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.     

Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

The WNK (With No Lysine K) serine-threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABA-responsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants.     

Arabidopsis EIN2 modulates stress response through abscisic acid response pathway

The nuclear protein ETHYLENE INSENSITIVE2 (EIN2) is a central component of the ethylene signal transduction pathway in plants, and plays an important role in mediating cross-links between several hormone response pathways, including abscisic acid (ABA). ABA mediates stress responses in plants, but there is no report on the role of EIN2 on plant response to salt and osmotic stresses. Here, we show that EIN2 gene regulates plant response to osmotic and salt stress through an ABA-dependent pathway in Arabidopsis. The expression of the EIN2 gene is down-regulated by salt and osmotic stress. An Arabidopsis EIN2 null mutant was supersensitive to both salt and osmotic stress conditions. Disruption of EIN2 specifically altered the expression pattern of stress marker gene RD29B in response to the stresses, but not the stress- or ABA-responsive genes RD29A and RD22, suggesting EIN2 modulates plant stress responses through the RD29B branch of the ABA response. Furthermore, disruption of EIN2 caused substantial increase in ABA. Lastly, our data showed that mutations of other key genes in ethylene pathway also had altered sensitivity to abiotic stresses, indicating that the intact ethylene may involve in the stress response. Taken together, the results identified EIN2 as a cross-link node in ethylene, ABA and stress signaling pathways, and EIN2 is necessary to induce developmental arrest during seed germination, and seedling establishment, as well as subsequent vegetative growth, thereby allowing the survival and growth of plants under the adverse environmental conditions. Youning Wang and Chuang Liu contributed equally to this work.     

Enhancement of abscisic acid sensitivity and reduction of water consumption in Arabidopsis by combined inactivation of the protein phosphatases type 2C ABI1 and HAB1

Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.     

HOS5–a negative regulator of osmotic stress-induced gene expression in Arabidopsis thaliana

Osmotic stress activates the expression of many plant genes through ABA-dependent as well as ABA-independent signaling pathways. We report here the characterization of a novel mutant of Arabidopsis thaliana, hos5-1, which exhibits increased expression of the osmotic stress responsive RD29A gene. The expression of several other stress genes are also enhanced by the hos5-1 mutation. The enhanced expression is specific to ABA and osmotic stress because low temperature regulation of these genes is not altered in the mutant. Genetic analysis indicated that hos5-1 is a recessive mutation in a single nuclear gene on chromosome III. Double mutant analysis of hos5-1 and the ABA-deficient aba1-1 as well as the ABA-insensitive abi1-1 mutant indicated that the osmotic stress hypersensitivity of hos5-1 is not affected by ABA deficiency or insensitivity. Furthermore, combined treatments of hos5-1 with ABA and osmotic stress had an additive effect on RD29A-LUC expression. These results suggest that the osmotic stress hypersensitivity in hos5-1 may be ABA-independent. The germination of hos5-1 seeds was more resistant to ABA. However, the hos5-1 mutation did not influence stomatal control and only slightly affected the regulation of growth and proline accumulation by ABA. The hos5-1 mutation reveals a negative regulator of osmotic stress-responsive gene expression shared by ABA-dependent and ABA-independent osmotic stress signaling pathways.     

Arabidopsis mutants with reduced response to NaCl and osmotic stress

We isolated 6 mutant lines of Arabidopsis thaliana that expressed reduced sensitivity to salt and osmotic stress during germination. All 6 lines cum recessive mutations in a single gene, designated reduced salt sensitivity (rss), linked to the ADH marker on chromosome 1. The rss mutants are less sensitive than wild type for NaCl and osmotic stress inhibition of germination, tolerating approximately 150 mM higher concentrations of NaCl and about 250 mM higher concentrations of sorbitol in the media. Germination assays on media containing various salts indicate that the rss mutations reduce sensitivity lo Na⁺ and Rh⁺ but also, to a much lesser degree, to K⁺ and Cs^s+. However, the rss mutation does not improve plant growth when plantlets are transferred to high salt or high osmotic pressure media after germination. The rss plantlets accumulate praline to a significantly lesser degree than wild type when they are exposed to either salt or osmotic stress. Thus, the rss mutants differ from wild type both at germination and during vegetative growth indicating that the rss mutations are pleiotropic and might affect perception of solutes or some aspect of stress-induced signaling. The rss mutations do not alter ABA sensitivity and therefore probably do not affect ABA-mediated signaling.     

Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.