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.     

Cloning and characterization of a novel CBL-interacting protein kinase from maize

A novel CBL-interacting protein kinase (CIPK) gene, ZmCIPK16, was isolated from maize (Zea mays), which has been certified to have two copies in the genome. The ZmCIPK16 is strongly induced in maize seedlings by PEG, NaCl, ABA, dehydration, heat and drought, but not by cold. A yeast two-hybrid assay demonstrated that ZmCIPK16 interacted with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8. Bimolecular fluorescence complementation (BiFC) assays prove that ZmCIPK16 can interact with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8 in vivo. Subcellular localization showed that ZmCIPK16 is distributed in the nucleus, plasma membrane and cytoplasm; this is different from the specific localization of ZmCBL3, ZmCBL4, and ZmCBL5, which are found in the plasma membrane. The results also showed that overexpression of ZmCIPK16 in the Arabidopsis sos2 mutant induced the expression of the SOS1 gene and enhanced salt tolerance. These findings indicate that ZmCIPK16 may be involved in the CBL-CIPK signaling network in maize responses to salt stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfeng Zhao and Zhenfei Sun are contributed equally to this work.     

A Putative Plasma Membrane Cation/proton Antiporter from Soybean Confers Salt Tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cation transport is thought to be an important process for ion homeostasis in plant cells. Here, we report that a soybean putative cation/proton antiporter GmCAX1 may be a mediator of this process. GmCAX1 is expressed in all tissues of the soybean plants but at a lower level in roots. Its expression was induced by PEG, ABA, Ca²⁺, Na⁺ and Li⁺ treatments. The GmCAX1-GFP fusion protein was mainly localized in plasma membrane of the transgenic Arabidopsis plant cells and onion epidermal cells. Transgenic Arabidopsis plants overexpressing GmCAX1 accumulated less Na⁺, K⁺, and Li⁺, and were more tolerant to elevated Li⁺ and Na⁺ levels during germination when compared with the controls. These results suggest that GmCAX1 may function as an antiporter for Na⁺, K⁺ and Li⁺. Modulation of this antiporter may be beneficial for regulation of ion homeostasis and thus plant salt tolerance.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Expression of a Suaeda salsa Vacuolar H+/Ca2+ Transporter Gene in Arabidopsis Contributes to Physiological Changes in Salinity

A cDNA (SsCAX1) encoding a tonoplast-localised Ca²⁺/H⁺ exchanger was isolated from a C₃ halophyte Suaeda salsa (L.). To clarify the role of SsCAX1 in plant salt tolerance, Arabidopsis plants expressing SsCAX1 were treated with NaCl. Transgenic Arabidopsis plants displayed decreased salt tolerance. Although Na⁺ content was close to wild-type plants, transgenic plants accumulated more Ca²⁺ and retained less K⁺ in leaves than the wild-type plants in salinity. Furthermore, transgenic lines held higher leaf membrane leakage than wild-type lines under NaCl treatment. In addition, transgenic plants showed a 23% increase in vacuolar H⁺-ATPase activity compared with wild-type plants in normal condition. But the leaf V-H⁺-ATPase activity had subtle changes in transgenic plants, while significantly increased in wild-type plants under saline condition. These results suggested that regulated expression of Ca²⁺/H⁺ antiport was critical for maintenance of cation homeostasis and activity of V-H⁺-ATPase under saline condition.     

Cation co-tolerance phenomenon in cell cultures of Oryza sativa adapted to LiCl and NaCl

Cell lines of Oryza sativa L. (cv. Taipei-309) were adapted to 30 mM LiCl and 150 mM NaCl. Both adapted lines were considerably more tolerant than non adapted line when grown on 200, 250 and 300 mM NaCl and 30 mM LiCl stresses. The tolerance of LiCl-adapted line to NaCl (150 to 300 mM) and the tolerance of NaCl-adapted cells line to LiCl (30 mM) indicated that there was a cross-adaptation towards alkali metals (Na⁺ and Li⁺) not the Cl^–. Na⁺ and K⁺ contents of all lines which increased with increasing medium salinity but to a different degree. The increase in Na⁺ and K⁺ content in NaCl-adapted and non-adapted lines were comparable, while LiCl-adapted line accumulated significantly lower Na⁺and higher K⁺ content. Proline content of all lines increased with the increase in NaCl-stress but the magnitude of increase was much higher in the LiCl-adapted than other lines. The differential response of adapted lines to NaCl stress in accumulating proline and maintaining the ionic contents reveals that adapted lines have evolved different features of adaptation to cope with NaCl stress.     

The wheat gene <Emphasis Type="Italic">TaST</Emphasis> can increase the salt tolerance of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

On the basis of the results of gene chip analysis of the salt-tolerant wheat mutant RH8706-49 under conditions of salt stress, we identified and cloned an unknown salt-induced gene TaST (Triticum aestivum salt-tolerant). Real-time quantitative PCR analysis showed that the expression of the gene was induced by salt stress. Transgenic Arabidopsis plants overexpressing the TaST gene showed higher salt tolerance than the wild-type controls. Subcellular localization studies revealed that the protein encoded by this gene was in the nucleus. In comparison with wild-type controls, transgenic Arabidopsis plants accumulated more Ca²⁺, soluble sugar, and proline and less Na⁺ under salt stress. Real-time quantitative PCR analysis showed that Arabidopsis plants overexpressing TaST also showed increased expression of many stress-related genes. All these findings indicated that TaST can enhance the salt tolerance of transgenic Arabidopsis plants.     

Overexpression ofENA1 from yeast increases salt tolerance inArabidopsis

In yeast, the plasma membrane Na⁺/H⁺ antiporter and Na⁺-ATPase are key enzymes for salt tolerance.Saccharomyces cerevisiae Na⁺-ATPase (Enalp ATPase) is encoded by theENA1/PMR2A gene; expression ofENA1 is tightly regulated by Na⁺ and depends on ambient pH. Although Enalp is active mainly at alkaline pH values inS. cerevisiae, no Na⁺-ATPase has been found in flowering plants. To test whether this yeast enzyme would improve salt tolerance in plants, we introducedENA1 intoArabidopsis (cv. Columbia) under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on a medium containing kanamyin. Southern blot analyses confirmed thatENA1 was transferred into theArabidopsis genome and northern blot analyses showed thatENA1 was expressed in the transformants. Several transgenic homozygous lines and wild-type (WT) plants were evaluated for salt tolerance. No obvious morphological or developmental differences existed between the transgenic and WT plants in the absence of stress. However, overexpression ofENA1 inArabidopsis improved seed germination rates and salt tolerance in seedlings. Under saline conditions, transgenic plants accumulated a lower amount of Na⁺ than did the wild type, and fresh and dry weights of the former were higher. Other experiments revealed that expression ofENA1 promoted salt tolerance in transgenicArabidopsis under both acidic and alkaline conditions. These authors contributed equally to this article.     

Radial transport of salt and water in roots of the common reed (Phragmites australis Trin. ex Steudel)

To understand the root function in salt tolerance, radial salt and water transport were studied using reed plants growing in brackish habitat water with an osmotic pressure (π_M) of 0.63 MPa. Roots bathed in this medium exuded a xylem sap with NaCl as the major osmolyte and did so even at higher salt concentration (π_M up to 1.3 MPa). Exudation was stopped after a small increase of π_M (0.26 MPa) using polyethylene glycol 600 as osmolyte. The endodermis of fine lateral roots was found to be the main barrier to radial solute diffusion on an apoplastic path. Apoplastic salt transfer was proven by rapid replacement of stelar Na⁺ by Li⁺ in an isomolar LiCl medium. Water fluxes did not exert a true solvent drag on NaCl. Xylem sap concentrations of NaCl in basal internodes of transpiring culms were more than five times higher than in medial and upper ones. It was concluded that the radial NaCl flux was mainly diffusion through the apoplast, and radial water transport, because of the resistance of the cell wall matrix to convective mass flow, was confined to the symplast. Radial salt permeation in roots reduced the water stress exerted by the brackish medium.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

Heterologous expression of Arabidopsis H+‐pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.)

The Arabidopsis vacuolar H⁺‐pyrophosphatase (AVP1), when over‐expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over‐expressing AVP1 exhibited improved resistance to salinity than wild‐type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mm NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mm NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na⁺ uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na⁺, K⁺, Cl^‐ and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H⁺‐pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

AtCPK23 functions in <Emphasis Type="Italic">Arabidopsis</Emphasis> responses to drought and salt stresses

Calcium-dependent protein kinases (CDPKs) are unique serine/threonine kinases in plants and there are 34 CDPKs in Arabidopsis genome alone. Although several CDPKs have been demonstrated to be critical calcium signaling mediators for plant responses to various environmental stresses, the biological functions of most CDPKs in stress signaling remain unclear. In this study, we provide the evidences to demonstrate that AtCPK23 plays important role in Arabidopsis responses to drought and salt stresses. The cpk23 mutant, a T-DNA insertion mutant for AtCPK23 gene, showed greatly enhanced tolerance to drought and salt stresses, while the AtCPK23 overexpression lines became more sensitive to drought and salt stresses and the complementary line of the cpk23 mutant displayed similar phenotype as wild-type plants. The results of stomatal aperture measurement showed that the disruption of AtCPK23 expression reduced stomatal apertures, while overexpression of AtCPK23 increased stomatal apertures. The alteration of stomatal apertures by changes in AtCPK23 expression may account, at least in partial, for the modified Arabidopsis response to drought stress. In consistent with the enhanced salt-tolerance by disruption of AtCPK23 expression, K⁺ content in the cpk23 mutant was not reduced under high NaCl stress compared with wild-type plants, which indicates that the AtCPK23 may also regulate plant K⁺-uptake. The possible mechanisms by which AtCPK23 mediates drought and salt stresses signaling are discussed.     

Constitutive overexpression of the calcium sensor CBL5 confers osmotic or drought stress tolerance in Arabidopsis

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of CBL-interacting protein kinases (CIPKs). In this study, we report the role of Arabidopsis CBL5 gene in high salt or drought tolerance. CBL5 gene is expressed significantly in green tissues, but not in roots. CBL5 was not induced by abiotic stress conditions such as high salt, drought or low temperature. To determine whether the CBL5 gene plays a role in stress response pathways, we ectopically expressed the CBL5 protein in transgenic Arabidopsis plants (35S-CBL5) and examined plant responses to abiotic stresses. CBL5-overexpressing plants displayed enhanced tolerance to high salt or drought stress. CBL5 overexpression also rendered plants more resistant to high salt or hyperosmotic stress during early development (i.e., seed germination) but did not alter their response to abiscisic acid (ABA). Furthermore, overexpression of CBL5 alters the gene expression of stress gene markers, such as RD29A, RD29B and Kin1 etc. These results suggest that CBL5 may function as a positive regulator of salt or drought responses in plants.     

Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium <Emphasis Type="Italic">Desulfonatronum thiodismutans</Emphasis>

It has been shown that the intracellular concentrations of Na⁺, K⁺, and Cl^? ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na⁺ ions. An increase in the extracellular concentration of Na⁺ results in the accumulation of K⁺ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCl added to the medium was increased to 4%, the concentration gradient of Cl^? ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase—periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na⁺, K⁺, and Li⁺ salts and pH. The enzymes were found to be resistant to high concentrations of Na⁺ and K⁺ chlorides and Na⁺ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na⁺ and Li⁺ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na⁺ concentrations, close to those typical of cells growing at optimal salt concentrations.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.