OsLOL1, a C2C2‐type zinc finger protein,interacts with OsbZIP58 to promote seed germination through the modulation of gibberellin biosynthesis in Oryza sativa

Seed germination is a key developmental process in the plant life cycle that is influenced by various environmental cues and phytohormones through gene expression and a series of metabolism pathways. In the present study, we investigated a C2C2‐type finger protein, OsLOL1, which promotes gibberellin (GA) biosynthesis and affects seed germination in Oryza sativa (rice). We used OsLOL1 antisense and sense transgenic lines to explore OsLOL1 functions. Seed germination timing in antisense plants was restored to wild type when exogenous GA₃ was applied. The reduced expression of the GA biosynthesis gene OsKO2 and the accumulation of ent‐kaurene were observed during germination in antisense plants. Based on yeast two‐hybrid and firefly luciferase complementation analyses, OsLOL1 interacted with the basic leucine zipper protein OsbZIP58. The results from electrophoretic mobility shift and dual‐luciferase reporter assays showed that OsbZIP58 binds the G‐box cis‐element of the OsKO2 promoter and activates LUC reporter gene expression, and that interaction between OsLOL1 and OsbZIP58 activates OsKO2 gene expression. In addition, OsLOL1 decreased SOD1 gene expression and accelerated programmed cell death (PCD) in the aleurone layer of rice grains. These findings demonstrate that the interaction between OsLOL1 and OsbZIP58 influences GA biosynthesis through the activation of OsKO2 via OsbZIP58, thereby stimulating aleurone PCD and seed germination.     

小麦锌指蛋白基因TaLOL2的克隆及特征分析

通过电子克隆与RT-PCR相结合的方法,在条锈菌诱导的小麦叶片中克隆获得1个新的LSD1型锌指蛋白基因TaLOL2,并用qRT-PCR技术分析了其转录表达特征。结果显示:(1)小麦锌指蛋白基因TaLOL2的cDNA全长1 095bp,编码179个氨基酸。(2)TaLOL2含有3个典型的zf-LSD1型(CxxCxRxxLMYxxGASxVxCxxC)保守结构域,与水稻、拟南芥、大麦等植物LSD1型锌指蛋白序列具有高度相似性,其中与水稻OsLOL2相似度达86.0%。(3)进化树分析表明,TaLOL2与水稻、拟南芥和大麦中部分含有3个保守zf-LSD1锌指结构的基因亲缘关系较近,而与其它包含不同数目的zf-LSD1锌指结构的基因亲缘关系较远。(4)qRT-PCR定量分析表明,TaLOL2在条锈菌侵染前期呈上调表达,在亲和及非亲和反应中差异表达。研究表明,TaLOL2参与了条锈菌诱导的小麦抗病防卫反应,很可能作为正调控因子参与了小麦-条锈菌非亲和互作中对条锈菌的抗性信号途径。     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C₁₉-GAs, including the bioactive GAs GA₁ and GA₄, by 2β-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA₁ and GA₄. Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds. C. Dijkstra, E. Adams, A. Bhattacharya and A. F. Page contributed equally to this paper.     

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA₃) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA₁ and real-time PCR quantitative assay analyses confirmed that the decrease of GA₁ in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice.     

Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T₀ plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T₁ generation. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T₂ plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T₂ plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T₃ plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

<Emphasis Type="Italic">OsMADS22</Emphasis>, an <Emphasis Type="Italic">STMADS11</Emphasis>-like MADS-box gene of rice,is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy

We report the cDNA sequence and gene expression patterns of OsMADS22, a novel member of the STMADS11-like family of MADS-box genes, from rice. In contrast to previously reported STMADS11-like genes, whose expression is detected in vegetative tissues, OsMADS22 is mainly expressed during embryogenesis and flower development. In situ hybridization analysis revealed that OsMADS22 expression is localized in the L1 layer of embryos and in developing stamen primordia. Ectopic expression of OsMADS22 in transgenic rice plants resulted in aberrant floral morphogenesis, characterized by a disorganized palea, an elongated glume, and a two-floret spikelet. The results are discussed in terms of rice spikelet development and a novel non-vegetative role for a STMADS11-like gene.     

Identification of gibberellins in the rice plant and quantitative changes of gibberellin A19 throughout its life cycle

The major endogenous gibberellin (GA) in shoots, roots and ears of the rice plant, Oryza sativa L. japonica cv. Nihonbare, was identified as GA₁₉ by combined gas liquid chromatography-mass spectrometry (GC-MS) and GC-selected ion current monitoring (GC-SICM). Another GA present in these tissues in small quantity was tentatively identified as GA₁ by GC-SICM, and GA₄ may be present in the seeds (kernels) of 3rd-leaf-stage seedlings. Using GC-SICM, the GA₁₉ content was quantified throughout the life cycle of rice plants. It was found to reach high levels (ca. 10–15 g/kg fresh weight) in 3rd-leaf seedlings, at panicle initiation (shoots), and during heading and anthesis (ears). The levels of GA₁₉ in Oryza sativa indica cv. T-136 underwent changes closely similar to those found in Nihonbare. The growth-promoting activity in rice of exogenous GA₁₉ is generally considerably less than that of GA₁. It therefore seems possible that GA₁₉ functions as a pool GA. The level of active GAs such as GA₁ may be regulated by the rate of biosynthesis of GA₁₉ or its metabolic conversions.Abbreviations GA(s) gibberellin(s) - GA_n gibberellin A_n - GA_n-MeTMS trimethylsilyl ether of GA_n methyl ester - GC-MS combined gas liquid chromatography-mass spectrometry - GC-SICM combined gas liquid chromatography-selected ion current monitoring - TLC thin-layer chromatography     

Gibberellin production and phosphate solubilization by newly isolated strain of <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> and its effect on plant growth

Plant growth-promoting rhizobacteria with gibberellins (GA)-producing potential were isolated from soil and screened for plant growth promotion. A new strain, Acinetobacter calcoaceticus SE370, produced extracellular GA and also had phosphate solubilising potential. It produced 10 different gibberellins, including the bioactive GA₁, GA₃ and GA₄ which were at, respectively, 0.45, 6.2 and 2.8 ng/100 ml. The isolate solubilised tricalcium phosphate and lowered pH of the medium during the process. Culture filtrates of the organism after growth on broth promoted growth of cucumber, Chinese cabbage and crown daisy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gibberellin production by pure cultures of a new strain of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Fungal endophytes produce a variety of favorable metabolites for plant growth and survival, but there is limited information on their gibberellin (GA) production capacity. In the current study, we isolated eight endophytic fungi from the roots of a drought stressed soybean cultivar Hwangkeumkong, and screened them on waito-c rice for plant growth promotion. Seven fungal isolates promoted plant growth, while one inhibited it. The culture filtrate (CF) of fungal isolate HK-5-2 provided the best results for growth promotion and was thus bioassayed on soybean. HK-5-2 CF enhanced plant length, plant fresh and dry weight and endogenous bioactive GA₁ and GA₄ contents of soybean as compared to control. The GA analysis of HK-5-2 CF showed the presence of bioactive GA₃ (8.38 ng/ml), GA₄ (2.16 ng/ml) and GA₇ (1.56 ng/ml) in conjunction with physiologically inactive GA₅, GA₁₉ and GA₂₄. Gibberella fujikuroi was used as positive control during this experiment. The fungal isolate HK-5-2 was identified as a new strain of Aspergillus fumigatus through molecular and phylogenetic analysis of 18S and 28S rDNA sequences.     

Gibberellin production and plant growth promotion by a newly isolated strain of <Emphasis Type="Italic">Gliomastix murorum</Emphasis>

Endophytic fungi are known to play a vital role in the growth and development of their host plants. We isolated eleven endophytic fungi from the roots of sand-dune plant Elymus mollis and their growth-promoting ability was studied on waito-c rice and Atriplex gemelinii. We found that eight fungal isolates promoted growth of both plants. Fungal isolate EM-7-1 induced maximum growth promotion in waito-c rice (9.25 cm) and Atriplex gemelinii (3.1 cm), which was higher than wild-type Gibberella fujikuroi. Gibberellin analysis of EM-7-1 culture filtrate showed the presence of bioactive gibberellins GA₁ (0.32 ng/ml), GA₃ (5.76 ng/ml), GA₄ (0.82 ng/ml) and GA₇: (0.1 ng/ml) along with physiologically inactive GA₅ (0.59 ng/ml), GA₉ (5.38 ng/ml), GA₂₀ (0.25 ng/ml) and GA₂₄ (2.03 ng/ml). The fungal isolate EM-7-1 was identified as new strain of Gliomastix murorum (G. murorum KACC43902) with 99% sequence homology. This study reports the plant growth-promoting ability of genus Gliomastix and the presence of GA₅ in the culture filtrate of fungi for the first time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .     

Pleiotropic effects of the<Emphasis Type="Italic"> male sterile33</Emphasis> (<Emphasis Type="Italic">ms33</Emphasis>) mutation in<Emphasis Type="Italic"> Arabidopsis</Emphasis> are associated with modifications in endogenous gibberellins,indole-3-acetic acid and abscisic acid

Earlier, we reported that mutation in the Male Sterile33 (MS33) locus in Arabidopsis thaliana causes inhibition of stamen filament growth and a defect in the maturation of pollen grains [Fei and Sawhney (1999) Physiol Plant 105:165–170; Fei and Sawhney (2001) Can J Bot 79:118–129]. Here we report that the ms33 mutant has other pleiotropic effects, including aberrant growth of all floral organs and a delay in seed germination and in flowering time. These defects could be partially or completely restored by low temperature or by exogenous gibberellin A₄ (GA₄), which in all cases was more effective than GA₃ Analysis of endogenous GAs showed that in wild type (WT) mature flowers GA₄ was the major GA, and that relative to WT the ms33 flowers had low levels of the growth active GAs, GA₁ and GA₄, and very reduced levels of GA₉, GA₂₄ and GA₁₅, precursors of GA₄. This suggests that mutation in the MS33 gene may suppress the GA biosynthetic pathway that leads to GA₄ via GA₉ and the early 13-H C₂₀ GAs. WT flowers also possessed a much higher level of indole-3-acetic acid (IAA), and a lower level of abscisic acid (ABA), relative to ms33 flowers. Low temperature induced partial restoration of male fertility in the ms33 flowers and this was associated with partial increase in GA₄. In contrast, in WT flowers GA₁ and GA₄ were very much reduced by low temperature. Low temperature also had little effect on IAA or ABA levels of ms33 flowers, but did reduce (>2-fold) IAA levels in WT flowers. The double mutants, ms33 aba1-1 (an ABA-deficient mutant), and ms33 spy-3 (a GA signal transduction mutant) had flower phenotypes similar to ms33. Together, the data suggest that the developmental defects in the ms33 mutant are unrelated to ABA levels, but may be causally associated with reduced levels of IAA, GA₁ and GA₄, compared to WT flowers.Abbreviations ABA Abscisic acid - GA Gibberellin - GC-MS-SIM Gas chromatography-mass spectrometry-selected ion monitoring - IAA Indole-3-acetic acid - ms33 Male sterile33 mutant - PP333 Paclobutrazol - WT Wild type