This study presents the first large-scale analysis of plant intact glycopeptides. Using wheat germ agglutinin lectin weak affinity chromatography to enrich modified peptides, followed by electron transfer dissociation (ETD)(1) fragmentation tandem mass spectrometry, glycan compositions on over 1100 glycopeptides from 270 proteins found in Arabidopsis inflorescence tissue were characterized. While some sites were only detected with a single glycan attached, others displayed up to 16 different glycoforms. Among the identified glycopeptides were four modified in nonconsensus glycosylation motifs. While most of the modified proteins are secreted, membrane, endoplasmic reticulum (ER), or Golgi-localized proteins, surprisingly, N-linked sugars were detected on a protein predicted to be cytosolic or nuclear.

Brassinosteroid (BR) homeostasis and signaling are crucial for normal growth and development of plants. BR signaling through cell-surface receptor kinases and intracellular components leads to dephosphorylation and accumulation of the nuclear protein BZR1. How BR signaling regulates gene expression, however, remains unknown. Here we show that BZR1 is a transcriptional repressor that has a previously unknown DNA binding domain and binds directly to the promoters of feed back-regulated BR biosynthetic genes. Microarray analyses identified additional potential targets of BZR1 and illustrated, together with physiological studies, that BZR1 coordinates BR homeostasis and signaling by playing dual roles in regulating BR biosynthesis and downstream growth responses.

Mutation of the immunophilin-like FK506-binding protein TWISTED DWARF1 (FKBP42/TWD1) causes dwarf and twisted-organ phenotypes in Arabidopsis. However, the function of FKBP42 is not fully understood at the molecular level. Using genetic, physiological, and immunological experiments, we show here that FKBP42/TWD1 is necessary for brassinosteroid (BR) signal transduction. The twd1 mutant showed reduced BR sensitivity in growth responses and activation of the BZR1 transcription factor. However, twd1 showed normal responses to an inhibitor of BIN2/GSK3, suggesting that twd1 has a defect upstream of BIN2 in the BR signaling pathway. In vitro and in vivo assays showed that TWD1 interacts physically with the kinase domains of the BR receptor kinases BRI1 and BAK1. TWD1 is not required for normal localization of BRI1-GFP to the plasma membrane or for activation of the flagellin receptor kinase FLS2. Our results suggest that FKBP42/TWD1 plays a specific role in the activation of BRI1 receptor

For maintenance of cellular homeostasis, the actions of growth-promoting hormones must be attenuated when nutrient and energy become limiting. The molecular mechanisms that coordinate hormone-dependent growth responses with nutrient availability remain poorly understood in plants [1, 2]. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates nutrient and energy signaling to regulate growth and homeostasis in both animals and plants [3-7]. Here, we show that sugar signaling through TOR controls the accumulation of the brassinosteroid (BR)-signaling transcription factor BZR1, which is essential for growth promotion by multiple hormonal and environmental signals [8-11]. Starvation, caused by shifting of light-grown Arabidopsis seedlings into darkness, as well as inhibition of TOR by inducible RNAi, led to plant growth arrest and reduced expression of BR-responsive genes. The growth arrest caused by TOR inactivation was partially recovered by BR treatment and the gain-of-function mutation bzr1-1D, which causes accumulation of active forms of BZR1 [12]. Exogenous sugar promoted BZR1 accumulation and seedling growth, but such sugar effects were largely abolished by inactivation of TOR, whereas the effect of TOR inactivation on BZR1 degradation is abolished by inhibition of autophagy and by the bzr1-1D mutation. These results indicate that cellular starvation leads sequentially to TOR inactivation, autophagy, and BZR1 degradation. Such regulation of BZR1 accumulation by glucose-TOR signaling allows carbon availability to control the growth promotion hormonal programs, ensuring supply-demand balance in plant growth.

Plant growth is controlled by integration of hormonal and light-signaling pathways. BZS1 is a B-box zinc finger protein previously characterized as a negative regulator in the brassinosteroid (BR)-signaling pathway and a positive regulator in the light-signaling pathway. However, the mechanisms by which BZS1/BBX20 integrates light and hormonal pathways are not fully understood. Here, using a quantitative proteomic workflow, we identified several BZS1-associated proteins, including light-signaling components COP1 and HY5. Direct interactions of BZS1 with COP1 and HY5 were verified by yeast two-hybrid and co-immunoprecipitation assays. Overexpression of BZS1 causes a dwarf phenotype that is suppressed by the hy5 mutation, while overexpression of BZS1 fused with the SRDX transcription repressor domain (BZS1-SRDX) causes a long-hypocotyl phenotype similar to hy5, indicating that BZS1's function requires HY5. BZS1 positively regulates HY5 expression, whereas HY5 negatively regulates BZS1 protein level, forming a feedback loop that potentially contributes to signaling dynamics. In contrast to BR, strigolactone (SL) increases BZS1 level, whereas the SL responses of hypocotyl elongation, chlorophyll and HY5 accumulation are diminished in the BZS1-SRDX seedlings, indicating that BZS1 is involved in these SL responses. These results demonstrate that BZS1 interacts with HY5 and plays a central role in integrating light and multiple hormone signals for photomorphogenesis in Arabidopsis. Copyright (C) 2016, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

Unlike many wild grasses, domesticated rice cultivars have uniform culm height and panicle size among tillers and the main shoot, which is an important trait for grain yield. However, the genetic basis of this trait remains unknown. Here, we report that DWARF TILLER1 (DWT1) controls the developmental uniformity of the main shoot and tillers in rice (Oryza sativa). Most dwt1 mutant plants develop main shoots with normal height and larger panicles, but dwarf tillers bearing smaller panicles compared with those of the wild type. In addition, dwt1 tillers have shorter internodes with fewer and un-elongated cells compared with the wild type, indicating that DWT1 affects cell division and cell elongation. Map-based cloning revealed that DWT1 encodes a WUSCHEL-related homeobox (WOX) transcription factor homologous to the Arabidopsis WOX8 and WOX9. The DWT1 gene is highly expressed in young panicles, but undetectable in the internodes, suggesting that DWT1 expression is spatially or temporally separated from its effect on the internode growth. Transcriptomic analysis revealed altered expression of genes involved in cell division and cell elongation, cytokinin/gibberellin homeostasis and signaling in dwt1 shorter internodes. Moreover, the non-elongating internodes of dwt1 are insensitive to exogenous gibberellin (GA) treatment, and some of the slender rice1 (slr1) dwt1 double mutant exhibits defective internodes similar to the dwt1 single mutant, suggesting that the DWT1 activity in the internode elongation is directly or indirectly associated with GA signaling. This study reveals a genetic pathway synchronizing the development of tillers and the main shoot, and a new function of WOX genes in balancing branch growth in rice.

Arabidopsis adapts to elevated temperature by promoting stem elongation and hyponastic growth through a temperature-responsive transcription factor PIF4. Here we show that the evening-expressed clock component TOC1 interacts with and inactivates PIF4, thereby suppressing thermoresponsive growth in the evening. We find that the expression of PIF4 target genes show circadian rhythms of thermosensitivity, with minimum responsiveness in the evening when TOC1 level is high. Loss of function of TOC1 and its close homologue PRR5 restores thermosensitivity in the evening, whereas TOC1 overexpression causes thermo insensitivity, demonstrating that TOC1 mediates the evening-specific inhibition of thermoresponses. We further show that PIF4 is required for thermoadaptation mediated by moderately elevated temperature. Our results demonstrate that the interaction between TOC1 and PIF4 mediates the circadian gating of thermoresponsive growth, which may serve to increase fitness by matching thermoresponsiveness with the day-night cycles of fluctuating temperature and light conditions.

Genetic studies have shown essential functions of O-linked N-acetyl-glucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana. Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

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.