Ascorbic acid and reactive oxygen species are involved in the inhibition of seed germination by abscisic acid in rice seeds

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. The possible links among ABA, reactive oxygen species (ROS), ascorbic acid (ASC), and GA during rice seed germination were investigated. Unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in the embryo region. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis.     

Inhibition of germination of dormant barley (Hordeum vulgare L.) grains by blue light as related to oxygen and hormonal regulation

Germination of primary dormant barley grains is promoted by darkness and temperatures below 20 °C, but is strongly inhibited by blue light. Exposure under blue light at 10 °C for periods longer than five days, results in a progressive inability to germinate in the dark, considered as secondary dormancy. We demonstrate that the inhibitory effect of blue light is reinforced in hypoxia. The inhibitory effect of blue light is associated with an increase in embryo abscisic acid (ABA) content (by 3.5‐ to 3.8‐fold) and embryo sensitivity to both ABA and hypoxia. Analysis of expression of ABA metabolism genes shows that increase in ABA mainly results in a strong increase in HvNCED1 and HvNCED2 expression, and a slight decrease in HvABA8′OH‐1. Among the gibberellins (GA) metabolism genes examined, blue light decreases the expression of HvGA3ox2, involved in GA synthesis, increases that of GA2ox3 and GA2ox5, involved in GA catabolism, and reduces the GA signalling evaluated by the HvExpA11 expression. Expression of secondary dormancy is associated with maintenance of high embryo ABA content and a low HvExpA11 expression. The partial reversion of the inhibitory effect of blue light by green light also suggests that cryptochrome might be involved in this hormonal regulation.     

Comparison of two proteomics techniques used to identify proteins regulated by gibberellin in rice

Proteomics has become an essential methodology for large-scale analysis of proteins in various fields of plant biology. We compared two proteomics techniques, two-dimensional liquid chromatography (2D-LC) and fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), for their ability to identify proteins regulated by gibberellin (GA) in rice. Two-week-old rice seedlings were treated with or without 5 microM GA3 for 48 h and proteins extracted from the basal region of the leaf sheath. After separation of the proteins by the two techniques, the amino acid sequences of GA3-responsive proteins were analyzed using a protein sequencer and mass spectrometry. 2D-LC and 2D-DIGE were able to resolve 1248 protein fractions and 1500 proteins, respectively. Out of these, 2D-LC identified 9 proteins that were up-regulated and 9 that were down-regulated by GA treatment; 2D-DIGE identified 4 up-regulated and 4 down-regulated proteins. The two techniques detected overlapping sets of proteins. For example, cytosolic glyceraldehyde-3-phosphate dehydrogenase and photosystem II oxygen-evolving complex protein were identified as GA3-regulated proteins by both methods. In addition, these two methods uncovered GA3-regulated unknown proteins which had not been reported previously, and novel proteins which are not detected in 2D-PAGE followed by Coomassie brilliant blue staining. These results suggest that these two methods are among some of the very useful tools for detecting proteins that may function in various physiological and developmental processes in plants.     

GAMT2 Encodes a Methyltransferase of Gibberellic Acid That is Involved in Seed Maturation and Germination in Arabidopsis

Salicylic acid methyltransferase （SAMT）, benzoic acid methyltransferase （BAMT） and theobromine methyltransferase （TH） （henceforth, SABATH） family proteins belong to a unique class of mehtyltransferase that can methylate small molecular compounds Including indole-3-acidic acid （IAA）, salicylic acid （SA） and jasmonic acid （JA）, in plants. Here we report that the GAMT2 protein, which has 34.2% similarity with IAMT1 in the amino acid sequence, can methylate gibberellic acid （GA）. Biolnformatics analysis suggests that GAMT2 may be able to methylate one molecule larger than SA. GAMT2 is predominantly expressed in the developing seed embryo and endosperm in Arabidopsis. During seed germination, the expression of GAMT2 decreases until the cotyledons expand out of the seed coat. Overexpression of GAMT2 in Arabidopsis resulted in multiple phenotypes, including dwarfism, retarded growth, late flowering, and reduced fertility, which are similar to the phenotypes of GA-deficient mutants. Seed germination assay showed that GAMT2 overexpression in plants was hypersensitive to GA biosynthesis inhibitor （ancymidol） and abscisic acid （ABA） treatments, whereas the GAMT2 null mutant （SALK_075450） was slightly Insensitive to such treatments, suggesting that GAMT2 may methylate GA or ABA. Enzyme activity analysis indicated that GAMT2 was able to methylate GA3 into Methyi-GA3 in vitro, but could not methylate ABA. Microarray analysis on GAMT2 overexpression plants suggested that Methyl-GA may be an Inactive form of GA in Arabidopsis. These data suggest that GAMT2 Is Involved in seed maturation and germination by modulating GA activity.     

Abscisic acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce

The ontogenetic course followed by somatic embryos of interior spruce is highly dependent on the media concentration of abscisic acid (ABA). Little or no organized development occurs in the absence of ABA and as the level of ABA is increased, a range of embryo types is produced. "Shooty embryo" structures predominate in many callus lines at low levels of ABA (1-10 μM), while 10-20 μM ABA promotes the formation of bipolar embryos that germinate precociously. When ABA is increased to 30-40 μM, precocious germination is inhibited and opaque cotyledonary embryos characteristic of their zygotic counterparts are formed which enter a period of quiescence. Only "mature" somatic embryos contain significant amounts of storage proteins and the level to which these proteins accumulate is dependent on the concentration of ABA. Indole-butyric acid (IBA) included with ABA increases the number of mature embryos. Root elongation, which was used as a measure of embryo quality, was never observed from shooty embryo Structures and was 2-3 fold higher in mature embryos compared to those that germinated precociously.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

水稻胚胎发生与萌发早期分离胚中内源激素的变化

以酶联免疫吸附检测技术分析了水稻(Oryza sativa ssp．iapoica)分离胚不同发育时期及萌发早期的内源激素含量的动态变化。GA1含量是所测激素中含量最高的。GA1的变化趋势基本上与ABA相反。花后4d的胚中GAl和ABA的含量最高;花后8d到18d,GA1的含量下降,而ABA含量增加。在早期萌发过程中,种子吸涨后2d的胚中GA,含量迅速上升,而ABA下降。GA1／ABA的最高比值也出现在吸涨后2d的胚中。iPAs和ZRs的最高含量也出现在开花后4d的胚中,但随后含量均下降到相当低的水平,并几乎没有变化。研究结果进一步证实了GAl在早期胚胎发生和萌发过程中起重要的作用;推测iPAs和ZRs可能仅在胚胎发生的早期起作用;GA1与ABA含量之间的相对平衡控制着胚胎发育的过程。用分离胚作为测试材料可以避免胚乳等其他组织成分的干扰,从而比较准确地反映了胚的内源激素变化。此外,本研究是首次用4d的水稻幼胚作为激素含量测定的起始材料。     

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non‐germinated (NG) seeds treated (+GA₃) or not treated (control) with gibberellins (GA). With GA₃ treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA₃ treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA₃ treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1‐aminocyclopropane‐1‐carboylic acid (ACC) decreased after imbibition. In addition, α‐tocopherol and α‐tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).     

Abscisic acid pretreatment enhances salt tolerance of rice seedlings: Proteomic evidence

Enhanced salt tolerance of rice seedlings by abscisic acid (ABA) pretreatment was observed from phenotypic and physiological analyses. Total proteins from rice roots treated with ABA plus subsequent salt stress were analyzed by using proteomics method. Results showed that, 40 protein spots were uniquely upregulated in the seedlings under the condition of ABA pretreatment plus subsequent salt stress, whereas only 16 under the condition of salt treatment. About 78% (31 spots) of the 40 protein spots were only upregulated in the presence of the subsequent salt stress, indicating that plants might have an economical strategy to prevent energy loss under a false alarm. The results also showed that more enzymes involved in energy metabolism, defense, primary metabolism, etc. were upregulated uniquely in ABA-pretreated rice seedlings, suggesting more abundant energy supply, more active anabolism (nitrogen, nucleotide acid, carbohydrate, etc), and more comprehensive defense systems in ABA-pretreated seedlings than in salt stressed ones.     

Osgstu3 and osgtu4, encoding tau class glutathione S-transferases, are heavy metal- and hypoxic stress-induced and differentially salt stress-responsive in rice roots

Glutathione S-transferases (GSTs) have poorly understood roles in plant responses to environmental stresses. A polyethylene glycol (PEG)-induced tau class GST was identified in rice roots by protein microsequencing. PEG and the heavy metals Cd (20 microM), Zn (30 microM), Co and Ni rapidly and markedly induced osgstu4 and osgstu3 in rice seedling roots. Osgstu4 and osgstu3 were also induced in roots by hypoxic stress but not by cold nor heat shock. Salt stress and abscisic acid (ABA) rapidly induced osgstu3 in rice roots, whereas osgstu4 exhibited a late salt stress and no ABA response. Salicylic acid, jasmonic acid and the auxin alpha-naphthalene acetic acid triggered osgstu4 and osgstu3 expression. Osgstu4 and osgstu3 were rapidly and markedly induced by the antioxidant dithiothreitol and the strong oxidant hydrogen peroxide, which suggested that redox perturbations and reactive oxygen species are involved in their stress response regulations.     

Antagonism between abscisic acid and gibberellins is partially mediated by ascorbic acid during seed germination in rice

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination,^1,2 but the mechanism of antagonism during this process is not known. In the associated study,³ we investigated the relationship among ABA, reactive oxygen species (ROS), ascorbic acid (ASC) and GA during rice seed germination. ROS production is reduced by ABA, which hence results in decreasing ASC accumulation during imbibition. GA accumulation was also suppressed by a reduced ROS and ASC level, whereas application of exogenous ASC can partially rescue seed germination from ABA treatment. Further results show that production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. These studies reveal a new role for ASC in mediating the antagonism between ABA and GA during seed germination in rice.     

Identification of rice root proteins regulated by gibberellin using proteome analysis

Gibberellin (GA) promoted rice (Oryza sativa L.) root elongation in a concentration-dependent manner with roots grown in 0.1 µm GA₃ being 13.8% longer than controls. On the other hand, the roots of Tan-ginbozu, a semi-dwarf cultivar were 69.8% shorter in comparison with Nipponbare, a normal cultivar. Treatments with 10 µm uniconazole-P and 10 µm abscisic acid (ABA) caused decreases in root length in Tan-ginbozu by 44.6 and 79.2%, respectively. To investigate how GA influences rice root growth, proteome analysis techniques were applied. Extracted proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analysed using an automated protein sequencer and mass spectrometer. Sixteen proteins show differences in accumulation levels as a result of treatment with GA₃, uniconazole-P and ABA treatment and/or the difference between the semi-dwarf cultivar, Tan-ginbozu, and normal cultivars. Among these proteins, fructose-bisphosphate aldolase (EC 4.1.2.13) increased in roots treated with GA₃, occurred in low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole-P or ABA. Moreover, roots from seedlings grown in 100 µm glucose were 9.1% longer than controls. These results indicate that increases in fructose-bisphosphate aldolase activity stimulate the glycolytic pathway and may play an important role in the GA-induced growth of roots.     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.     

Proteome analysis of rice root proteins regulated by gibberellin

To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.     

The slender rice mutant,with constitutively activated gibberellin signal transduction,has enhanced capacity for abscisic acid level

The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible alpha-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.