Interactions between ethylene,abscisic acid and cytokinin during germination and seedling establishment in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to investigate the interaction of the plant hormones ethylene, abscisic acid (ABA) and cytokinin in seed germination and early seedling development, we studied germination in ethylene-related mutants of Arabidopsis. Mutations in the genes etr1 and ein2, which reduce ethylene responses, showed increased dormancy and a delay in germination in comparison with wild type. Mutations in etr1, ein2 and ein6 also resulted in increased sensitivity to ABA with respect to inhibition of germination. Conversely, mutations in ctr1 and eto3, which lead to an increased ethylene response and overproduction of ethylene, respectively, decreased sensitivity to ABA during germination. Increased ABA sensitivity was also effected in wild type seeds by the presence during germination of AgNO₃, an inhibitor of ethylene action. The addition of the cytokinin N-6 benzyl adenine (BA) reversed the increased sensitivity of ethylene-resistant mutants to ABA. The action of cytokinin in reversing increased ABA sensitivity of ethylene-resistant mutants also suggests that at least part of the action of cytokinin in promoting germination is independent of its role in stimulating ethylene production. These observations further extend the evidence in support of interaction between ethylene, ABA and cytokinin signalling in controlling seed germination and early seedling development in Arabidopsis.     

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.     

The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination

In Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moist-chilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moist-chilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.     

The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.     

DEXH box RNA helicase-mediated mitochondrial reactive oxygen species production in Arabidopsis mediates crosstalk between abscisic acid and auxin signaling

It is well known that abscisic acid (ABA) promotes reactive oxygen species (ROS) production through plasma membrane-associated NADPH oxidases during ABA signaling. However, whether ROS from organelles can act as second messengers in ABA signaling is largely unknown. Here, we identified an ABA overly sensitive mutant, abo6, in a genetic screen for ABA-mediated inhibition of primary root growth. ABO6 encodes a DEXH box RNA helicase that is involved in regulating the splicing of several genes of complex I in mitochondria. The abo6 mutant accumulated more ROS in mitochondria, as established using a mitochondrial superoxide indicator, circularly permuted yellow fluorescent protein. Two dominant-negative mutations in ABA insensitive1 (abi1-1) and abi2-1 greatly reduced ROS production in mitochondria. The ABA sensitivity of abo6 can also be compromised by the atrbohF mutation. ABA-mediated inhibition of seed germination and primary root growth in abo6 was released by the addition of reduced GSH and exogenous auxin to the medium. Expression of auxin-responsive markers ProDR5:GUS (for synthetic auxin response element D1-4 with site-directed mutants in the 5'-end from soybean):β-glucuronidase) and Indole-3-acetic acid inducible2:GUS was greatly reduced by the abo6 mutation. Hence, our results provide molecular evidence for the interplay between ABA and auxin through the production of ROS from mitochondria. This interplay regulates primary root growth and seed germination in Arabidopsis thaliana.     

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     

BAK1 plays contrasting roles in regulating abscisic acid-induced stomatal closure and abscisic acid-inhibited primary root growth in Arabidopsis

The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid (ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined the responses of Arabidopsis thaliana protein kinase mutants to ABA treatment. We found that mutants of BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) were hypersensitive to the effects of ABA on both seed germination and primary root growth. The kinase OPEN STOMATA 1 (OST1) was more highly activated by ABA in bak1 mutant than the wild type. BAK1 was not activated by ABA treatment in the dominant negative mutant abi1-1 or the pyr1 pyl4 pyl5 pyl8 quadruple mutant, but it was more highly activated by this treatment in the abi1-2 abi2-2 hab1-1 loss-of-function triple mutant than the wild type. BAK1 phosphorylates OST1 T146 and inhibits its activity. Genetic analyses suggested that BAK1 acts at or upstream of core components in the ABA signaling pathway, including PYLs, PP2Cs, and SnRK2s, during seed germination and primary root growth. Although the upstream brassinosteroid (BR) signaling components BAK1 and BR INSENSITIVE 1 (BRI1) positively regulate ABA-induced stomatal closure, mutations affecting downstream components of BR signaling, including BRASSINOSTEROID-SIGNALING KINASEs (BSKs) and BRASSINOSTEROID-INSENSITIVE 2 (BIN2), did not affect ABA-mediated stomatal movement. Thus, our study uncovered an important role of BAK1 in negatively regulating ABA signaling during seed germination and primary root growth, but positively modulating ABA-induced stomatal closure, thus optimizing the plant growth under drought stress.     

Abscisic acid rescues the root meristem defects of the Medicago truncatula latd mutant

The LATD gene of the model legume, Medicago truncatula, is required for the normal function of three meristems, i.e. the primary root, lateral roots and nitrogen-fixing nodules. In latd mutants, primary root growth eventually arrests, resulting in a disorganized root tip lacking a presumptive meristem and root cap columella cells. Lateral root organs are more severely affected; latd lateral roots and nodules arrest immediately after emerging from the primary root, and reveal a lack of organization. Here we show that the plant hormone, abscisic acid (ABA), can rescue the latd root, but not nodule, meristem defects. Growth on ABA is sufficient to restore formation of small, cytoplasm-rich cells in the presumptive meristem region, rescue meristem organization and root growth and formation of root cap columella cells. In contrast, inhibition of ethylene synthesis or signaling fails to restore latd primary root growth. We find that latd mutants have normal levels of ABA, but exhibit reduced sensitivity to the hormone in two other ABA-dependent processes: seed germination and stomatal closure. Together, these observations demonstrate that the latd mutant is defective in the ABA response and indicate a role for LATD-dependent ABA signaling in M. truncatula root meristem function.     

Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway

The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid. The accuracy of this approximation is discussed. Considering its limitations, it allows a geometric approximation to the analysis of changes occurring in seed shape during imbibition prior to seed germination as well as the differences in shape between genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2, etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show different response to imbibition than the wild type. Imbibed seeds of the wild type increase their asymmetry compared with the dry seeds. This is detected by the relative changes in the curvature values in both poles. Thus, during imbibition of the wild-type seeds, the reduction in curvature values observed in the basal pole gives them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in curvature values occurs in both basal and apical poles, and its shape remains as a prolate spheroid. Our data indicate that the ethylene signaling pathway is involved, in general, in the complex regulation of seed shape and, in particular, in the establishment of polarity in seeds, controlling curvature values in the seed poles.     

Maternal Effects Govern Variable Dominance of Two Abscisic Acid Response Mutations in Arabidopsis thaliana

Three abscisic acid (ABA)-controlled responses (seed dormancy, inhibition of germination by applied ABA, and stomatal closure) were compared in wild-type versus homo- and heterozygotes of two Arabidopsis thaliana ABA-insensitive mutants, abi1 and abi2. We found that sensitivity of seeds to applied ABA is partially maternally controlled but that seed dormancy is determined by the embryonic genotype. The effects of the abi1 and abi2 mutations on ABA sensitivity of seed germination ranged from recessive to nearly fully dominant, depending on the parental source of the mutant allele. This maternal effect disappeared during vegetative growth. Stomatal regulation in heterozygotes showed substantial variability, but the average water loss was intermediate between that of homozygous mutants and wild type.     

Mutational analysis of <Emphasis Type="Italic">Arabidopsis PP2CA2</Emphasis> involved in abscisic acid signal transduction

The homozygous T-DNA mutant of the PP2CA2 gene in Arabidopsis thaliana was identified at DNA and RNA levels. The semi-quantitative RT-PCR analysis showed expression of PP2CA2 was induced by NaCl and ABA. When grown in presence of increasing concentration of exogenous ABA the pp2ca2 mutant showed a significant loss of ABA sensitivity in terms of seed germination, efficiency of post-germination growth and root growth. In presence of all ABA and NaCl concentrations tested the germination percentage of wild-type seeds was lower than that of mutant ppca2 seeds. Furthermore, in the presence of exogenous ABA, the pp2ca2 seeds showed higher germination percentages than wild-type at different stages of development and the pp2ca2 seedlings showed a reduced inhibition of root growth compared with wild-type plants. The above results indicated that the pp2ca2 was an ABA-hyposensitive mutant.     

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.     

Inhibition of Amaranthus caudatus seed germination by polyethylene glycol-6000 and abscisic acid and its reversal by ethephon or 1-aminocyclopropane-1-carboxylic acid

The effect of polyethylene glycol (PEG-6000), abscisic acid (ABA), 2-chloroethylphosphonic acid (ethephon) and 1-aminocyclopropane-1-carboxylic acid (ACC) on the germination of Amaranthus caudatus L. seeds was examined. Both PEG-6000 and ABA inhibited the rate and percentage of seed germination. ABA potentiated the effect of PEG. Ethephon was highly effective in reversing the inhibitory effect of PEG and ABA or combinations of both. ACC relieved inhibition by ABA and the combined effect of ABA and PEG. Aminoethoxyvinylglycine (AVG) increased the inhibition of seed germination caused by ABA. The inhibition of seed germination by ABA seems to be related more or less to ethylene biosynthesis or is associated with a change of tissue sensitivity to ethylene. The possibility of ethylene control of water uptake by seeds is also considered.     

Arabidopsis CPR5 independently regulates seed germination and postgermination arrest of development through LOX pathway and ABA signaling

The phytohormone abscisic acid (ABA) and the lipoxygenases (LOXs) pathway play important roles in seed germination and seedling growth and development. Here, we reported on the functional characterization of Arabidopsis CPR5 in the ABA signaling and LOX pathways. The cpr5 mutant was hypersensitive to ABA in the seed germination, cotyledon greening and root growth, whereas transgenic plants overexpressing CPR5 were insensitive. Genetic analysis demonstrated that CPR5 gene may be located downstream of the ABI1 in the ABA signaling pathway. However, the cpr5 mutant showed an ABA independent drought-resistant phenotype. It was also found that the cpr5 mutant was hypersensitive to NDGA and NDGA treatment aggravated the ABA-induced delay in the seed germination and cotyledon greening. Taken together, these results suggest that the CPR5 plays a regulatory role in the regulation of seed germination and early seedling growth through ABA and LOX pathways independently.     

Nitric oxide suppresses the inhibitory effect of abscisic acid on seed germination by S-nitrosylation of SnRK2 proteins

Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.