Proteome-metabolome profiling of wax gland complex reveals functional changes in honeybee, Apis mellifera L.

Wax gland complex (WGC) serves as the primary generator of beeswax; however, the dynamic biological function in wax secretion remains unclear. To elucidate the developmental mechanism of WGC, we conducted a comprehensive analysis to reveal the variations in proteins and metabolites among the newly emerged bee (NEB), wax-secreting bee (WSB), and overaged bee (OAB). We identified 3,295 proteins and 159 metabolites in WGC. Specifically, NEB elevated the expression of ribosomal proteins for preparing the glandular organ. While WSB promoted the size of epidermal cells and oenocytes, the enrichment of fatty acids and energy metabolism in WSB suggested a strong ability in wax synthesis. In OAB, disorganized wax tubules, and up-regulated cysteine proteases reflected the gland degeneration. These findings highlight the dynamic changes in the level of molecule and morphological structure in WGC, offering valuable insights into the development and mechanism of wax secretion in honeybees and other wax insects.

The Rice Aspartyl-tRNA Synthetase YLC3 Regulates Amino Acid Homeostasis and Chloroplast Development Under Low Temperature.

Aminoacyl tRNA synthetases primarily function to attach specific amino acids to the corresponding tRNAs during protein translation. However, their roles in regulating plant growth and development still remain elusive. Here we reported a rice thermo-sensitive mutant yellow leaf chlorosis3 (ylc3) with reduced chlorophyll content, altered thylakoid structure, and substantially elevated levels of free aspartate, asparagine and glutamine in leaves under low temperature condition. Map-based cloning identified that YLC3 encodes an aspartyl-tRNA synthetase which is localized in cytosol and mitochondria. In addition, quantitative proteomics analysis revealed that both nuclear and chloroplast-encoded thylakoid proteins were significantly down-regulated in the mutant. On the other hand, proteins involved in amino acid metabolism and the process of protein synthesis were up-regulated in ylc3, particularly for key enzymes that convert aspartate to asparagine. Moreover, uncharged tRNA-Asp accumulation and phosphorylation of the translation initiation factor eIF2α was detected in the mutant, suggesting that YLC3 regulates the homeostasis of amino acid metabolism and chloroplast thylakoid development through modulation of processes during protein synthesis.

OsTGAL1 suppresses the resistance of rice to bacterial blight disease by regulating the expression of salicylic acid glucosyltransferase OsSGT1.

Many TGA transcription factors participate in immune responses in the SA-mediated signaling pathway in Arabidopsis. This study identified a transcription factor OsTGAL1, which is induced upon infection by Xoo. Overexpression of OsTGAL1 increased the susceptibility of rice to Xoo. Plants overexpressing OsTGAL1 could affect the expression of many SA signaling-related genes. OsTGAL1 was able to interact with the promoter of OsSGT1, which encodes a key enzyme for SA metabolism. The transcript of OsSGT1 was induced by Xoo and this responsive expression was further increased in plants overexpressing OsTGAL1. OsSGT1 knockout lines had enhanced resistance to Xoo, and knocking out OsSGT1 in plants overexpressing OsTGAL1 blocked the susceptibility caused by OsTGAL1. Altered expression levels of several OsPRs in all the transgenic plants demonstrated that SA-mediated signaling had been affected. Furthermore, we identified an oxidoreductase of CC-type glutaredoxin, OsGRX17, which interacted with OsTGAL1. OsGRX17 reduced the regulation of OsTGAL1 on OsSGT1, and this may be due to its redox modulation. Thus, our results demonstrate that OsTGAL1 negatively regulates resistance to Xoo by its effects on SA metabolism via the activation of OsSGT1, which provides valuable targets for plant breeders in developing new cultivars that are resistant to Xoo.

VDAC1 Negatively Regulates Floral Transition in Arabidopsis thaliana.

Voltage-dependent anion channels (VDACs) are the most important proteins in mitochondria. They localize to the outer mitochondrial membrane and contribute to the metabolite transport between the mitochondria and cytoplasm, which aids plant growth regulation. Here, we report that Arabidopsis thaliana VDAC1 is involved in the floral transition, with the loss of AtVDAC1 function, resulting in an early-flowering phenotype. AtVDAC1 is expressed ubiquitously in Arabidopsis. To identify the flowering pathway integrators that may be responsible for AtVDAC1's function during the floral transition, an RNA-seq analysis was performed. In total, 106 differentially expressed genes (DEGs) were identified between wild-type and atvdac1-5 mutant seedlings. However, none were involved in flowering-related pathways. In contrast, AtVDAC1 physically associated with FLOWERING LOCUS T. Thus, in the floral transition, AtVDAC1 may function partly through the FLOWERING LOCUS T protein.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control.

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a ß-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct ß-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

pGP-B2E, a Recombinant Compatible TA/TB-Ligation Vector for Rapid and Inexpensive Gene Cloning.

DNA cloning is the basic step for different fields of life science, and many efforts have been made to simplify this procedure. In this study, we report two general purpose plasmids (pGP), pGP-XB2E and pGP-B2E, for rapid and cost-effective construct of basic clones. The BciVI and XcmI cleavage sites are designed in pGP-XB2E to test plasmid linearization efficiency. The plasmid has better linearization efficiency by using BciVI which could almost completely digest 2 µg plasmid in 10 min with only one-tenth the recommended enzyme concentration. In order to further optimize the pGP-XB2E, a new plasmid, pGP-B2E, which removed XcmI cleavage site was designed. This vector is highly efficient for cloning PCR products up to 1812 bp, and the number of colonies was about five times that of pGP-XB2E. In addition to TA cloning, blunt-end PCR products with T ended in the primer could be positively linked to the T-vector pGP-B2E without A-tailing treatment (TB cloning). Moreover, as an entry vector, pGP-B2E was also compatible for gateway recombination reaction without frameshift mutations. In general, this vector provides a universal, quick, and cost-efficient method for basic molecular cloning.