Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling

SPINDLY (SPY) is a negative regulator of gibberellin (GA) responses; however, spy mutants exhibit various phenotypic alterations not found in GA-treated plants. Assaying for additional roles for SPY revealed that spy mutants are resistant to exogenously applied cytokinin. GA also repressed the effects of cytokinin, suggesting that there is cross talk between the two hormone-response pathways, which may involve SPY function. Two spy alleles showing severe (spy-4) and mild (spy-3) GA-associated phenotypes exhibited similar resistance to cytokinin, suggesting that SPY enhances cytokinin responses and inhibits GA signaling through distinct mechanisms. GA and spy repressed numerous cytokinin responses, from seedling development to senescence, indicating that cross talk occurs early in the cytokinin-signaling pathway. Because GA3 and spy-4 inhibited induction of the cytokinin primary-response gene, type-A Arabidopsis response regulator 5, SPY may interact with and modify elements from the phosphorelay cascade of the cytokinin signal transduction pathway. Cytokinin, on the other hand, had no effect on GA biosynthesis or responses. Our results demonstrate that SPY acts as both a repressor of GA responses and a positive regulator of cytokinin signaling. Hence, SPY may play a central role in the regulation of GA/cytokinin cross talk during plant development.     

Mutations at the SPINDLY locus of Arabidopsis alter gibberellin signal transduction.

Three independent recessive mutations at the SPINDLY (SPY) locus of Arabidopsis confer resistance to the gibberellin (GA) biosynthesis inhibitor paclobutrazol. Relative to wild type, spy mutants exhibit longer hypocotyls, leaves that are a lighter green color, increased stem elongation, early flowering, parthenocarpy, and partial male sterility. All of these phenotypes are also observed when wild-type Arabidopsis plants are repeatedly treated with gibberellin A3 (GA3). The spy-1 allele is partially epistatic to the ga1-2 mutation, which causes GA deficiency. In addition, the spy-1 mutation can simultaneously suppress the effects of the ga1-2 mutation and paclobutrazol treatment, which inhibit different steps in the GA biosynthesis pathway. This observation suggests that spy-1 activates a basal level of GA signal transduction that is independent of GA. Furthermore, results from GA3 dose-response experiments suggest that GA3 and spy-1 interact in an additive manner. These results are consistent with models in which the SPY gene product regulates a portion of the GA signal transduction pathway.     

SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana pathways involved in light responses, flowering, and rhythms in cotyledon movements

SPINDLY (SPY) is a negative regulator of gibberellin signaling in Arabidopsis thaliana that also functions in previously undefined pathways. The N terminus of SPY contains a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPRs). GIGANTEA (GI) was recovered from a yeast two-hybrid screen for proteins that interact with the TPR domain. GI and SPY also interacted in Escherichia coli and in vitro pull-down assays. The phenotypes of spy and spy-4 gi-2 plants support the hypothesis that SPY functions with GI in pathways controlling flowering, circadian cotyledon movements, and hypocotyl elongation. GI acts in the long-day flowering pathway upstream of CONSTANS (CO) and FLOWERING LOCUS T (FT). Loss of GI function causes late flowering and reduces CO and FT RNA levels. Consistent with SPY functioning in the long-day flowering pathway upstream of CO, spy-4 partially suppressed the reduced abundance of CO and FT RNA and the late flowering of gi-2 plants. Like gi, spy affects the free-running period of cotyledon movements. The free-running period was lengthened in spy-4 mutants and shortened in plants that overexpress SPY under the control of the 35S promoter of Cauliflower mosaic virus. When grown under red light, gi-2 plants have a long hypocotyl. This hypocotyl phenotype was suppressed in spy-4 gi-2 double mutants. Additionally, dark-grown and far-red-light-grown spy-4 seedlings were found to have short and long hypocotyls, respectively. The different hypocotyl length phenotypes of spy-4 seedlings grown under different light conditions are consistent with SPY acting in the GA pathway to inhibit hypocotyl elongation and also acting as a light-regulated promoter of elongation.     

Altered Expression of SPINDLY Affects Gibberellin Response and Plant Development

Gibberellins (GAs) are plant hormones with diverse roles in plant growth and development. SPINDLY (SPY) is one of several genes identified in Arabidopsis that are involved in GA response and it is thought to encode an O-GlcNAc transferase. Genetic analysis suggests that SPY negatively regulates GA response. To test the hypothesis that SPY acts specifically as a negatively acting component of GA signal transduction, spy mutants and plants containing a 35S:SPY construct have been examined. A detailed investigation of the spy mutant phenotype suggests that SPY may play a role in plant development beyond its role in GA signaling. Consistent with this suggestion, the analysis of spy er plants suggests that the ERECTA (ER) gene, which has not been implicated as having a role in GA signaling, appears to enhance the non-GA spy mutant phenotypes. Arabidopsis plants containing a 35S:SPY construct possess reduced GA response at seed germination, but also possess phenotypes consistent with increased GA response, although not identical to spy mutants, during later vegetative and reproductive development. Based on these results, the hypothesis that SPY is specific for GA signaling is rejected. Instead, it is proposed that SPY is a negative regulator of GA response that has additional roles in plant development.     

SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.     

SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis

Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1-beta-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein-SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling.     

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.     

Salt tolerance in crop plants: new approaches through tissue culture and gene regulation

Recent approaches to study of salinity tolerance in crop plants have ranged from genetic mapping to molecular characterization of gene products induced by salt/drought stress. Transgenic plant design has allowed to test the effects of overexpression of specific prokaryotic or plant genes that are known to be up-regulated by salt/drought stress. This review summarizes current progress in the field in the context of adaptive metabolic and physiological responses to salt stress and their potential role in long term tolerance. Specifically considered are gene activation by salt, in view of proposed avenues for improved salt tolerance and the need to ascertain the additional influences of developmental regulation of such genes. Discussion includes the alternate genetic strategy we have pursued for improving salinity tolerance in alfalfa (Medicago sativa L.) and rice (Oryza sativa L.). This strategy combines single-step selection of salt-tolerant cells in culture, followed by regeneration of salt-tolerant plants and identification of genes important in conferring salt tolerance. We have postulated that activation or improved expression of a subset of genes encoding functions that are particularly vulnerable under conditions of salt-stress could counteract the molecular effects of such stress and could provide incremental improvements in tolerance. We have proceeded to identify the acquired specific changes in gene regulation for our salt-tolerant mutant cells and plants. One particularly interesting and novel gene isolate from the salt-tolerant cells is Alfin1, which encodes a putative zinc-finger regulatory protein, expressed predominantly in roots. We have demonstrated that this protein binds DNA in a sequence specific manner and may be potentially important in gene regulation in roots in response to salt and an important marker for salt tolerance in crop plants.     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.     

The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis

SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。