SUMMARY Plants encounter a variety of stresses and must fine-tune their growth and stress-response programs to best suit their environment. BES1 functions as a master regulator in the brassinosteroid (BR) pathway that promotes plant growth. Here, we show that BES1 interacts with the ubiquitin receptor protein DSK2 and is targeted to the autophagy pathway during stress via the interaction of DSK2 with ATG8, a ubiquitin-like protein directing autophagosome formation and cargo recruitment. Additionally, DSK2 is phosphorylated by the GSK3-like kinase BIN2, a negative regulator in the BR pathway. BIN2 phosphorylation of DSK2 flanking its ATG8 interacting motifs (AIMs) promotes DSK2-ATG8 interaction, thereby targeting BES1 for degradation. Accordingly, loss-of-function dsk2 mutants accumulate BES1, have altered global gene expression profiles, and have compromised stress responses. Our results thus reveal that plants coordinate growth and stress responses by integrating BR and autophagy pathways and identify the molecular basis of this crosstalk.

Fundamental and quantitative understanding of the interactions between nanoparticles and plant leaves is crucial for advancing the field of nanoenabled agriculture. Herein, we systematically investigated and modeled how ζ potential (−52.3 mV to +36.6 mV) and hydrodynamic size (1.7−18 nm) of hydrophilic nanoparticles influence delivery efficiency and pathways to specific leaf cells and organelles. We studied interactions of nanoparticles of agricultural interest including carbon dots (CDs, 0.5 and 5 mg/mL), cerium oxide (CeO 2 , 0.5 mg/mL), and silica (SiO 2 , 0.5 mg/mL) nanoparticles with leaves of two major crop species having contrasting leaf anatomies: cotton (dicotyledon) and maize (monocotyledon). Biocompatible CDs allowed real-time tracking of nanoparticle translocation and distribution in planta by confocal fluorescence microscopy at high spatial (∼200 nm) and temporal (2−5 min) resolution. Nanoparticle formulations with surfactants (Silwet L-77) that reduced surface tension to 22 mN/m were found to be crucial for enabling rapid uptake (<10 min) of nanoparticles through the leaf stomata and cuticle pathways. Nanoparticle−leaf interaction (NLI) empirical models based on hydrodynamic size and ζ potential indicate that hydrophilic nanoparticles with <20 and 11 nm for cotton and maize, respectively, and positive charge (>15 mV), exhibit the highest foliar delivery efficiencies into guard cells (100%), extracellular space (90.3%), and chloroplasts (55.8%). Systematic assessments of nanoparticle−plant interactions would lead to the development of NLI models that predict the translocation and distribution of nanomaterials in plants based on their chemical and physical properties.

Plant steroid hormones, brassinosteroids (BRs), play important roles in growth and development. BR signaling controls the activities of BRASSINOSTERIOD INSENSITIVE1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 (BES1/BZR1) family transcription factors. Besides the role in promoting growth, BRs are also implicated in plant responses to drought stress. However, the molecular mechanisms by which BRs regulate drought response have just begun to be revealed. The functions of WRKY transcription factors in BR-regulated plant growth have not been established, although their roles in stress responses are well documented. Here, we found that three group III WRKY transcription factors, WRKY46, WRKY54, and WRKY70, are involved in both BR-regulated plant growth and drought response as the triple mutant has defects in BR-regulated growth and is more tolerant to drought stress. RNA-sequencing analysis revealed global roles of WRKY46, WRKY54, and WRKY70 in promoting BR-mediated gene expression and inhibiting drought responsive genes. WRKY54 directly interacts with BES1 to cooperatively regulate the expression of target genes. In addition, WRKY54 is phosphorylated and destabilized by GSK3-like kinase BR-INSENSITIVE2, a negative regulator in the BR pathway. Our results therefore establish WRKY46/54/70 as important signaling components that are positively involved in BR-regulated growth and negatively involved in drought responses.

SUMMARYCotton is an important cash crop worldwide, and is a significant source of fiber, feed, foodstuff, oil and biofuel products. Considerable effort has been expended to increase sustainable yield and quality through molecular breeding and genetic engineering of new cotton cultivars. Given the recent availability of the whole-genome sequence of cotton, it is necessary to develop molecular tools and resources for large-scale analysis of gene functions at the genome-wide level. We have successfully developed an Agrobacterium-mediated virusinduced gene silencing (VIGS) assay in several cotton cultivars with various genetic backgrounds. The genes of interest were potently and readily silenced within 2 weeks after inoculation at the seedling stage. Importantly, we showed that silencing GhNDR1 and GhMKK2 compromised cotton resistance to the infection by Verticillium dahliae, a fungal pathogen causing Verticillium wilt. Furthermore, we developed a cotton protoplast system for transient gene expression to study gene functions by a gain-of-function approach. The viable protoplasts were isolated from green cotyledons, etiolated cotyledons and true leaves, and responded to a wide range of pathogen elicitors and phytohormones. Remarkably, cotton plants possess conserved, but also distinct, MAP kinase activation with Arabidopsis upon bacterial elicitor flagellin perception. Thus, using gene silencing assays, we have shown that GhNDR1 and GhMKK2 are required for Verticillium resistance in cotton, and have developed high throughput loss-of-function and gain-of-function assays for functional genomic studies in cotton.

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