Mutants at the Slender1 locus of barley cv Himalaya. Molecular and physiological characterization

A dominant dwarf mutant of barley (Hordeum vulgare) that resembles dominant gibberellin (GA) "-insensitive" or "-nonresponsive" mutants in other species is described. alpha-Amylase production by endosperm half-grains of the mutant required GA3 at concentrations about 100 times that of the WT. The mutant showed only a slight growth response to GA3, even at very high concentrations. However, when additionally dwarfed, growth rate responded to GA3 over the normal concentration range, although only back to the original (dwarf) elongation rate. Genetic studies indicated that the dominant dwarf locus was either closely linked or identical to the Sln1 (Slender1) locus. A barley sequence related to Arabidopsis GAI/RGA was isolated, and shown to represent the Sln1 locus by the analysis of sln1 mutants. The dominant dwarf mutant was also altered in this sequence, indicating that it too is an allele at Sln1. Thus, mutations at Sln1 generate plants of radically different phenotypes; either dwarfs that are largely dominant and GA "-insensitive/-nonresponsive," or the recessive slender types in which GA responses appear to be constitutive. Immunoblotting studies showed that in growing leaves, SLN1 protein localized almost exclusively to the leaf elongation zone. In mutants at the Sln1 locus, there were differences in both the abundance and distribution of SLN1 protein, and large changes in the amounts of bioactive GAs, and of their metabolic precursors and catabolites. These results suggest that there are dynamic interactions between SLN1 protein and GA content in determining leaf elongation rate.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

Involvement of Abscisic Acid in Mesocotyl Growth in Etiolated Seedlings of a Foxtail Millet Dwarf Mutant

Etiolated seedlings of foxtail millet (Setaria italica Beauv.) dwarf mutant CH84113 were treated with various concentrations of abscisic acid (ABA), mefluidide, mannitol, or polyethylene glycol (PEG) 6000. It was found that these chemicals, at suitable concentrations, could increase mesocotyl length significantly, whereas these chemicals at higher concentrations had an inhibitory effect. Endogenous levels of ABA in mesocotyl were measured by enzyme-linked immunosorbent assay. It was found that endogenous ABA increased progressively in a chemical (ABA, mefluidide, mannitol, or PEG 6000) concentration-dependent manner, indicating that the effects of these chemicals on mesocotyl growth may be mediated by increased endogenous ABA levels. On the other hand, S-3307, an inhibitor of the oxidative reactions in gibberellin (GA) biosynthesis, inhibited the elongation of mesocotyl significantly. When ABA and GA₃ were applied simultaneously, the effect on mesocotyl growth was additive. These results imply that ABA and GA may control different processes in the regulation of mesocotyl growth. Received October 27, 1997; accepted May 11, 1998     

Genetic analysis and fine mapping of a novel semidominant dwarfing gene LB4D in rice

A dwarf mutant, designated LB4D, was obtained among the progeny of backcrosses to a wild rice introgression line. Genetic analysis of LB4D indicated that the dwarf phenotype was controlled by a single semidominant dwarfing gene, which was named LB4D. The mutants were categorized as dn-type dwarf mutants according to the pattern of internode reduction. In addition, gibberellin (GA) response tests showed that LB4D plants were neither deficient nor insensitive to GA. This study found that tiller formation by LB4D plants was decreased by 40% compared with the wild type, in contrast to other dominant dwarf mutants that have been identified, indicating that a different dwarfing mechanism might be involved in the LB4D dominant mutant. The reduction of plant height in F(1) plants ranged from 27.9% to 38.1% in different genetic backgrounds, showing that LB4D exerted a stronger dominant dwarfing effect. Using large F(2) and F(3) populations derived from a cross between heterozygous LB4D and the japonica cultivar Nipponbare, the LB4D gene was localized to a 46 kb region between the markers Indel 4 and Indel G on the short arm of chromosome 11, and four predicted genes were identified as candidates in the target region.     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

The Metabolism of Gibberellin A20 to Gibberellin A1 by Tall and Dwarf Mutants of Oryza sativa and Arabidopsis thaliana

The purpose of this study was to demonstrate the metabolism of gibberellin A20 (GA20) to gibberellin A1 (GA1) by tall and mutant shoots of rice (Oryza sativa L.) and Arabidopsis thaliana (L.) Heynh. The data show that the tall and dx mutant of rice and the tall and ga5 mutant of Arabidopsis metabolize GA20 to GA1. The data also show that the dy mutant of rice and the ga4 mutant of Arabidopsis block the metabolism of GA20 to GA1. [17-13C,3H]GA20 was fed to tall and the dwarf mutants, dx and dy, of rice and tall and the dwarf mutants, ga5 and ga4, of Arabidopsis. The metabolites were analyzed by high-performance liquid chromatography and full-scan gas chromatography-mass spectrometry together with Kovats retention index data. For rice, the metabolite [13C]GA, was identified from tall and dx seedlings; [13C]GA1 was not identified from the dy seedlings. [13C]GA29 was identified from tall, dx, and dy seedlings. For Arabidopsis, the metabolite [13C]GA1 was identified from tall, ga5, and ga4 plants. The amount of [13C]GA1 from ga4 plants was less than 15% of that obtained from tall and ga5 plants. [13C]GA29 was identified from tall, ga5, and ga4 plants. [13C]GA5 and [13C]GA3 were not identified from any of the six types of plant material.     

水稻矮秆突变体dtl1的分离鉴定及其突变基因的精细定位

文章通过对所构建的水稻突变体库进行大规模筛选,获得一个稳定遗传的矮秆突变体,与野生型日本晴相比,该突变体表现为植株矮化、叶片卷曲、分蘖减少和不育等性状,命名为dtl1(dwarf and twist leaf 1)。dtl1属于nl型矮秆,激素检测表明,矮秆性状与赤霉素和油菜素内酯无关。遗传分析显示,突变性状受单一隐性核基因控制。利用dtl1与籼稻品种Taichung Native 1杂交构建F2群体,将该突变基因DTL1定位于水稻第10染色体长臂2个SSR标记RM25923和RM6673之间约70.4 kb区域内,并与InDel标记Z10-29共分离,在该区域预测有13个候选基因,但未见调控水稻株高相关基因的报道,因此,认为DTL1基因是一个新的控制水稻株高的基因。     

Growth and development of Brassica genotypes differing in endogenous gibberellin content. I. Leaf and reproductive development

Leaf and reproductive development were compared in 3 rapid cycling Brassica rapa genotypes grown for 4 weeks under greenhouse conditions. The dwarf mutant, rosette ( ros ), is gibberellin (GA)-deficient, while the tall mutant, elongated internode ( ein ), has enhanced endogenous GA levels. Germination was delayed in ros and a selection of a more severe form of ros , named dormant ( do ), has even more retarded germination and some seeds entirely fail to germinate. Seeds of do and ros respond to exogenous GA, by rapid germination.
The 3 genotypes, ros , normal and ein , displayed similar developmental sequences, although floral bud formation and subsequent floral development and anthesis were delayed in ros. Conversely, anthesis was slightly accelerated in ein . Individual leaf areas were reduced in both ros and ein relative to the normal genotype, but leaf numbers were similar in all 3 genotypes. Differences in leaf morphology (heterophylly) were also observed; the normal genotype and ein plants possessed uniform leaf shapes and relatively smooth leaf margins, although petiole length was increased in ein . The mutant ros had scalloped leaf margins and convoluted leaf blades in addition to shortened petioles. These phenotypes suggest a role for GA in the regulation of germination and reproductive and leaf development in Brassica.     

A gibberellin insensitive mutant of Arabidopsis thaliana

A dwarf mutant of Arabidopsis thaliana (L.) Heynh. was found to be less sensitive to applied gibberellins than the wild type, and this character was controlled by one partially-dominant gene (denoted Gai) located on chromosome 1. This mutant resembled gibberellin-deficient mutants since not only stem growth, but also apical dominanace and seed germination were reduced. However, in contrast to the latter mutants, gibberellin does not reverse these effects in the Gai mutant. The insensitivity of the mutant could be quantified in much more detail in the recombinant of this mutation with the GA deficient mutant ga-1/ga-1 . Endogenous gibberellins of the Gai mutant did not differ from the wild type either in quantity or composition. The data suggest that the gene controls a step involved in gibberellin action.     

Auxin–cytokinin and auxin–gibberellin interactions during morphogenesis of the compound leaves of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

A number of mutations that alter the form of the compound leaf in pea (Pisum sativum) has proven useful in elucidating the role that auxin might play in pea leaf development. The goals of this study were to determine if auxin application can rescue any of the pea leaf mutants and if gibberellic acid (GA) plays a role in leaf morphogenesis in pea. A tissue culture system was used to determine the effects of various auxins, GA or a GA biosynethesis inhibitor (paclobutrazol) on leaf development. The GA mutant, nana1 (na1) was analyzed. The uni-tac mutant was rescued by auxin and GA and rescue involved both a conversion of the terminal leaflet into a tendril and an addition of a pair of lateral tendrils. This rescue required the presence of cytokinin. The auxins tested varied in their effectiveness, although methyl-IAA worked best. The terminal tendrils of wildtype plantlets grown on paclobutrazol were converted into leaflets, stubs or were aborted. The number of lateral pinna pairs produced was reduced and leaf initiation was impaired. These abnormalities resembled those caused by auxin transport inhibitors and phenocopy the uni mutants. The na1 mutant shared some morphological features with the uni mutants; including, flowering late and producing leaves with fewer lateral pinna pairs. These results show that both auxin and GA play similar and significant roles in pea leaf development. Pea leaf morphogenesis might involve auxin regulation of GA biosynthesis and GA regulation of Uni expression.     

Multiple loss-of-function of Arabidopsis gibberellin receptor AtGID1s completely shuts down a gibberellin signal

Arabidopsis carries three receptor genes for the phytohormone gibberellin (GA), AtGID1a, AtGID1b and AtGID1c. Expression of each gene in the rice gid1-1 mutant for GA receptors causes reversion of its severely dwarfed phenotype and GA insensitivity to a normal level, even though each loss-of-function mutant shows no clear phenotype in Arabidopsis (Nakajima et al., 2006). In this paper, we report the functional redundancy and specificity of each AtGID1 by analyzing the multiple mutants for loss of function. Seeds of the double knockout mutants atgid1a atgid1b, atgid1a atgid1c and atgid1b atgid1c germinated normally. The double knockout mutant atgid1a atgid1c showed a dwarf phenotype, while other double mutants were of normal height compared to the wild-type. The stamens of the double knockout mutant atgid1a atgid1b were significantly shorter than those of the wild-type, and this leads to low fertility. A severe disarrangement of the pattern on its seed surface was also observed. The triple knockout mutant atgid1a atgid1b atgid1c did not germinate voluntarily, and only started to grow when the seed coat was peeled off after soaking. Seedlings of the triple knockout mutants were severe dwarfs, only a few millimeters high after growing for 1 month. Moreover, the triple knockout seedlings completely lost their ability to respond to exogenously applied GA. These results show that all AtGID1s function as GA receptors in Arabidopsis, but have specific role(s) for growth and development.     

The pea gene NA encodes ent-kaurenoic acid oxidase

The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.     

Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice

The rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein that belongs to a subfamily of the GRAS protein superfamily and that functions as a repressor of gibberellin (GA) signaling. Based on the constitutive GA response phenotype of slr1 mutants, SLR1 has been thought to be the sole DELLA-type protein suppressing GA signals in rice. However, in rice genome databases we identified two sequences homologous to SLR1: SLR1-like1 and -2 (SLRL1 and -2). SLRL1 and SLRL2 contain regions with high similarity to the C-terminal conserved domains in SLR1, but lack the N-terminal conserved region of the DELLA proteins. The expression of SLRL1 was positively regulated by GA at the mRNA level and occurred preferentially in reproductive organs, whereas SLRL2 was moderately expressed in mature leaf organs and was not affected by GA. Transformation of SLRL1 into the slr1 mutant rescued the slender phenotype of this mutant. Moreover, overexpression of SLRL1 in normal rice plants induced a dwarf phenotype with an increased level of OsGA20ox2 gene expression and diminished the GA-induced shoot elongation, suggesting that SLRL1 acts as a repressor of GA signaling. Consistent with the fact that SLRL1 does not have a DELLA domain, which is essential for degradation of DELLA proteins, a level of SLRL1 protein was not degraded by application of gibberellic acid. However, the repressive activity of SLRL1 against GA signaling was much weaker than a truncated SLR1 lacking the DELLA domain. Based on these characteristics of SLRL1, the functional roles of SLRL1 in GA signaling in rice are discussed.     

蒺藜苜蓿株高控制基因COSTIN的克隆及功能研究

株高是影响植物株型建成的重要农艺性状之一,直接决定作物的倒伏性和生物产量,但目前关于苜蓿等豆科牧草株高性状形成的分子调控机制尚不清楚。通过定向筛选豆科模式植物蒺藜苜蓿Tnt1逆转座子插入突变体库,分离鉴定了一个蒺藜苜蓿矮化突变体compact stalk internodes(costin),该突变体的矮化表型是由于茎节伸长受到抑制所致。通过基因表型连锁分析成功克隆了COSTIN基因,该基因编码一个钙离子交换蛋白,与拟南芥的CALCIUM EXCHANGER 7(CAX7) 基因高度同源。qRT-PCR检测发现COSTIN基因在茎、叶和果荚等组织中有较高的表达。进一步研究发现在costin突变体中赤霉素合成途径关键基因MtCPS、MtKAO1、MtGA20ox4、MtGA20ox7和MtGA3ox1表达下调;外施赤霉素GA3可以恢复costin突变体的矮化表型。上述研究表明COSTIN基因通过影响植物激素赤霉素的生物合成来调控蒺藜苜蓿的茎节伸长。     

Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants

Degradation of active C(19)-gibberellins (GAs) by dioxygenases through 2beta-hydroxylation yields inactive GA products. We identified two genes in Arabidopsis (AtGA2ox7 and AtGA2ox8), using an activation-tagging mutant screen, that encode 2beta-hydroxylases. GA levels in both activation-tagged lines were reduced significantly, and the lines displayed dwarf phenotypes typical of mutants with a GA deficiency. Increased expression of either AtGA2ox7 or AtGA2ox8 also caused a dwarf phenotype in tobacco, indicating that the substrates for these enzymes are conserved. AtGA2ox7 and AtGA2ox8 are more similar to each other than to other proteins encoded in the Arabidopsis genome, indicating that they may constitute a separate class of GA-modifying enzymes. Indeed, enzymatic assays demonstrated that AtGA2ox7 and AtGA2ox8 both perform the same GA modification: 2beta-hydroxylation of C(20)-GAs but not of C(19)-GAs. Lines containing increased expression of AtGA2ox8 exhibited a GA dose-response curve for stem elongation similar to that of the biosynthetic mutant ga1-11. Double loss-of-function Atga2ox7 Atga2ox8 mutants had twofold to fourfold higher levels of active GAs and displayed phenotypes associated with excess GAs, such as early bolting in short days, resistance to the GA biosynthesis inhibitor ancymidol, and decreased mRNA levels of AtGA20ox1, a gene in the GA biosynthetic pathway.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.