Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.     

The microfilament cytoskeleton plays a vital role in salt and osmotic stress tolerance in Arabidopsis

Although recent studies have suggested that the microfilament (MF) cytoskeleton of plant cells participates in the response to salt stress, it remains unclear as to whether the MF cytoskeleton actually plays an active role in a plant's ability to withstand salt stress. In the present study, we report for the first time the role of MFs in salt tolerance of Arabidopsis thaliana . Our experiments revealed that Arabidopsis seedlings treated with 150 m m NaCl maintained MF assembly and bundle formation, whereas treatment with 250 m m NaCl initially induced MF assembly but subsequently caused MF disassembly. A corresponding change in the fluorescence intensity of MFs was also observed; that is, a sustained rise in fluorescence intensity in seedlings exposed to 150 m m NaCl and an initial rise and subsequent fall in seedlings exposed to 250 m m NaCl. These results suggest that MF assembly and bundles are induced early after salt stress treatment, while MF polymerization disappears after high salt stress. Facilitation of MF assembly with phalloidin rescued wild-type seedlings from death, whereas blocking MFs assembly with latrunculin A and cytochalasin D resulted in few survivors under salt stress. Pre-treatment of seedlings with phalloidin also clearly increased plant ability to withstand salt stress. MF assembly increased survival of Arabidopsis salt-sensitive sos2 mutants under salt stress and rescued defective sos2 mutants. Polymerization of MFs and its role in promoting survival was also found in plants exposed to osmotic stress. These findings suggest that the MF cytoskeleton participates and plays a vital role in responses to salt and osmotic stress in Arabidopsis .     

Excessive expression of the plant kinesin TBK5 converts cortical and perinuclear microtubules into a radial array emanating from a single focus

TBK5 is a plant-specific kinesin constantly expressed in tobacco BY-2 cells. An analysis of the distribution of green fluorescent protein-tagged TBK5 (GFP-TBK5) transiently expressed in BY-2 protoplasts revealed that TBK5 could associate with microtubules in vivo. GFP-TBK5 often assembled to form a single particle when accumulated in cells. The particle was located in close proximity to the nucleus, and its formation was accompanied by the development of a radial array of microtubules emanating from it and the loss of cortical microtubules. Microtubule depolymerization by treatment with propyzamide inhibited particle formation and stimulated the formation of dispersed aggregates of GFP-TBK5. Through expression of different TBK5 mutants as GFP fusions, the motor domain, two separated coiled-coil domains and the C-terminal domain of TBK5 were identified as the domains playing essential roles in particle formation. Mutants with putatively non-motile motor domains or lacking the C-terminal domain were localized to cortical and perinuclear microtubules, whereas those lacking either of the coiled-coil domains were preferentially distributed around the nucleus and along perinuclar microtubules. Further, the deletion of one of the coiled-coil domains or the C-terminal domain was sufficient to inhibit the propyzamide-induced formation of dispersed aggregates, whereas the mutation in the motor domain was not. These results led us to propose a model in which the particle is formed through the microtubule-based movement of GFP-TBK5 toward the nucleus and subsequent microtubule-independent aggregation based on coiled-coil interactions. The dramatic microtubule rearrangement would be explained if GFP-TBK5 relocated and gathered newly formed microtubules and/or microtubule-nucleating units.     

Effect of water and salt stresses on growth, chlorophyll, mineral ions and organic solutes contents, and enzymes activity in mung bean seedlings

The experiment was made by using different concentrations of polyethylene glycol (PEG) or salt solutions to decrease the osmotic potential of the growth medium to reveal the response of mung bean (Vigna radiata) to water and salt stresses. No germination (emergence of the seedling) occurred at medium osmotic potential lower than -1.0 MPa in all treatments. It was found that the activity of α-amylase and protease, and contents of proline, saccharides and the soluble proteins decreased in the germinating seeds during 3-d stress. However, after 10-d stress, the contents of organic solutes and the activity of the hydrolytic enzymes increased. Growth, chlorophyll content and mineral uptake were also significantly reduced under stress. The seedlings under water stress induced by PEG were affected much more than under salinity. This may be due to the maintenance of a higher succulence under salt stress than under PEG-induced water stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

A ROP2‐RIC1 pathway fine‐tunes microtubule reorganization for salt tolerance in Arabidopsis

The reorganization of microtubules induced by salt stress is required for Arabidopsis survival under high salinity conditions. RIC1 is an effector of Rho‐related GTPase from plants (ROPs) and a known microtubule‐associated protein. In this study, we demonstrated that RIC1 expression decreased with long‐term NaCl treatment, and ric1‐1 seedlings exhibited a higher survival rate under salt stress. We found that RIC1 reduced the frequency of microtubule transition from shortening to growing status and knockout of RIC1 improved the reassembly of depolymerized microtubules caused by either oryzalin treatment or salt stress. Further investigation showed that constitutively active ROP2 promoted the reassembly of microtubules and the survival of seedlings under salt stress. A rop2‐1 ric1‐1 double mutant rescued the salt‐sensitive phenotype of rop2‐1, indicating that ROP2 functions in salt tolerance through RIC1. Although ROP2 did not regulate RIC1 expression upon salt stress, a quick but mild increase of ROP2 activity was induced, led to reduction of RIC1 on microtubules. Collectively, our study reveals an ROP2‐RIC1 pathway that fine‐tunes microtubule dynamics in response to salt stress in Arabidopsis. This finding not only reveals a new regulatory mechanism for microtubule reorganization under salt stress but also the importance of ROP signalling for salinity tolerance.     

MDP25, a novel calcium regulatory protein, mediates hypocotyl cell elongation by destabilizing cortical microtubules in Arabidopsis

The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation.     

Production of Polyamines Is Enhanced by Endogenous Abscisic Acid in Maize Seedlings Subjected to Salt Stress

It is known that salt stress and exogenously applied abscisic acid （ABA） can enhance the polyamine content in plants and that salt stress itself can lead to an increase in endogenous ABA production. In the present study, the relationships between salt-induced ABA and polyamine accumulation were inves- tigated using ABA-deficient mutant （vp5/vp5） maize （Zea mays L.） seedlings and ABA and polyamine biosynthesis inhibitors. The results show that reduced endogenous ABA levels, as a result of either the mutation or by using a chemical inhibitor （sodium tungstate）, also reduced the accumulation of polyamines in salt-stressed leaves of maize seedlings. The polyamine synthesis inhibitors D-arginine and α- difluoromethylornithine also reduced the polyamine content of the leaves of maize seedling under salt stress. Both ABA and polyamine enhanced the dry weight accumulation of salt-stressed seedlings and also increased the activities of the two dominant tonoplast membrane enzymes, H^＋-ATPase and H^＋-PPase, when plants were under salt stress. The results suggest that salt stress induces an increase in endogenous ABA levels, which then enhances polyamine synthesis. Such responses may increase a plant＇s tolerance to salt.     

不同盐胁迫下小麦叶片渗透性调节和叶绿素荧光特性

盐胁迫对植物伤害机理受到普遍关注。本试验以‘西旱3号’小麦幼苗为材料,通过比较钠盐(150 mmol·L^-1)、钙盐(5、30 mmol·L^-1)单独及其复合胁迫对叶片渗透调节和光合特性的影响,揭示不同盐胁迫对小麦的伤害机理。结果表明: 钠盐或钙盐单独胁迫显著抑制了小麦幼苗根、茎的生长,使叶片可溶性糖和脯氨酸含量、调节性能量耗散电子产量、非光化学猝灭及玉米黄质相对含量均显著增加,而叶绿素a和叶绿素b含量、最大光化学效率、PSⅡ实际光化学效率、光化学猝灭及光合电子传递效率均显著下降。此外,钙盐对小麦幼苗生长的抑制作用更强,钠盐处理下叶片叶绿素含量减少和叶绿素荧光参数降低更显著。除了可溶性蛋白、叶黄素和玉米黄质相对含量以外,低浓度钙盐有效缓解了钠盐诱导其他各指标的变化,而高浓度钙盐进一步增大了钠盐处理小麦幼苗各参数的变化幅度。总之,钠盐和钙盐显著抑制了小麦幼苗的生长,低浓度钙盐能有效缓解钠盐对小麦幼苗的伤害,而高浓度钙盐加剧了钠盐的毒害作用。这均与叶片光合色素含量、光能捕获及光合电子传递的改变有关。此外,渗透调节物质在增强钠盐或钙盐环境中小麦幼苗的抗性方面发挥着重要作用。     

Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1

The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress-induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H^＋-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响。结果表明,0.1 mmol/L SNP处理显著缓解了150 mmol/L NaCl 胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性。进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜 H -ATPase和焦磷酸酶活性诱导有关。此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响。应用外源CaSO4 和 EGTA 处理也证实,Ca2 可能在NO诱导的质膜 H -ATPase和焦磷酸酶活性的提高过程中起信号作用。另外,分析盐胁迫下小麦幼苗根部 Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一。     

Reorganization of interphase microtubules in root cells of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during acclimation to osmotic and salt stress

We examined the organization of microtubule system of interphase cells in roots of Medicago sativa L. during acclimation to salt and osmotic stress at different concentrations of NaCl, Na₂SO₄, and mannitol. We identified morphological changes of tubulin cytoskeleton in different root tissues during the acclimation to salt and osmotic stress: (1) decreased density of the cortical microtubule network, (2) random orientation of cortical microtubule bundles, (4) thickening of the bundles, (3) nonuniform density of the bundles, (4) fragmentation of the bundles, and (5) formation of microtubule converging centers. Network thinning and thickening of the bundles were observed both under osmotic and salt stress. Random orientation of cortical microtubules was visualized under osmotic stress but not during salt stress. Fragmentation of microtubule bundles took place under salt stress with a high concentration of mannitol. Formation of microtubule converging centers was common under prolonged action of sodium sulfate, less evident under sodium chloride, and not found after mannitol treatment. Our data show that, in alfalfa root cells, cortical microtubules rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered by sodium cations, as well as by sulfate and chloride anions at concentrations that do not cause irreversible cell damage.     

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

MDP25 mediates the fine-tuning of microtubule organization in response to salt stress

Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca²⁺ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca²⁺ signaling was blocked by BAPTA-AM or LaCl₃, microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.     

外源亚精胺对盐胁迫下马铃薯幼苗生长和生理生化特征的影响

盐胁迫是影响作物生长的主要非生物胁迫类型，引起离子毒害和渗透胁迫，导致植物生长减弱、失绿、萎蔫甚至死亡。前期研究表明，适宜浓度的外源亚精胺能够缓解盐胁迫条件下植物叶片受损伤程度，提升生物膜抵抗盐离子伤害的能力，促进植物生长。该试验采用营养液培养法，以100mmol·L-1、200mmol·L-1、300 mmol· L-1NaCl溶液模拟不同盐胁迫程度，以中度耐盐品种晋薯16号、轻度耐盐品种冀张薯12号为试材，当马铃薯脱毒幼苗长至 4～5 片真叶时，连续叶面喷施0.9mmol·L-1外援亚精胺 7 d，2次/d。分析叶面喷施外源亚精胺（Spd）对不同盐胁迫程度条件下马铃薯幼苗生长、叶片抗氧化酶活性、渗透调节物质含量的影响。结果表明：（1）叶面喷施Spd缓解了盐胁迫对幼苗生长的抑制作用，提高了叶绿素含量和根系活力，提升超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）、抗坏血酸(ASA)和谷胱甘肽(GSH)等抗氧化酶活性，以及脯氨酸、可溶性糖、氨基酸含量；（2）200 mmol· L-1NaCl胁迫条件下，Spd对“晋薯16号”缓解作用最显著。研究表明Spd通过提高马铃薯幼苗根系活力、叶绿素含量、抗氧化酶活性、渗透调节能力，提高马铃薯幼苗对盐胁迫的适应性，促进马铃薯幼苗生长。     

Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress

The effects of 24-epibrassinolide (24-epiBL) on seedling growth, antioxidative system, lipid peroxidation, proline and soluble protein content were investigated in seedlings of the salt-sensitive rice cultivar IR-28. Seedling growth of rice plants was improved by 24-epiBL treatment under salt stress conditions. When seedlings treated with 24-epiBL were subjected to 120 mM NaCl stress, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) did not show significant difference, whereas the activity of ascorbate peroxidase (EC 1.11.1.11) significantly increased. Increased activity of peroxidase (EC 1.11.1.7) under NaCl stress showed remarkable decrease in the 24-epiBL+NaCl-applied group. Lipid peroxidation level significantly increased under salt stress but decreased with 24-epiBL application revealing that less oxidative damage occurred in this group (24-epiBL+NaCl). In addition, increased proline content in the NaCl-applied group was decreased by 24-epiBL application in the 24-epiBL+NaCl-applied group. Soluble protein content was increased by 24-epiBL application even under NaCl stress, being also higher than control conditions (no 24-epiBL or NaCl treatment). 24-epiBL treatment considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings.     

Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis

The molecular mechanism governing the response of plants to salinity stress, one of the most significant limiting factors for agriculture worldwide, has just started to be revealed. Here, we report AtSZF1 and AtSZF2, two closely related CCCH-type zinc finger proteins, involved in salt stress responses in Arabidopsis. The expression of AtSZF1 and AtSZF2 is quickly and transiently induced by NaCl treatment. Mutants disrupted in the expression of AtSZF1 or AtSZF2 exhibit increased expression of a group of salt stress-responsive genes in response to high salt. Significantly, the atszf1-1/atszf2-1 double mutant displays more sensitive responses to salt stress than the atszf1-1 or atszf2-1 single mutants and wild-type plants. On the other hand, transgenic plants overexpressing AtSZF1 show reduced induction of salt stress-responsive genes and are more tolerant to salt stress. We also showed that AtSZF1 is localized in the nucleus. Taken together, these results demonstrated that AtSZF1 and AtSZF2 negatively regulate the expression of salt-responsive genes and play important roles in modulating the tolerance of Arabidopsis plants to salt stress.