drought stress in negative and redundant manner. Loss of function of these three AHP genes resulted in a strong drought-tolerant phenotype that was associated with the stimulation of protective mechanisms. Specifically, cell membrane integrity was improved as well as an increased sensitivity to abscisic acid (ABA) was observed rather than an alteration in ABA-mediated stomatal closure and density. Consistent with their negative regulatory functions, all three AHP genes' expression was down-regulated by dehydration, which most likely resulted from a stress-induced reduction of endogenous CK levels. Furthermore, global transcriptional analysis of ahp2,3,5 leaves revealed down-regulation of many well-known stress- and/or ABA-responsive genes, suggesting that these three AHPs may control drought response in both ABA-dependent and ABA-independent manners. The discovery of mechanisms of activation and the targets of the downstream components of CK signaling involved in stress responses is an important and challenging goal for the study of plant stress regulatory network responses and plant growth. The knowledge gained from this study also has broad potential for biotechnological applications to increase abiotic stress tolerance in plants.

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis.

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.