Abscisic acid and gibberellic acid-regulated responses of embryos and aleurone layers isolated from dormant and nondormant barley grains

Dormant and nondormant isogenic barley grains were obtained by maturing grains under short day (SD) or long day (LD) growth conditions, respectively. Hormonal responses of isolated embryos and aleurone layers from these grains were studied. Addition of abscisic acid (ABA) reduced germination rate and percentage of embryos, and induced Rab (ABA-responsive) mRNA in aleurone layers from both types of grain. Embryos and aleurone layers from dormant grains responded stronger to ABA than those from nondormant grains. Gibberellic acid (GA₃) increased the germination rate and percentage of embryos from dormant grains and counteracted the ABA-induced inhibition of embryo germination. GA₃ did not affect the amount of Rab mRNA in aleurone layers, suggesting that expression of the Rab gene has no direct correlation with germination. The stronger response of embryos and aleurone layers from dormant grains to ABA may not be explained by higher endogenous ABA levels, but might be due to differences in hormone signal transduction. Aleurone protoplasts from dormant grains had a higher cytosolic pH than those from nondormant grains. To inhibit the ABA-induced Rab mRNA, a much higher concentration of weak acid was required for aleurone layers from dormant grains than for those from nondormant grains. A possible difference in ABA signal transduction between dormant and nondormant grains is discussed.     

PM19, a barley (Hordeum vulgare L.) gene encoding a putative plasma membrane protein, is expressed during embryo development and dormancy.

A barley (Hordeum vulgare L.) cDNA, PM19, encoding a putative plasma membrane protein was isolated through differential screening of a dormant wild oat embryo library. PM19 is expressed in barley embryos from mid-embryogenesis up to maturity. PM19 mRNA levels decline upon germination, whereas dormant embryos retained high levels of message for up to 72 h of imbibition. PM19 mRNA levels also remained high or were reinduced in non-dormant embryos by treatments that prevented germination (250 mm NaCl, 10% sorbitol, or 50 microm ABA). The PM19 protein sequence is highly conserved in monocotyledonous and dicotyledonous plants.     

赤霉素与脱落酸对番茄种子萌发中细胞周期的调控

利用细胞流检仪检测番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．） ＧＡ－缺陷型、ＡＢＡ－缺陷型和相应的正常品种（野生型）成熟种子胚根尖细胞倍性水平时发现：ＧＡ－缺陷型和野生型种子绝大多数细胞ＤＮＡ 水平为２Ｃ,而ＡＢＡ－缺陷型种子则含有较多的４Ｃ细胞。在标准发芽条件下,ＡＢＡ－缺陷型和野生型种子浸种１ ｄ 后胚根尖细胞ＤＮＡ 开始复制,随后胚根突破种皮而发芽。然而ＧＡ－缺陷型种子除非加入外源ＧＡ,否则既不发生细胞ＤＮＡ 复制,也不发芽。这说明内源ＧＡ 是启动番茄种子胚根尖细胞ＤＮＡ 复制的关键因素,同时也说明番茄根尖细胞ＤＮＡ 复制是种子发芽的必要条件。实验证明：ＡＢＡ 不抑制细胞ＤＮＡ 合成,但阻止Ｇ２ 细胞进入到Ｍ 期。外源ＡＢＡ处理野生型种子与渗控处理结果相似,可以大幅度提高胚根尖４Ｃ／２Ｃ细胞的比例,但抑制种子的最终发芽     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

GA and ABA Regulation on the Cell Cycle in Germination of Tomato Seeds

Flow cytometric determination of ploidy levels in embryos of GA-deficient, ABA-deficient mutant and isogenic wild type tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds revealed that, large amount of 2C DNA signals existed both in wild type and GA-deficient mutant seeds, showing that most cells had arrested in the cell cycle at presynthesis Gl, whereas a relative amount of 4C proportion which is a sign of seed germination was found in ABA-deficient mutant seeds, indicating that endogenous ABA play a role in regulating the switch from development to germination in seeds. DNA replication was stimulated 1 d after the seed was imbibed in water and a visible germination occurred subsequently either in wild type GA-deficient mutant seeds. But it was not the case for ABA-deficient mutant seeds unless an exogenous GA was supplemented. This demonstrated that DNA replication in embryo root tips cells was subjected to be a compulsory factor for seed germination, whereas endogenous GA triggered DNA synthesis. It was evident that exogenous ABA could inhibit seed germination not by suppressing DNA synthesis but by bloking the route leading to mitosis since a great amount of 4C proportion was found in the germinating wild type and GA-deficient mutant seeds in the ABA solution when visible ger mination did not occur. Finally a simple mode of hormonal regulation on cell cycle in high plants was hypothesized.     

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.     

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.     

Onset of desiccation tolerance during development of the barley embryo

We have investigated events which take place in the developing barley (Hordeum vulgare L.) embryo during its acquisition of desiccation tolerance. Excised embryos are capable of precocious germination as early as 8 d after pollination (DAP). At this age, however, they are not capable of resisting a desiccation treatment which induces a loss of 96–98% of their initial water content. At 16 DAP the embryos germinate despite the drastic drying treatment. The pattern of in-vivo and in-vitro proteins synthesized by the developing embryos from 12 DAP (desiccation-intolerant) and 16 DAP (desiccation-tolerant) were compared. A set of 25–30 proteins was identified which is denovo synthesized or enhanced during the developmental period leading to desiccation tolerance. Abscisic acid (ABA; 100 M) applied in vitro for 5 d to 12-DAP embryos induces desiccation tolerance and represses a subset of polypeptides preferentially associated with 16-DAP embryos. During in vitro culture of barley embryos ABA stimulates the appearance of a set of proteins and prevents the precocious germination allowing embryogenesis to continue in vitro. It also suppresses a set of germination-related proteins which appear 4 h after the incubation of the dissected embryo on a germination medium without ABA. Almost all mRNAs remain functional for translation when isolated embryos are dried at the desiccation-intolerant and tolerant stages of embryo development.Abbreviations ABA abscisic acid - DAP days after pollination - GM germination medium - poly(A)RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism

In a wide range of plant species, seed germination is regulated antagonistically by two plant hormones, abscisic acid (ABA) and gibberellin (GA). In the present study, we have revealed that ABA metabolism (both biosynthesis and inactivation) was phytochrome-regulated in an opposite fashion to GA metabolism during photoreversible seed germination in Arabidopsis. Endogenous ABA levels were decreased by irradiation with a red (R) light pulse in dark-imbibed seeds pre-treated with a far-red (FR) light pulse, and the reduction in ABA levels in response to R light was inhibited in a phytochrome B (PHYB)-deficient mutant. Expression of an ABA biosynthesis gene, AtNCED6, and the inactivation gene, CYP707A2, was regulated in a photoreversible manner, suggesting a key role for the genes in PHYB-mediated regulation of ABA metabolism. Abscisic acid-deficient mutants such as nced6-1, aba2-2 and aao3-4 exhibited an enhanced ability to germinate relative to wild type when imbibed in the dark after irradiation with an FR light pulse. In addition, the ability to synthesize GA was improved in the aba2-2 mutant compared with wild type during dark-imbibition after an FR light pulse. Activation of GA biosynthesis in the aba2-2 mutant was also observed during seed development. These data indicate that ABA is involved in the suppression of GA biosynthesis in both imbibed and developing seeds. Spatial expression patterns of the AtABA2 and AAO3 genes, responsible for last two steps of ABA biosynthesis, were distinct from that of the GA biosynthesis gene, AtGA3ox2, in both imbibed and developing seeds, suggesting that biosynthesis of ABA and GA in seeds occurs in different cell types.     

The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination

Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth. To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants. Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant. Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated. These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development. Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed. Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants. These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development. We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type. The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed.     

Barley strigolactone signalling mutant hvd14.d reveals the role of strigolactones in abscisic acid-dependent response to drought

Strigolactones (SLs) are a group of plant hormones involved in many aspects of plant development and stress adaptation. Here, we investigated the drought response of a barley (Hordeum vulgare L.) mutant carrying a missense mutation in the gene encoding the SL-specific receptor HvD14. Our results clearly showed that hvd14.d mutant is hyper-sensitive to drought stress. This was illustrated by a lower leaf relative water content (RWC), impaired photosynthesis, disorganization of chloroplast structure, altered stomatal density and slower closure of stomata in response to drought in the mutant compared to the wild type parent cultivar Sebastian. Although the content of abscisic acid (ABA) and its derivatives remained unchanged in the mutant, significant differences in expression of genes related to ABA biosynthesis were observed. Moreover, hvd14.d was insensitive to ABA during seed germination. Analysis of Arabidopsis thaliana mutant atd14-1 also demonstrated that mutation in the SL receptor resulted in increased sensitivity to drought. Our results indicate that the drought-sensitive phenotype of barley SL mutant might be caused by a disturbed ABA metabolism and/or signalling pathways. These results together uncovered a link between SL signalling and ABA-dependent drought stress response in barley.     

Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single‐nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild‐type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.     

The rea (red embryonic axis) phenotype describes a new mutation affecting the response of maize embryos to abscisic acid and osmotic stress

During a screen for mutants with defective germination, a newphenotype was observed consisting of red pigmentation of theembryonic axis in the dormant seed. Segregation ratios, as determinedin F₂ and back-crossed progeny, indicate that the phenotypeis due to a recessive single gene mutation that has been symbolizedrea to denote red embryonic axis. A closer inspection of therea phenotype revealed that the mutant is occasionally viviparous,indicating a defect in abscisic acid (ABA) metabolism. The mutationprobably affects ABA sensitivity since no difference in ABAcontent was detected in mutant versus normal tissues. Moreover,when immature mutant and wild-type embryos were incubated onmedia containing 10 M ABA, only the mutants germinated. ABA-regulatedgene expression in rea embryos differed from that of embryosof the viviparous mutant vp1 which does not respond to the inhibitoryaction of ABA at the level of immature embryo germination. Theseresults, therefore, indicate that the two genes exert a differentrole in the control of embryogenesis. Key words: Zea mays L, embryo dormancy, ABA     

Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts

Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.     

Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog

Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.     

Determination of endogenous abscisic acid levels in immature cereal embryos during in vitro culture

Levels of endogenous abscisic acid (ABA) in immature wheat (Triticum aestivum cv. Timmo) and barley (Hordeum vulgare cv. Golden Promise) embryos have been determined by enzyme-linked immunosorbent assay. Embryos of both cereal species showed an increase in ABA content during development on the parent plant. Immature embryos were excised and cultured in vitro on nutrient media that led to precocious germination or on media containing 9% (w/v) mannitol that maintained their developmental arrest. Barley and wheat embryos responded to these culture conditions in an identical manner with respect to changes in morphology, fresh weight, protein and lectin content. However, in complete contrast, the ABA content of barley embryos increased by an order of magnitude during culture on mannitol, whereas that of wheat embryos showed no significant change. The results are discussed within the context of the role of ABA in the regulation of embryo development.Abbreviations ABA abscisic acid - BGA barley-germ agglutinin - dpa days post anthesis - ELISA enzyme-linked immunosorbent assay - GC-MS gas chromatography-mass spectrometry - WGA wheat-germ agglutinin     

拟南芥prr5突变体对ABA的响应

以拟南芥为材料,统计PRRs （pseudo-response regulators）突变体 prr5及其野生型经ABA处理后的萌发率、根长和NaCl处理后的萌发率,并采用实时定量PCR方法,对不同浓度ABA处理的拟南芥幼苗中的PRR5基因表达进行分析.结果表明：prr5突变体对ABA弱敏感,其种子萌发率比野生型显著或极显著增高,主根比野生型长,且PRR5基因表达受ABA抑制.同时,NaCl处理后,prr5的萌发率比野生型极显著增高.因此,推测prr5可能为ABA信号通路相关基因.     

Maize VP1 complements Arabidopsisabi3 and confers a novel ABA/auxin interaction in roots

The maize Vp1 gene and abi3 gene of Arabidopsis are believed to be orthologs based on similarities of the mutant phenotypes and amino acid sequence conservation. Here we show that expression of VP1 driven by the 35S promoter can partially complement abi3-6, a deletion mutant allele of abi3. The visible phenotype of seed produced from VP1 expression in the abi3 mutant background is nearly indistinguishable from wild type. VP1 fully restores abscisic acid (ABA) sensitivity of abi3 during seed germination and suppresses the early flowering phenotype of abi3. The temporal regulation of C1-beta-glucuronidase (GUS) and chlorophyll a/b binding protein (cab3)-GUS reporter genes in developing seeds of 35S-VP1 lines were similar to wild type. On the other hand, two qualitative differences are observed between the 35S-VP1 line and wild type. The levels of CRC and C1-GUS expression are markedly lower in the seeds of 35S-VP1 lines than in wild type suggesting incomplete complementation of gene activation functions. Similar to ectopic expression of ABI3 (Parcy et al., 1994), ectopic expression of VP1 in vegetative tissue enhances ABA inhibition of root growth. In addition, 35S-VP1 confers strong ABA inducible expression of the normally seed-specific cruciferin C (CRC) gene in leaves. In contrast, ectopic ABA induction of C1-GUS is restricted to a localized region of the root elongation zone. The ABA-dependent C1-GUS expression expanded to a broader area in the root tissues treated with exogenous application of auxin. Interestingly, auxin-induced lateral root formation is completely suppressed by ABA in 35S-VP1 plants but not in wild type. These results indicate VP1 mediates a novel interaction between ABA and auxin signaling that results in developmental arrest and altered patterns of gene expression.