Level of glutathione is regulated by ATP-dependent ligation of glutamate and cysteine through photosynthesis in Arabidopsis thaliana: mechanism of strong interaction of light intensity with flowering

Glutathione (GSH) is associated with flowering in Arabidopsis thaliana, but how GSH biosynthesis is regulated to control the transition to flowering remains to be elucidated. Since the key reaction of GSH synthesis is catalyzed by gamma-glutamylcysteine synthetase (gamma-ECS) and all the gamma-ECS cDNAs examined contained extra sequences for plastid targeting, we investigated the relationships among GSH levels, photosynthesis and flowering. The GSH level in Arabidopsis increased with the light intensity. The ch1 mutants defective in a light-harvesting antenna in photosystem II showed reduced GSH levels with accumulation of the GSH precursor cysteine, and introduction of the gamma-ECS gene GSH1 under the control of the cauliflower mosaic virus 35S promoter (35S-GSH1) into the ch1 mutant altered the GSH level in response to the gamma-ECS mRNA level. These indicate that photosynthesis limits the gamma-ECS reaction to regulate GSH biosynthesis. Like the glutathione-biosynthesis-defective cad2-1 mutant, the ch1 mutants flowered late under weak-light conditions, and this late-flowering phenotype was rescued by supplementation of GSH. Introduction of the 35S-GSH1 construct into the ch1 mutant altered flowering in response to the gamma-ECS mRNA and GSH levels. These findings indicate that flowering in A. thaliana is regulated by the gamma-ECS reaction of GSH synthesis that is coupled with photosynthesis.     

Association of glutathione with flowering in Arabidopsis thaliana

In order to study the relationship between GSH and flowering, wild-type and late-flowering mutant, fca-1, of Arabidopsis thaliana were treated with L-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, under long-day conditions. BSO treatment of the fca-1 mutant starting at 17 d after imbibition promoted flowering. However, when the treatment was started at 12 d after imbibition, BSO treatment at 10(-4) M resulted in an inhibition of flowering. This inhibitory effect of BSO on flowering was abolished by GSH treatment at 10(-4) M, although GSH treatment at an increased concentration of 10(-3) M clearly delayed flowering. In contrast, BSO treatment of wild-type plants starting at 12 d after imbibition promoted flowering, whose effect was abolished by GSH application. In the fca-1 mutant, whose endogenous GSH levels were high, chilling treatment lowered the GSH levels and promoted flowering, as was the case in the BSO treatment. An A. thaliana mutant, cad2-1, which has a defect in GSH biosynthesis also exhibited late flowering. The late-flowering phenotype of this mutant tended to be strengthened by BSO and abolished by GSH treatment. These results suggest that flowering is associated with the rate of GSH biosynthesis and/or the levels of GSH in A. thaliana.     

Opposing effects of reduced DNA methylation on flowering time in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Demethylation of DNA promotes flowering in plants from the vernalization-responsive ecotype C24 of Arabidopsis thaliana (L.) Heynh., but delays flowering in the ecotype Landsberg erecta which is not responsive to vernalization. To investigate these contrasting effects of low methylation we have monitored flowering times and expression of two repressors of flowering, FLC and FWA, in low-methylation plants from three late-flowering mutants in the ecotype Landsberg erecta. Demethylation of DNA decreased FLC expression in the vernalization-responsive mutants, but was not associated with a promotion of flowering; rather, in some lines, demethylation delayed flowering. The opposing effects of demethylation could be explained by its differential effect on the expression of two repressors of flowering. FLC was down-regulated in plants with low methylation, promoting flowering, while FWA was activated in response to demethylation, which probably delays the transition to flowering. Expression of the FWA gene did not delay flowering in plants of ecotype C24; our data suggest that the FWA protein of C24 may be non-functional.     

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

Proteomic analysis of seed dormancy in Arabidopsis

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.     

Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis

Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis (Arabidopsis thaliana), the histone acetyltransferase AtHAC1 is homologous to animal p300/CREB (cAMP-responsive element-binding protein)-binding proteins, which are the main histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. The functions of p300/CREB-binding proteins in animals are well characterized, whereas little is known about the roles of AtHAC1 in developmental control in Arabidopsis. Lesions in AtHAC1 caused pleiotropic developmental defects, including delayed flowering, a shortened primary root, and partially reduced fertility. Analysis of the molecular basis of late flowering in hac1 mutants showed that the hac1 plants respond normally to day length, gibberellic acid treatment, and vernalization. Furthermore, the expression level of the flowering repressor FLOWERING LOCUS C (FLC) is increased in hac1 mutants, indicating that the late-flowering phenotype of hac1 mutants is mediated by FLC. Since histone acetylation is usually associated with the activation of gene expression, histone modifications of FLC chromatin are not affected by mutations in HAC1 and expression levels of all known autonomous pathway genes are unchanged in hac1 plants, we propose that HAC1 affects flowering time by epigenetic modification of factors upstream of FLC.     

NOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis and Rubisco formation in rice

NITRIC OXIDE-ASSOCIATED1 (NOA1) encodes a circularly permuted GTPase (cGTPase) known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-suppressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30°C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22°C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30°C or 15°C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30°C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15°C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A), OsrbcL (Rubisco large subunit), OsRALyase (Ribosomal RNA apurinic site specific lyase) and OsPuf4 (RNA-binding protein of the Puf family), which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice.     

Resetting and regulation of FLOWERING LOCUS C expression during Arabidopsis reproductive development

The epigenetic regulation of the floral repressor FLOWERING LOCUS C ( FLC ) is one of the critical factors that determine flowering time in Arabidopsis thaliana . Although many FLC regulators, and their effects on FLC chromatin, have been extensively studied, the epigenetic resetting of FLC has not yet been thoroughly characterized. Here, we investigate the FLC expression during gametogenesis and embryogenesis using FLC::GUS transgenic plants and RNA analysis. Regardless of the epigenetic state in adult plants, FLC expression disappeared in gametophytes. Subsequently, FLC expression was reactivated after fertilization in embryos, but not in the endosperm. Both parental alleles contributed equally to the expression of FLC in embryos. Surprisingly, the reactivation of FLC in early embryos was independent of FRIGIDA (FRI) and SUPPRESSOR OF FRIGIDA 4 (SUF4) activities. Instead, FRI , SUF4 and autonomous-pathway genes determined the level of FLC expression only in late embryogenesis. Many FLC regulators exhibited expression patterns similar to that of FLC , indicating potential roles in FLC reprogramming. An FVE mutation caused ectopic expression of FLC in the endosperm. A mutation in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 caused defects in FLC reactivation in early embryogenesis, and maintenance of full FLC expression in late embryogenesis. We also show that the polycomb group complex components, Fertilization-Independent endosperm and MEDEA, which mediate epigenetic regulation in seeds, are not relevant for FLC reprogramming. Based on our results, we propose that FLC reprogramming is composed of three phases: (i) repression in gametogenesis, (ii) reactivation in early embryogenesis and (iii) maintenance in late embryogenesis.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.     

Salicylic acid regulates flowering time and links defence responses and reproductive development

Flowering relies on signaling networks that integrate endogenous and external cues. Normally, plants flower at a particular season, reflecting day length and/or temperature cues. However, plants can surpass this seasonal regulation and show precocious flowering under stress environmental conditions. Here, we show that UV-C light stress activates the transition to flowering in Arabidopsis thaliana through salicylic acid (SA). Moreover, SA also regulates flowering time in non-stressed plants, as SA-deficient plants are late flowering. The regulation of flowering time by SA seems to involve the photoperiod and autonomous pathways, but it does not require the function of the flowering time genes CONSTANS (CO), FCA, or FLOWERING LOCUS C (FLC).