Project description:Plants adapt to environmental light conditions by photoreceptor-mediated physiological responses, but the mechanism by which photoreceptors perceive and transduce the signals is still unresolved. Here, we used 2D difference gel electrophoresis (2D DIGE) and mass spectrometry to characterize early molecular events induced by short blue light exposures in etiolated Arabidopsis seedlings. We observed the phosphorylation of phototropin 1 (phot1) and accumulation of weak chloroplast movement under blue light 1 (WEB1) in the membrane fraction after blue light irradiation. Over 50 spots could be observed for the two rows of phot1 spots in the 2-DE gels, and eight novel phosphorylated Ser/Thr sites were identified in the N-terminus and Hinge 1 regions of phot1 in vivo. Blue light caused ubiquitination of phot1, and K526 of phot1 was identified as a putative ubiquitination site. Our study indicates that post-translational modification of phot1 is more complex than previously reported.
Project description:Arabidopsis bZIP transcription factor, GBF1, acts as a differential regulator of cryptochrome-mediated blue light signaling. Whereas the bZIP proteins, HY5 (elongated hypocotyl 5) and HYH (HY5 homologue), are degraded by COP1-mediated proteasomal pathways, GBF1 is degraded by a proteasomal pathway independent of COP1. In this study, we have investigated the functional interrelations of GBF1 with HY5 and HYH in Arabidopsis seedling development. The genetic studies using double and triple mutants reveal that GBF1 largely acts antagonistically with HY5 and HYH in Arabidopsis seedling development. Further, GBF1 and HY5 play more important roles than HYH in blue light-mediated photomorphogenic growth. This study reveals that GBF1 is able to form a G-box-binding heterodimer with HY5 but not with HYH. The in vitro and in vivo studies demonstrate that GBF1 co-localizes with HY5 or HYH in the nucleus and physically interacts with both of the proteins. The protein-protein interaction studies further reveal that the bZIP domain of GBF1 is essential and sufficient for the interaction with HY5 or HYH. Taken together, these data demonstrate the functional interrelations of GBF1 with HY5 and HYH in Arabidopsis seedling development.
Project description:In 2009, the draft genome of the reference inbred line of maize (Zea mays L. spp. mays cv. B73) was published so that, using this specific corn variety, molecular analyses of physiological processes became possible. However, the morphology and developmental patterns of B73 maize, compared with that of the more frequently used hybrid varieties, have not yet been analyzed. Here, we describe organ development in seedlings of B73 maize and in those of six other hybrid cultivars, and document significant morphological as well as quantitative differences between these varieties of Z. mays. In a second set of experiments, we used etiolated seedlings of B73 maize to analyze the effect of blue light (BL) on the patterns of proteins in the tip vs. growing region of this sheath-like organ. By using two-dimensional difference gel electrophoresis (2D DIGE), coupled with tandem mass spectrometry, we detected, in the microsomal fraction of maize coleoptile tips, rapid changes in the abundance of protein spots of maize phototropin 1 and several metabolic enzymes. In the sub-apical (growing) region of the coleoptile, proteomic changes were less pronounced. These results suggest that the tip of the coleoptile of B73 maize may serve as a unique model system for dissecting BL responses in a light-sensitive plant organ of known function.
Project description:Karrikins are a class of seed germination stimulants identified in smoke from wildfires. Microarray analysis of imbibed Arabidopsis thaliana seeds was performed to identify transcriptional responses to KAR(1) before germination. A small set of genes that are regulated by KAR(1), even when germination is prevented by the absence of gibberellin biosynthesis or light, were identified. Light-induced genes, putative HY5-binding targets, and ABRE-like promoter motifs were overrepresented among KAR(1)-up-regulated genes. KAR(1) transiently induced the light signal transduction transcription factor genes HY5 and HYH. Germination of afterripened Arabidopsis seed was triggered at lower fluences of red light when treated with KAR(1). Light-dependent cotyledon expansion and inhibition of hypocotyl elongation were enhanced in the presence of germination-active karrikins. HY5 is important for the Arabidopsis hypocotyl elongation, but not seed germination, response to karrikins. These results reveal a role for karrikins in priming light responses in the emerging seedling, and suggest that the influence of karrikins on postfire ecology may not be limited to germination recruitment.
Project description:Plants have evolved mechanisms to improve utilization efficiency or acquisition of inorganic phosphate (Pi) in response to Pi deficiency, such as altering root architecture, secreting acid phosphatases, and activating the expression of genes related to Pi uptake and recycling. Although many genes responsive to Pi starvation have been identified, transcription factors that affect tolerance to Pi deficiency have not been well characterized. We show here that the ectopic expression of B-BOX32 (BBX32) and the mutation of ELONGATED HYPOCOTYL 5 (HY5), whose transcriptional activity is negatively regulated by BBX32, resulted in the tolerance to Pi deficiency in Arabidopsis. The primary root lengths of 35S:BBX32 and hy5 plants were only slightly inhibited under Pi deficient condition and the fresh weights were significantly higher than those of wild type. The Pi deficiency-tolerant root phenotype of hy5 was similarly observed when grown on the medium without Pi. In addition, a double mutant, hy5 slr1, without lateral roots, also showed a long primary root phenotype under phosphate deficiency, indicating that the root phenotype of hy5 does not result from an increase of external Pi uptake. Moreover, we found that blue light may regulate Pi deficiency-dependent primary root growth inhibition through activating peroxidase gene expression, suggesting the Pi-deficiency tolerant root phenotype of hy5 may be due to blockage of blue light responses. Altogether, this study points out light quality may play an important role in the regulation of Pi deficiency responses. It may contribute to regulate plant growth under Pi deficiency through proper illumination.
Project description:A tuberization inhibitor has long been postulated, but not yet found. We found that blue light inhibits tuberization in Norland, a day-neutral variety of Solanum tuberosum L. Tissue-cultured plants formed tubers within 8 weeks under continuous darkness, and white, red, or far-red light. Preliminary experiments indicated that a one- or two-day exposure to blue light after 3-4 weeks of dark treatment will inhibit tuber formation in ‘Norland’ plants. Using this system and expression profiling, we may be able to identify candidate tuberization inhibitors. 'Norland' plants (subcultured from existing cultures and grown for two weeks under continuous 100 umol/m2-s white fluorescent light) were placed in tuber-inducing media containing 6% sucrose, vitamins, MS salts, and kinetin (2.5 mg/L). Tubes containing plants were wrapped in two layers of aluminum foil. After 3 weeks and 2 days, half of the tubes were exposed to 6-7 umol/m2-s blue light. The other half of the tubes were left in darkness (controls). After 2 days, all plants were harvested and frozen in liquid nitrogen. Plants exposed to blue light were harvested under blue light. Control plants were harvested under < 2 umol/m2-s light conditions. All plant transfers were done at 1700 (5 PM) to avoid possible complications due to circadian effects. Experiments were performed four times, from subculture to harvest. RNA was extracted from stem and leaf tissue of plants using the Qiagen RNeasy Plant Mini kit. Extracted RNA was then converted to dsDNA using the Invitrogen protocol and reagents for double stranded cDNA synthesis. The resulting dsDNA was in vitro transcribed into amplified RNA using the Ambion procedure and reagents for in vitro transcription. cDNA was purified using Qiagen MinElute columns and protocol. Amplified RNA was purified using Ambion columns or Qiagen RNeasy columns and the Ambion protocol, and quantified using RiboGreen dye fluorometry. Keywords: Direct comparison
Project description:To understand the transcript regulation of early Arabidopsis seedlings developments with different chemicals under continuous blue light irradiation.
Project description:Plant growth and development is often regulated by the interaction of environmental factors such as light and various phytohormones. Arabidopsis FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT 1 (JAR1) participates in phytochrome A-mediated far-red (FR) light signaling and interacts with different light signaling regulators. FIN219/JAR1 is a jasmonic acid (JA)-conjugating enzyme responsible for the formation of JA-isoleucine. However, how FIN219/JAR1 integrates FR light and JA signaling remains largely unknown. We used a microarray approach to dissect the effect of fin219 mutation on the interaction of FR light and JA signaling. The fin219-2 mutant was less sensitive than the wild type to various concentrations of methyl jasmonate (MeJA) under low and high FR light. High FR light reduced the sensitivity of Arabidopsis seedlings to MeJA likely through FIN219. Intriguingly, in response to MeJA, FIN219 levels showed a negative feedback regulation. Further microarray assay revealed that FR light could regulate gene expression by FIN219-dependent or -independent pathways. The expression profiles affected in fin219-2 indicated that FIN219/JAR1 plays a critical role in the integration of multiple hormone-related signaling. In particular, FIN219 regulates a number of transcription factors (TFs), including 94 basic helix-loop-helix (bHLH) TFs, in response to FR light and MeJA. Loss-of-function mutants of some bHLH TFs affected by FIN219 showed altered responses to MeJA in the regulation of hypocotyl and root elongation. Thus, FIN219/JAR1 is tightly regulated in response to exogenous MeJA. It also interacts with multiple plant hormones to modulate hypocotyl and root elongation of Arabidopsis seedlings likely by regulating a group of TFs.
Project description:Plants have evolved mechanisms to improve utilization efficiency or acquisition of inorganic phosphate (Pi) in response to Pi deficiency, such as altering root architecture, secreting acid phosphatases, and activating the expression of genes related to Pi uptake and recycling. Although many genes responsive to Pi starvation have been identified, transcription factors that affect tolerance to Pi deficiency have not been well characterized. We show here that the ectopic expression of B-BOX32 (BBX32) and the mutation of ELONGATED HYPOCOTYL 5 (HY5), whose transcriptional activity is negatively regulated by BBX32, resulted in the tolerance to Pi deficiency in Arabidopsis. The primary root lengths of 35S:BBX32 and hy5 plants were only slightly inhibited under Pi deficient condition and the fresh weights were significantly higher than those of wild type. The Pi deficiency-tolerant root phenotype of hy5 was similarly observed when grown on the medium without Pi. In addition, a double mutant, hy5 slr1, without lateral roots also showed a long primary root phenotype under phosphate deficiency, indicating that the root phenotype of hy5 does not result from increase of external Pi uptake. Moreover, we found that blue light may regulate Pi deficiency-dependent primary root growth inhibition through activating peroxidase gene expression, suggesting the Pi-deficiency tolerant root phenotype of hy5 may be due to blockage of blue-light responses. Altogether, this study points out light quality may play an important role in the regulation of Pi deficiency responses. It may contribute to regulate plant growth under Pi deficiency through a proper illumination.