Identification and Quantitative Analysis of Significantly Accumulated Proteins During the Arabidopsis Seedling De-etiolation Process

Proteomic analysis was performed on seedlings after different light treatments. A total of （1 350±31） protein spots was separated and visualized on each silver nitrate-stained two-dimensional gel using protein samples prepared from light-grown or etiolated seedlings with or without 6-9 h light treatment. Twenty-five protein spots （encoded by 19 genes） that were significantly accumulated upon light treatment were identified using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method. Functional proteomics indicated that these proteins involved mainly in chloroplast development, energy metabolism, cell cycle progression and membrane electron transport. For 18 of the protein-coding genes we identified through an internet search, the transcript levels of 17 genes matched roughly with their protein content in etiolated and green seedlings, suggesting that these genes were regulated by light mainly at the transcriptional level. Despite a very significant increase in the amount of proteins upon light treatment, similar RNA levels were found in dark-grown or green seedlings for the carbonic anhydrase gene At3g05100, indicating a possible post-transcriptional regulatory mechanism. Elucidation of light-induced protein accumulation will undoubtedly enhance our understanding of plant photomorphogenesis.

Large-scale Proteomic and Phosphoproteomic Analyses of Maize Seedling Leaves During De-etiolation

De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light.During this process light,an important environmental signal,triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts,and etiolated leaves transition from the"sink"stage to the"source"stage.De-etiolation has been extensively studied in maize(Zea mays L.).However,little is known about how this transition is regulated.In this study,we described a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize.We identified 16,420 proteins in proteome,among which 14,168 proteins were quantified.In addition,8746 phosphorylation sites within 3110 proteins were identified.From the combined proteomic and phosphoproteomic data,we identified a total of 17,436 proteins.Only 7.0%(998/14,168)of proteins significantly changed in abundance during de-etiolation.In contrast,26.6%of phosphorylated proteins exhibited significant changes in phosphorylation level;these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthetic light and carbon reactions.Based on phosphoproteomic analysis,34.0%(1057/3110)of phosphorylated proteins identified in this study contained more than 2 phosphorylation sites,and 37 proteins contained more than 16 phosphorylation sites,indicating that multi-phosphorylation is ubiquitous during the de-etiolation process.Our results suggest that plants might preferentially regulate the level of posttranslational modifications(PTMs)rather than protein abundance for adapting to changing environments.The study of PTMs could thus better reveal the regulation of de-etiolation.

HYPOSENSITIVE TO LIGHT, an Alpha/Beta Fold Protein, Acts Downstream of ELONGATED HYPOCOTYL 5 to Regulate Seedling De-Etiolation

Ambient light has profound effects on early seedling de-etiolation through red and far-red light-absorbing phytochromes and blue and UV-A light-absorbing cryptochromes. Subsequent integration of various light signal trans- duction pathways leads to changes in gene expression and morphogenic responses. Here, we report the isolation of a new Arabidopsis light-signaling component, HYPOSENSITIVE TO LIGHT or HTL. Both htl-1 and htl-2 alleles displayed a long hypocotyl phenotype under red, far-red, and blue light, whereas overexpression of HTL caused a short hypocotyl pheno- type under similar light conditions. The mutants also showed other photomorphogenic defects such as elongated petioles, retarded cotyledon and leaf expansion, reduced accumulation of chlorophyll and anthocyanin pigments, and attenuated expression of light-responsive CHLOROPHYLL A/B BINDING PROTEIN 3 and CHALCONE SYNTHASE genes. HTL belongs to an alpha/beta fold protein family and is localized strongly in the nucleus and weakly in the cytosol. The expression of HTL was strongly induced by light of various wavelengths and this light induction was impaired in elongated hypocotyl 5. HY5 directly bound to both a C/G-box and a G-box in the HTL promoter but with a greater affinity toward the C/G-box. HTL, therefore, represents a new signaling step downstream of HY5 in phy- and cry-mediated de-etiolation responses.

Large-scale Identification and Time-course Quantification of Ubiquitylation Events During Maize Seedling De-etiolation

The ubiquitin system is crucial for the development and fitness of higher plants.De-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L.(exposed to light for 1, 6, or 12 h)achieved through immunoprecipitation-based high-resolution mass spectrometry(MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites(fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and histones.Meanwhile, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.