Molecular cloning and expression analysis of a RGA-like gene responsive to plant hormones in Brassica napus

DELLA proteins are negative regulators of GA-induced growth. DELLA protein family is characterized by a DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. A full-length cDNA encoding a putative DELLA protein with high sequence homology to Arabidopsis thaliana RGA (AtRGA), designated as BnRGA, was isolated from Brassica napus. The full-length cDNA of BnRGA contained a 1,740 bp open reading frame (ORF) encoding a precursor protein of 579 amino acid residues. Comparative and bioinformatics analyses revealed that BnRGA showed a high degree of homology with DELLA proteins and contained the DELLA domain, TVHYNP domain, VHIID domain and RVER domain. Using real-time PCR, the expression patterns of BnRGA and two our previously isolated genes, BnGID1a and BnSLY1 in B. napus, were analyzed by adding exogenous gibberellins acid-3 (GA₃), GA biosynthetic inhibitor paclobutrazol (PAC) and abscisic acid (ABA). The results showed that the expression of BnGID1a and BnSLY1 was down-regulated after treated by GA₃ and induced by PAC and ABA. These results suggest that the expression of BnGID1a and BnSLY1 may be negatively regulated by the level of endogenous GA in B. napus. Moreover, BnRGA was not significantly regulated by GA₃, PAC and ABA in the low concentrations. These suggest that GA-GID1-SCF-DELLA complex may have a mechanism of self-regulation, thereby preserving the stability of the expression level of BnRGA in B. napus.     

Cloning and Characterization of a G Protein β Subunit Gene Responsive to Plant Hormones and Abiotic Stresses in <Emphasis Type="Italic">Brassica napus</Emphasis>

A full-length cDNA encoding a putative G protein β subunit (Gβ), designated as BnGB1, was isolated from Brassica napus. BnGB1 was predicted to encode a precursor protein of 378-amino acid residues. The expression of BnGB1 in different B. napus tissues and developmental stages was analyzed using real-time polymerase chain reaction. The results showed that BnGB1 expressed was high at the seventh day, the bolting stage, and fruiting stage. Moreover, BnGB1 was analyzed under four different plant hormones. All of the four tested hormones, abscisic acid (ABA), GA₃, brassinosteroid (BR), and indole-3-acetic acid (IAA), triggered an induction of BnGB1 at different hormone concentrations. The expression of BnGB1 was induced by low ABA concentrations, but high ABA concentrations inhibited the expression, which demonstrated that there might be an ABA-mediated feedback repression of the BnGB1 expression. BnGB1 was more prominently induced by high concentrations of GA₃ than by other plant hormones. The positive responses stimuli suggested that BnGB1 might be involved in signaling pathways for response to high concentrations of GA. However, the expression of BnGB1 did not significantly regulated by exogenous BR and IAA compared with the level regulated by ABA and GA₃. In addition, BnGB1 was responsive to different abiotic stresses and displayed differential expression patterns in B. napus. The BnGB1 was upregulated in salt and drought stress and downregulated in heat and cold stress. These results suggest that BnGB1 plays an important role in ABA and GA signal pathways and may also be involved in plant defense system against environmental stresses in B. napus.     

Isolation and characterization of a G protein γ subunit gene responsive to plant hormones and abiotic stresses in <Emphasis Type="Italic">Brassica napus</Emphasis> L

G protein plays an important role in signaling pathways and is involved in various signal transduction systems in plant. A full-length cDNA encoding a putative G protein γ subunit (Gγ), designated as BnGG2, was isolated from Brassica napus L. BnGG2 is predicted to encode a precursor protein of 100 amino acid residues. The expression of BnGG2 in different B. napus tissues and developmental stages was analyzed by using real-time PCR. The results showed that BnGG2 expression level was high at the 7-day-old seedling stage, the bolting stage, and the fruiting stage. Moreover, BnGG2 was analyzed under four different plant hormones. All of the four tested hormones, abscisic acid (ABA), gibberellins acid 3 (GA₃), brassinosteroid (BR) and indole-3-acetic acid (IAA), triggered an induction of BnGG2 expression at different hormone concentrations. The expression of BnGG2 was significantly induced by the high concentrations of ABA, BR, and IAA. The expression of BnGG2 was also induced by low GA₃ concentrations and inhibited by high GA₃ concentrations. In addition, BnGG2 was responsive to different abiotic stresses. The BnGG2 was up-regulated in salt and drought stress and down-regulated in heat and cold stress. These results suggested that BnGG2 plays an important role in plant hormone signaling pathways and may be also involved in plant defense systems against environmental stresses in B. napus.     

Changes in levels of gibberellins,their putative conjugates,and ABA in zygotic embryos during late maturation and dry seed stages of <Emphasis Type="Italic">Brassica napus</Emphasis> cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA₁ and even in levels of the inactive GA catabolites, GA₈ and GA₂₉. Levels of GA₄ and putative GA₈₅ also dropped appreciably, though not significantly. In contrast, the levels of GA₂₀ and GA₉ (the immediate precursors of GA₁ and GA₄, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA₉, GA₂₀) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA₂₀ and 23 and 5 ng/g DW, of GA₉, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA₈₅ aglycone, which increased sixfold (free GA₈₅ decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.     

Isolation and characterization of gene encoding G protein α subunit protein responsive to plant hormones and abiotic stresses in Brassica napus

G protein plays an important role in signal pathways and involved in various signal transduction systems in plant. A full-length cDNA encoding a putative G protein α subunit (Gα), designated as BnGA1, was isolated from Brassica napus. The expression of BnGA1 in different B. napus tissues and developmental stags was analyzed using real-time PCR. The results showed that BnGA1 expressed was high in root, cotyledon and shoot apex. Stage expression pattern analysis revealed that BnGA1 expressed strongly at the 7th day, the bolting stage and fruiting stage. In addition, the expression of BnGA1 was analyzed under different concentrations of four plant hormones. The expression of BnGA1 was significantly induced by the high concentrations of abscisic acid (ABA) and brassinosteroid (BR). The expression of BnGA1 was also induced by low gibberellins acid 3 (GA₃) concentrations and higher GA₃ concentrations inhibit the expression of BnGA1. However, the expression of BnGA1 did not significantly regulated by exogenous indole-3-acetic acid (IAA). Moreover, the expression of BnGA1 under different abiotic stresses was analyzed at different time points. The BnGA1 was up-regulated in salt and drought stress and down-regulated in heat and cold stress. These expression results suggested that BnGA1 play an important role in plant hormones signal pathways and BnGA1 may be involved in plant defense system against environmental stresses in B. napus.     

Germination and <Emphasis Type="Italic">In Vitro</Emphasis> Growth of Petunia Male Gametophyte Are Affected by Exogenous Hormones and Involve the Changes in the Endogenous Hormone Level

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia (sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA₃ considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA₃ produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C₃-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA₃ did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 584–590.Original Russian Text Copyright © 2005 by Kovaleva, Zakharova, Minkina, Timofeeva, Andreev.     

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA₃) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.     

2,4‐D‐induced parthenocarpy in pear is mediated by enhancement of GA4 biosynthesis

Parthenocarpy, the productions of seedless fruit without pollination or fertilization, is a potentially desirable trait in many commercially grown fruits, especially in pear, which is self‐incompatible. Phytohormones play important roles in fruit set, a process crucial for parthenocarpy. In this study, 2,4‐dichlorophenoxyacetic acid (2,4‐D), an artificially synthesized plant growth regulator with functions similar to auxin, was found to induce parthenocarpy in pear. Histological observations revealed that 2,4‐D promoted cell division and expansion, which increased cortex thickness, but the effect was weakened by paclobutrazol (PAC), a gibberellin (GA) biosynthesis inhibitor. Phenotypic differences in pear may therefore be due to different GA contents. Hormone testing indicated that 2,4‐D mainly induced the production of bioactive GA₄, rather than GA_3. Three key oxidase genes function in the GA biosynthetic pathway: GA20ox, GA3ox and GA2ox. In a pear group treated with only 2,4‐D, PbGA20ox2‐like and PbGA3ox‐1 were significantly upregulated. When treated with 2,4‐D supplemented with PAC, however, expression levels of these genes were significantly downregulated. Additionally, PbGA2ox1‐like and PbGA2ox2‐like expression levels were significantly downregulated in pear treated with either 2,4‐D only or 2,4‐D supplemented with PAC. We thus hypothesize that 2,4‐D can induce parthenocarpy by enhancing GA₄ biosynthesis.     

Molecular expression of <Emphasis Type="Italic">PsPIN1</Emphasis>, a putative auxin efflux carrier gene from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

A cDNA coding for a putative auxin efflux carrier was amplified from Pisum sativum seedling shoot tips by RT-PCR and the corresponding full-length cDNA, PsPIN1, was subsequently obtained by RACE-PCR. The deduced amino acid sequence (599 residues) showed the three domain topology typical of the other PIN proteins. The PsPIN1 protein structure prediction possessed five transmembrane domains at both the N-(7-150) and C-(450-575) termini and a hydrophylic region in the middle. PsPIN1 showed highest similarity to Medicago, MtPIN4. Using the Genome Walking technique, a 1511 bp upstream region for PsPIN1 gene was sequenced. This PsPIN1 upstream region possessed multiple putative auxin, GA and light regulatory elements. The PsPIN1 mRNA was ubiquitously expressed throughout the pea plant, especially in growing tissues. Auxin induced PsPIN1 mRNA in dark grown pea seedling shoot tips. It was induced by 4-chloro-IAA, which is also an active auxin in pea, and by gibberellin (GA₃). Interestingly, the PsPIN1 mRNA was down-regulated by light treatment, possibly because light negatively regulates auxin and, especially GA levels in pea. Thus PIN1-mediated auxin efflux is a highly regulated process, not only at the level of protein localization, but also at the level of mRNA accumulation.     

Isolation and characterization of a homologous to lipase gene from Brassica napus

Lipase is an important lipolytic enzyme involved in plant lipid metabolism. To analyze its function and roles during seed germination and growth, a full-length cDNA encoding a homologous to lipase gene named BnLIP1 was cloned from Brassica napus, cv. Huyou 15, by rapid amplification of cDNA ends. The BnLIP1 gene had a total length of 1318 bp, with an open reading frame of 1170 bp encoding 389 amino acid residues. Sequence analysis revealed that BnLIP1 protein belonged to the GDSL family of serine esterases/lipases. In B. napus genome, BnLIP1 is represented by several copies with the length of 1601 bp, the gene comprises five exons and four introns. RT-PCR analysis indicated that BnLIP1 showed no tissue-specific expression during reproductive growth and is strongly expressed during seed germination. No expression could be detected until three days after germination, and its peak was registered at the fifth day after germination. In conclusion, BnLIP1-encoded protein is predicted to be a lipolytic enzyme widely expressed at various stages of oilseed rape germination and development. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 410–417. The text was submitted by the authors in English.     

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     

矮化香蕉及其野生型GA3ox基因的结构特点和表达分析

香蕉的矮化突变是香蕉无性繁殖后代最常见的表型变异之一,但其变异的分子调控机理目前尚未研究清楚; 而内源赤霉素是影响植物株高的重要激素之一,GA3-氧化酶是赤霉素生物合成后期的关键酶。为探究GA3-氧化酶编码基因对香蕉矮化的分子调控机理,该研究以威廉斯B6矮化突变体及其野生型亲本为材料,通过RT-PCR技术克隆得到矮化香蕉及其野生型亲本GA3ox基因的全长cDNA序列,并对其推测的氨基酸序列进行比对分析,同时利用qRT-PCR技术对GA3ox基因在不同组织中的表达水平差异进行分析。结果表明:(1)矮化香蕉GA3ox-A和野生型香蕉GA3ox-G的ORF长度均为864 bp,均编码287个氨基酸,经序列比对分析发现两条氨基酸序列之间存在5个位点的差异,从而产生具有不同性质的蛋白质。(2)氨基酸序列同源性分析表明,矮化香蕉GA3ox的氨基酸序列与油棕、海枣、椰子的同源性最高。(3)qRT-PCR显示,GA3ox基因在矮化香蕉叶片和茎秆中的表达水平整体上低于野生型,其中GA3ox在野生型茎秆中的表达水平是矮化植株的2.2～32倍。综上推测,GA3ox基因可能对香蕉茎杆的矮化变异具有重要的调控作用。该研究结果为揭示香蕉矮化突变的分子机制与筛选优良矮化香蕉株系奠定了基础。     

甘蓝型油菜BnFLA基因的克隆及表达分析

利用同源克隆法从甘蓝型油菜中获得了1个类成束阿拉伯半乳聚糖蛋白基因(FLA),命名为BnFLA。BnFLA基因开放阅读框长为1 200bp,编码399个氨基酸,分子量为42 885.9Da,等电点为6.37。预测的BnFLA蛋白包含N-端信号肽、2个AGP-like结构域、2个fasciclin-like结构域和C-端GPI-anchor序列。系统进化分析表明BnFLA氨基酸序列与BrFLA17和AtFLA2进化关系较近,一致性分别为98%和87%。qRT-PCR分析表明,BnFLA基因在油菜各组织均有表达,并以下胚轴中表达量最高,其次为子叶,茎秆中表达最少;BnFLA基因的表达受到GA3、BR、IAA、ABA和NaCl的诱导,但受6-BA、蔗糖、低温和PEG抑制。研究认为,油菜中BnFLA基因可能参与激素信号转导途径和非生物胁迫应答。     

The rice <Emphasis Type="Italic">OsLOL2</Emphasis> gene encodes a zinc finger protein involved in rice growth and disease resistance

Arabidopsis LSD1-related proteins that contain LSD1-like zinc finger domains have been identified to be involved in disease resistance and programmed cell death. To investigate the potential role of LSD1-related gene in rice (Oryza sativa L.), we cloned an LSD1 ortholog, OsLOL2, from the rice cDNA plasmid library. The OsLOL2 gene is predicted to encode a polypeptide of 163 amino acids with two LSD1-like zinc finger domains with 74.5% identity to those of LSD1. Southern blot analysis indicated that OsLOL2 was a single-copy gene in the rice genome. Transgenic rice lines carrying the antisense strand of OsLOL2 with decreased expression of OsLOL2 had dwarf phenotypes, and the dwarfism could be restored by exogenous GA₃ treatment, suggesting that the dwarfism was the result of a deficiency in bioactive gibberellin (GA). In agreement with this possibility, the content of endogenous bioactive GA₁ decreased in the antisense transgenic lines. Expression of OsKS1, one of the genes encoding for GA biosynthetic enzymes, was suppressed in the antisense transgenic lines. Sense transgenic lines with increased expression of OsLOL2 were more resistant to rice bacterial blight, while antisense transgenic lines were less resistant to rice bacterial blight. The OsLOL2-GFP (green fluorescence protein) fusion protein was localized in the nucleus of cells of transgenic BY2 tobacco (Nicotiana tabacum L.). These data suggest that OsLOL2 is involved in rice growth and disease resistance.     

Over-expression of a tobacco homeobox gene, NTH15 , decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase

Ectopic expression of the homeobox gene, NTH15 ( Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA₁. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA₁₉ to GA₂₀ was blocked in transgenic tobacco plants overexpressing NTH15 . To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3β-hydroxylase. RNA gel blot analysis revealed that the expression of one gene ( Ntc12 , encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA₂₀ or GA₁ but not by GA₁₉. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12 , resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.