Stress physiology functions of the Arabidopsis histidine kinase cytokinin receptors

Cytokinin signaling has complex effects on abiotic stress responses that remain to be fully elucidated. The Arabidopsis histidine kinases (AHKs), AHK2, AHK3 and CRE1 (cytokinin response1/AHK4) are the principle cytokinin receptors of Arabidopsis. Using a set of ahk mutants, we found dramatic differences in response to low water potential and salt stress among the AHKs. ahk3‐3 mutants had increased root elongation after transfer to low water potential media. Conversely ahk2‐2 was hypersensitive to salt stress in terms of root growth and fresh weight and accumulated higher than wild‐type levels of proline specifically under salt stress. Strongly reduced proline accumulation in ahk double mutants after low water potential treatment indicated a more general role of cytokinin signaling in proline metabolism. Reduced P5CS1 (Δ¹‐pyrroline‐5‐carboxylate synthetase1) gene expression may have contributed to this reduced proline accumulation. Low water potential phenotypes of ahk mutants were not caused by altered abscisic acid (ABA) accumulation as all ahk mutants had wild‐type ABA levels, despite the observation that ahk double mutants had reduced NCED3 (9‐cis‐epoxycartenoid dioxygenase3) expression when exposed to low water potential. No difference in osmoregulatory solute accumulation was detected in any of the ahk mutants indicating that they do not affect drought responsive osmotic adjustment. Overall, our examination of ahk mutants found specific phenotypes associated with AHK2 and AHK3 as well as a general function of cytokinin signaling in proline accumulation and low water potential induction of P5CS1 and NCED3 expression. These results show the stress physiology function of AHKs at a new level of detail.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

A maize calcineurin B‐like interacting protein kinase ZmCIPK42 confers salt stress tolerance

Calcineurin B‐like (CBL) and CBL‐interacting protein kinase (CIPK) play a crucial role in biotic and abiotic stress responses. However, the roles of different CIPKs in biotic and abiotic stress responses are less well characterized. In this study, we identified a mutation leading to an early protein termination of the maize CIPK gene ZmCIPK42 that undergoes a G to A mutation at the coding region via searching for genes involved in salt stress tolerance and ion homeostasis from maize with querying the EMS mutant library of maize B73. The mutant zmcipk42 plants have less branched tassel and impaired salt stress tolerance at the seedling stage. Quantitative real‐time PCR analysis revealed that ZmCIPK42was expressed in diverse tissues and was induced by NaCl stress. A yeast two‐hybrid screen identified a proteinase inhibitor (ZmMPI) as well as calcineurin B‐like protein 1 and protein 4 (ZmCBL1, ZmCBL4) as interaction partners of ZmCIPK42. These interactions were further confirmed by bimolecular fluorescence complementation in plant cells. Moreover, over‐expressing ZmCIPK42 resulted in enhanced tolerance to high salinity in both maize and Arabidopsis. These findings suggest that ZmCIPK42 is a positive regulator of salt stress tolerance and is a promising candidate gene to improve salt stress tolerance in maize through genetic manipulation.     

ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions

In maize (Zea mays), the mitogen‐activated protein kinase ZmMPK5 has been shown to be involved in abscisic acid (ABA)‐induced antioxidant defence and to enhance the tolerance of plants to drought, salt stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, using ZmMPK5 as bait in yeast two‐hybrid screening, a protein interacting with ZmMPK5 named ZmABA2, which belongs to a member of the short‐chain dehydrogenase/reductase family, was identified. Pull‐down assay and bimolecular fluorescence complementation analysis and co‐immunoprecipitation test confirmed that ZmMPK5 interacts with ZmABA2 in vitro and in vivo. Phosphorylation of Ser173 in ZmABA2 by ZmMPK5 was shown to increase the activity of ZmABA2 and the protein stability. Various abiotic stimuli induced the expression of ZmABA2 in leaves of maize plants. Pharmacological, biochemical and molecular biology and genetic analyses showed that both ZmMPK5 and ZmABA2 coordinately regulate the content of ABA. Overexpression of ZmABA2 in tobacco plants was found to elevate the content of ABA, regulate seed germination and root growth under drought and salt stress and enhance the tolerance of tobacco plants to drought and salt stress. These results suggest that ZmABA2 is a direct target of ZmMPK5 and is involved in ABA biosynthesis and functions.     

Over-expression of the water and salt stress-regulated Asr1 gene confers an increased salt tolerance

ASR1 is a plant‐specific, highly charged, low molecular weight polypeptide. Purified ASR1 was shown to posses sequence specific Zn²⁺‐dependent DNA binding activity (Kalifa et al. Biochemical Journal 381, 373–378, 2004). Steady‐state levels of tomato Asr1 mRNA and protein are transiently increased following exposure of plants to polyethylene glycol, NaCl or abscisic acid. The biological role of ASR1 could not be deduced from sequence analyses or sequence homologies. Tobacco plants over‐expressing tomato ASR1 have a decreased rate of water loss and improved salt tolerance. Upon exposure to salt, ASR1‐over‐expressing plants accumulate less Na⁺ and proline than wild‐type plants, and also results in increased steady‐state levels of other gene products under non‐stressed plant growth conditions. Therefore, ASR1 is probably involved in the regulation of water‐ or salt‐stress‐modulated gene expression.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Nitric Oxide Enhances Salt Tolerance in Tomato Seedlings by Regulating Endogenous S-nitrosylation Levels

Salinity impairs plant growth and development, thereby leading to low yield and inferior quality of crops. Nitric oxide (NO) has emerged as an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. In this study, tomato seedlings of Lycopersicum esculentum L. “Micro-Tom” treated with 150 mM sodium chloride (NaCl) conducted decreased plant height, total root length, and leaf area by 25.43%, 24.87%, and 33.67%, respectively. While nitrosoglutathione (GSNO) pretreatment ameliorated salt toxicity in a dose-dependent manner and 10 µM GSNO exhibited the most significant mitigation effect. It increased the plant height, total root length, and leaf area of tomato seedlings, which was 31.44%, 20.56%, and 51.21% higher than NaCl treatment alone, respectively. However, NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO) treatment reversed the positive effect of NO under salt stress, implying that NO is essential for the enhancement of salt tolerance. Additionally, NaCl?+?GSNO treatment effectively decreased O^2? production and H₂O₂ content, increased the levels of soluble sugar, glycinebetaine, proline, and chlorophyll, and enhanced the activities of antioxidant enzymes and the content of antioxidants in tomato seedlings in comparison with NaCl treatment, whereas NaCl?+?cPTIO treatment significantly reversed the effect of NO under salt stress. Moreover, we found that GSNO treatment increased endogenous NO content, S-nitrosoglutathione reductase (GSNOR) activity, GSNOR expression and total S-nitrosylated level, and decreased S-nitrosothiol (SNO) content under salt stress, implicating that S-nitrosylation might be involved in NO-enhanced salt tolerance in tomatoes. Altogether, these results suggest that NO confers salt tolerance in tomato seedlings probably by the promotion of photosynthesis and osmotic balance, the enhancement of antioxidant capability and the increase of protein S-nitrosylation levels.

     

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

     

Salt hypersensitivity is associated with excessive xylem development in a thermospermine‐deficient mutant of Arabidopsis thaliana

In Arabidopsis, spermine is produced in most tissues and has been implicated in stress response, while its structural isomer thermospermine is only in xylem precursor cells. Studies on acaulis5 (acl5), a mutant defective in the biosynthesis of thermospermine, have revealed that thermospermine plays a repressive role in xylem development through enhancement of mRNA translation of the SAC51 family. In contrast, the pao5 mutant defective in the degradation of thermospermine has high levels of thermospermine and shows increased salt tolerance, suggesting a role of thermospermine in salt stress response. Here we compared acl5 with a mutant of spermine synthase, spms, in terms of abiotic stress tolerance and found that acl5 was much more sensitive to sodium than the wild‐type and spms. A double‐mutant of acl5 and sac51‐d, which suppresses the excessive xylem phenotype of acl5, recovered normal sensitivity, while a quadruple T‐DNA insertion mutant of the SAC51 family, which has an increased thermospermine level but shows excessive xylem development, showed increased salt sensitivity, unlike pao5. Together with the result that the salt tolerance of both wild‐type and acl5 seedlings was improved by long‐term treatment with thermospermine, we suggest a correlation of the salt tolerance with reduced xylem development rather than with the thermospermine level. We further found that the mutants containing high thermospermine levels showed increased tolerance to drought and heat stress, suggesting another role of thermospermine that may be common with that of spermine and secondary to that in restricting excess xylem development associated with salt hypersensitivity.