Gregorio Nicolas

Evidence for a Role of Gibberellins in Salicylic Acid-Modulated Early Plant Responses to Abiotic Stress in Arabidopsis Seeds

Exogenous application of gibberellic acid (GA3) was able to reverse the inhibitory effect of salt, oxidative, and heat stresses in the germination and seedling establishment of Arabidopsis (Arabidopsis thaliana), this effect being accompanied by an increase in salicylic acid (SA) levels, a hormone that in recent years has been implicated in plant responses to abiotic stress. Furthermore, this treatment induced an increase in the expression levels of the isochorismate synthase1 and nonexpressor of PR1 genes, involved in SA biosynthesis and action, respectively. In addition, we proved that transgenic plants overexpressing a gibberellin (GA)-responsive gene from beechnut (Fagus sylvatica), coding for a member of the GA3 stimulated in Arabidopsis (GASA) family (FsGASA4), showed a reduced GA dependence for growth and improved responses to salt, oxidative, and heat stress at the level of seed germination and seedling establishment. In 35S:FsGASA4 seeds, the improved behavior under abiotic stress was accompanied by an increase in SA endogenous levels. All these data taken together suggest that this GA-responsive gene and exogenous addition of GAs are able to counteract the inhibitory effects of these adverse environmental conditions in seed germination and seedling growth through modulation of SA biosynthesis. Furthermore, this hypothesis is supported by the fact that sid2 mutants, impaired in SA biosynthesis, are more sensitive to salt stress than wild type and are not affected by exogenous application of GA3.Salicylic acid (SA) is a plant hormone mainly associated with the induction of defense mechanism in plants, although in the last years there is increasing evidence on the role of SA in plant responses to abiotic stress. We recently reported that an increase in endogenous SA levels are able to counteract the inhibitory effects of several abiotic stress conditions during germination and seedling establishment of Arabidopsis thaliana and that this effect is modulated by gibberellins (GAs) probably through a member of the GASA (Giberellic Acid Stimulated in Arabidopsis) gene family, clearly showing the existence of a cross talk between these two plant hormones in Arabidopsis.

Negative Regulation of Abscisic Acid Signaling by the Fagus sylvatica FsPP2C1 Plays A Role in Seed Dormancy Regulation and Promotion of Seed Germination

FsPP2C1 was previously isolated from beech (Fagus sylvatica) seeds as a functional protein phosphatase type-2C (PP2C) with all the conserved features of these enzymes and high homology to ABI1, ABI2, and PP2CA, PP2Cs identified as negative regulators of ABA signaling. The expression of FsPP2C1 was induced upon abscisic acid (ABA) treatment and was also up-regulated during early weeks of stratification. Furthermore, this gene was specifically expressed in ABA-treated seeds and was hardly detectable in vegetative tissues. In this report, to provide genetic evidence on FsPP2C1 function in seed dormancy and germination, we used an overexpression approach in Arabidopsis because transgenic work is not feasible in beech. Constitutive expression of FsPP2C1 under the cauliflower mosaic virus 35S promoter confers ABA insensitivity in Arabidopsis seeds and, consequently, a reduced degree of seed dormancy. Additionally, transgenic 35S:FsPP2C1 plants are able to germinate under unfavorable conditions, as inhibitory concentrations of mannitol, NaCl, or paclobutrazol. In vegetative tissues, Arabidopsis FsPP2C1 transgenic plants show ABA-resistant early root growth and diminished induction of the ABA-response genes RAB18 and KIN2, but no effect on stomatal closure regulation. Seed and vegetative phenotypes of Arabidopsis 35S:FsPP2C1 plants suggest that FsPP2C1 negatively regulates ABA signaling. The ABA inducibility of FsPP2C1 expression, together with the transcript accumulation mainly in seeds, suggest that it could play an important role modulating ABA signaling in beechnuts through a negative feedback loop. Finally, we suggest that negative regulation of ABA signaling by FsPP2C1 is a factor contributing to promote the transition from seed dormancy to germination during early weeks of stratification.

The nuclear interactor PYL8/RCAR3 of Fagus sylvatica FsPP2C1 is a positive regulator of abscisic acid signaling in seeds and stress

The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two closely related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis (Arabidopsis thaliana) BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion (Allium cepa) cells and agroinfiltration in tobacco (Nicotiana benthamiana) as green fluorescent protein fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by bimolecular fluorescence complementation assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds and, consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl, or paclobutrazol, which are not inhibiting conditions to the wild type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses.

Overexpression of a protein phosphatase 2C from beech seeds in arabidopsis shows phenotypes related to abscisic acid responses and gibberellin biosynthesis

A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis.

Evidence for Positive Regulation by Gibberellins and Ethylene of ACC Oxidase Expression and Activity During Transition From Dormancy to Germination in Fagus sylvatica L. Seeds

Abstract1-aminocyclopropane-1-carboxylic acid (ACC) oxidase is a key enzyme in the ethylene biosynthesis pathway. In the present report the changes in ACC content, ACC oxidase activity and ethylene production have been examined in Fagus sylvatica L. dormant seeds after stratification and different treatments that maintain or release dormancy. Additionally, a cDNA encoding an ACC oxidase (ACO) from Fagus sylvatica has been isolated and characterized. This clone, named FsACO1, exhibits high homology to ACC oxidases from several plant species and the corresponding enzyme, expressed in Escherichia coli as a fusion protein, is active in converting ACC into ethylene. The transcript levels of FsACO1 are correlated with the ACC content, the ethylene production and the ACC oxidase activity measured in vitro as well as with the germination percentages observed in the seeds under the different treatments used in this study. There is a drastic increase in all these parameters when seeds are treated with GA3 or ethephon (which releases ethylene in solution), hormones previously proven to be efficient in the breaking of dormancy of beech seeds. The stimulatory effect of ethephon is reverted by paclobutrazol, a well known GA biosynthesis inhibitor. These results indicate that ethylene biosynthesis is positively regulated by both gibberellins and ethylene and suggest a cross-talk regulation by these two hormones of the processes involved in the transition from seed dormancy to germination.

Ethylene regulates the expression of a cysteine proteinase gene during germination of chickpea (Cicer arietinum L.)

Synthetic oligonucleotides corresponding to conserved regions of cysteine proteinases were used as primers in the RT-PCR amplification of a fragment of cDNA corresponding to a region of a cysteine proteinase gene expressed during germination of chickpea (cac for Cicer arietinum cysteine proteinase). The identity of the PCR-amplified fragment was confirmed by sequencing and the fragment used as a probe to investigate the pattern of cac gene expression during germination and its hormonal regulation. The corresponding transcript is undetected in the seed during embryogenesis and before imbibition, being detected 24 h after imbibition. Ablation of the embryonic axis before imbibition results in a dramatic decrease in the amount of transcript detected. Expression of the cac transcript in excised cotyledons is restored in the presence of aqueous extracts from embryonic axes and also by incubating the excised cotyledons in 1 mM ethephon. Experiments with various known inhibitors of ethylene action indicate that ethylene activates the expression of cac gene in the cotyledons of chickpea during normal germination.

Transcripts of a gene, encoding a small GTP-binding protein from Fagus sylvatica, are induced by ABA and accumulated in the embryonic axis of dormant seeds

A cDNA clone was selected from a cDNA library constructed using mRNA from ABA-treated Fagus sylvatica L. dormant seeds as a template. The clone is highly expressed in the presence of ABA and tends to disappear in stratified seeds. A search of sequence databases showed that the clone encodes a small GTP-binding protein. By means of in situ hybridization, the mRNA has been located in the apical meristem of the embryonic axis and in the central vascular cylinder. Its possible involvement in growth regulation in the embryonic axis of F. sylvatica is discussed.

Cross-talk between gibberellins and salicylic acid in early stress responses in Arabidopsis thaliana seeds.

Salicylic acid (SA) is a plant hormone mainly associated with the induction of defense mechanism in plants, although in the last years there is increasing evidence on the role of SA in plant responses to abiotic stress. We recently reported that an increase in endogenous SA levels are able to counteract the inhibitory effects of several abiotic stress conditions during germination and seedling establishment of Arabidopsis thaliana and that this effect is modulated by gibberellins (GAs) probably through a member of the GASA (Giberellic Acid Stimulated in Arabidopsis) gene family, clearly showing the existence of a cross talk between these two plant hormones in Arabidopsis.

Evidence of a role for tyrosine dephosphorylation in the control of postgermination arrest of development by abscisic acid in Arabidopsis thaliana L

In the present paper evidence is presented indicating that tyrosine dephosphorylation is a key regulatory mechanism in postgermination arrest of Arabidopsis thaliana L. seed development mediated by abscisic acid (ABA). By using phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, the sensitivity to the inhibitory effect of ABA on seed germination is enhanced. Consistent with this finding, we demonstrate that the ABA-responsive gene, RAB18, is hyperinduced in seeds imbibed in ABA plus PAO, compared with seeds imbibed only with ABA.

Variations of cell wall peroxidases in epicotyls of Cicer arietinum during growth

Abstract Fractionation and partial purification of the proteins extracted from cell walls of Cicer arietinum epicotyl reveal the presence of two fractions with peroxidase activity, denominated Px-1 and Px-2 according to their order of elution from a SP-Sephadex chromatography column. The specific activity of the Px-2 fraction increases with the age of the epicotyls, with an inverse relationship to their growth capacity. No such a relationship could be found with the peroxidase activity of the Px-1 fractions. These results indicate that the cell wall peroxidase fraction Px-2 could be related to cell wall stiffening processes. According to this, acid pHs-values, which induce cell wall loosening processes, diminish the enzymatic activity of the Px-2 fraction to a considerable extent.