BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis

SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways

FTO Is Expressed in Neurones throughout the Brain and Its Expression Is Unaltered by Fasting

Single-nucleotide polymorphisms in the first intron of the ubiquitously expressed FTO gene are associated with obesity. Although the physiological functions of FTO remain unclear, food intake is often altered when Fto expression levels are manipulated. Furthermore, deletion of FTO from neurones alone has a similar effect on food intake to deletion of FTO in all tissues. These results indicate that FTO expression in the brain is particularly important. Considerable focus has been placed on the dynamic regulation of Fto mRNA expression in the hypothalamus after short-term (16–48 hour) fasting, but results have been controversial. There are no studies that quantify FTO protein levels across the brain, and assess its alteration following short-term fasting. Using immunohistochemistry, we found that FTO protein is widely expressed in mouse brain, and present in the majority of neurones. Using quantitative Western blotting and RT-qPCR we show that FTO protein and mRNA levels in the hypothalamus, cerebellum and rostral brain are relatively uniform, and levels in the brain are higher than in skeletal muscles of the lower limbs. Fasting for 18 hours does not alter the expression pattern, or levels, of FTO protein and mRNA. We further show that the majority of POMC neurones, which are critically involved in food intake regulation, also express FTO, but that the percentage of FTO-positive POMC neurones is not altered by fasting. In summary, we find no evidence that Fto/FTO expression is regulated by short-term (18-hour) fasting. Thus, it is unlikely that the hunger and increased post-fasting food intake caused by such food deprivation is driven by alterations in Fto/FTO expression. The widespread expression of FTO in neurones also suggests that physiological studies of this protein should not be limited to the hypothalamus

The Arabidopsis Abscisic Acid Catabolic Gene CYP707A2 Plays a Key Role in Nitrate Control of Seed Dormancy.

Nitrate releases seed dormancy in Arabidopsis thaliana Col-0 seeds in part by reducing abscisic acid (ABA) levels. Nitrate led to lower levels of ABA in imbibed seeds when included in the germination media (exogenous nitrate). Nitrate also reduced ABA levels in dry seeds when provided to the mother plant during seed development (endogenous nitrate). Transcript profiling of imbibed seeds treated with or without nitrate revealed that exogenous nitrate led to a higher expression of nitrate responsive genes whereas endogenous nitrate led to a profile similar to stratified or after-ripened seeds. Profiling experiments indicated that the expression of the ABA catabolic gene CYP707A2 was regulated by exogenous nitrate. The cyp707a2-1 mutant failed to reduce seed ABA levels in response to both endogenous and exogenous nitrate. In contrast both endogenous and exogenous nitrate reduced ABA levels of the wild type and cyp707a1-1 mutant seeds. The CYP707A2 mRNA levels in developing siliques were positively correlated with different nitrate doses applied to the mother plants. This was consistent with a role of the CYP707A2 gene in controlling seed ABA levels in response to endogenous nitrate. The cyp707a2-1 mutant was less sensitive to exogenous nitrate for breaking seed dormancy. Altogether our data underline the central role of the CYP707A2 gene in the nitrate-mediated control of ABA levels during seed development and germination

Effects of light and wounding on jasmonates in rice phyAphyC mutants

Jasmonates (JA) are lipid-derived plant hormones. They have been shown to be important regulators of photomorphogenesis, a developmental program in plants, which is activated by light through different red and blue light sensitive photoreceptors. In rice, inhibition of coleoptile growth by light is a central event in photomorphogenesis. This growth inhibition is impaired, when jasmonate biosynthesis is knocked out. Previously, we found that JASMONATE RESISTANT 1 (OsJAR1) transcripts were not induced in the phytochrome (phy) mutant phyAphyC. Therefore, in the current study we investigated the regulation of JA and its highly bioactive derivative (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), as well as the transcriptional regulation of several JA-dependent genes both in wild type and phyAphyC mutant. JA and JA-Ile levels increased in the mutant seedlings in response to blue light. However, in phyAphyC mutant leaves, which were continuously wounded, JA and JA-Ile levels were lower compared to those in the wild type. Hence, the mutation of phyA and phyC has differential effects on jasmonate levels depending on the tissue and developmental stage. Our results suggest that the contribution of JA-Ile to signaling during photomorphogenesis of rice is minor, as coleoptile phenotypes of phyAphyC mutants resemble those of jasmonate-deficient mutants despite the fact that induction by blue light leads to higher levels of JA-Ile compared to the wild type. We postulate that phyA and phyC could control the activity of specific enzymes metabolizing JA to active derivatives

Arabidopsis PIZZA has the capacity to acylate brassinosteroids

Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively hi gh endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis

Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2 and -3 are the dominant paralogs

Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA12 is converted to GA9. GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:β-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.This work was supported by a Rothamsted quota studentship to A.R.G.P. and by Grant P16508, both funded by the Biotechnology and Biological Sciences Research Council of the UK, which also provides strategic support to Rothamsted Research. We thank the Japanese Society for the Promotion of Science for a fellowship to P.H., the Spanish Ministry of Education and Science for a fellowship to N.F.-G. (Ref: EX2004-0398), and the Czech Ministry of Education (Grants CZ.1.07/2.3.00/09.0035. and 522/08/4003) and Academy of Sciences (Grant KAN 2003 80801) to T.U.Peer reviewe