Molecular mechanism of mulberry response to drought stress revealed by complementary transcriptomic and iTRAQ analyses.

BACKGROUND: The use of mulberry leaves has long been limited to raising silkworms, but with the continuous improvement of mulberry (Morus alba) resource development and utilization, various mulberry leaf extension products have emerged. However, the fresh leaves of mulberry trees have a specific window of time for picking and are susceptible to adverse factors, such as drought stress. Therefore, exploring the molecular mechanism by which mulberry trees resist drought stress and clarifying the regulatory network of the mulberry drought response is the focus of the current work. RESULTS: In this study, natural and drought-treated mulberry grafted seedlings were used for transcriptomic and proteomic analyses (CK vs. DS9), aiming to clarify the molecular mechanism of the mulberry drought stress response. Through transcriptome and proteome sequencing, we identified 9889 DEGs and 1893 DEPs enriched in stress-responsive GO functional categories, such as signal transducer activity, antioxidant activity, and transcription regulator activity. KEGG enrichment analysis showed that a large number of codifferentially expressed genes were enriched in flavonoid biosynthesis pathways, hormone signalling pathways, lignin metabolism and other pathways. Through subsequent cooperation analysis, we identified 818 codifferentially expressed genes in the CK vs. DS9 comparison group, including peroxidase (POD), superoxide dismutase (SOD), aldehyde dehydrogenase (ALDHs), glutathione s-transferase (GST) and other genes closely related to the stress response. In addition, we determined that the mulberry gene MaWRKYIII8 (XP_010104968.1) underwent drought- and abscisic acid (ABA)-induced expression, indicating that it may play an important role in the mulberry response to drought stress. CONCLUSIONS: Our research shows that mulberry can activate proline and ABA biosynthesis pathways and produce a large amount of proline and ABA, which improves the drought resistance of mulberry. MaWRKYIII8 was up-regulated and induced by drought and exogenous ABA, indicating that MaWRKYIII8 may be involved in the mulberry response to drought stress. These studies will help us to analyse the molecular mechanism underlying mulberry drought tolerance and provide important gene information and a theoretical basis for improving mulberry drought tolerance through molecular breeding in the future.

Correction: Wang, Y.T., et al. Selenite Reduction and the Biogenesis of Selenium Nanoparticles by Alcaligenes faecalis Se03 Isolated from the Gut of Monochamus alternatus (Coleoptera: Cerambycidae). Int. J. Mol. Sci. 2018, 19, 2799.

The authors wish to make the following corrections to this paper [...].

Application of mass spectrometry in silkworm research-Review.

In this first mass spectrometry-focused review paper, we will review current applications of mass spectrometry in the area of silkworm research. We will focus our review on the following two most important areas as they are currently being researched by scientists. Firstly, the proteomics of proteins in the process of silkworm lifecycle has generated knowledge about previous undetected proteins, some of which might possess therapeutic effects. Secondly, fatty acids, which are the other major components in silkworm, have several potential medical applications. We will also highlight potential areas warranting further investigation.

Aluminum toxicity related to SOD and expression of presenilin and CREB in Bombyx mori.

Aluminum (Al) is an important environmental metal factor that can be potentially associated with pathological changes leading to neurotoxicity. The silkworm, Bombyx mori, is an important economic insect and has also been used as a model organism in various research areas. However, the toxicity of Al on silkworm physiology has not been reported. Here, we comprehensively investigate the toxic effects of Al on the silkworm, focusing on its effects on viability and development, superoxide dismutase (SOD) activity, and the expression of presenilin and cAMP response element-binding protein (CREB) in BmE cells and silkworm larvae. BmE cell viability decreased after treatment with aluminum chloride (AlCl3 ) in both dose- and time-dependent manners. When AlCl3 solution was injected into newly hatched fifth instar larvae, both larval weight gain and survival rate were significantly decreased in a manner correlating with AlCl3 dose and developmental stage. Furthermore, when BmE cells and silkworm larvae were exposed to AlCl3 , SOD activity decreased significantly relative to the control group, whereas presenilin expression increased more than twofold. Additionally, CREB and phosphorylated CREB (p-CREB) expression in the heads of fifth instar larvae decreased by 28.0% and 50.0%, respectively. These results indicate that Al inhibits the growth and development of silkworms in vitro and in vivo, altering SOD activity and the expressions of presenilin, CREB, and p-CREB. Our data suggest that B. mori can serve as a model animal for studying Al-induced neurotoxicity or neurodegeneration.

Selenite Reduction and the Biogenesis of Selenium Nanoparticles by Alcaligenesfaecalis Se03 Isolated from the Gut of Monochamus alternatus (Coleoptera: Cerambycidae).

In this study, a bacterial strain exhibiting high selenite (Na2SeO3) tolerance and reduction capacity was isolated from the gut of Monochamus alternatus larvae and identified as Alcaligenes faecalis Se03. The isolate exhibited extreme tolerance to selenite (up to 120 mM) when grown aerobically. In the liquid culture medium, it was capable of reducing nearly 100% of 1.0 and 5.0 mM Na2SeO3 within 24 and 42 h, respectively, leading to the formation of selenium nanoparticles (SeNPs). Electron microscopy and energy dispersive X-ray analysis demonstrated that A. faecalis Se03 produced spherical electron-dense SeNPs with an average hydrodynamic diameter of 273.8 ± 16.9 nm, localized mainly in the extracellular space. In vitro selenite reduction activity and real-time PCR indicated that proteins such as sulfite reductase and thioredoxin reductase present in the cytoplasm were likely to be involved in selenite reduction and the SeNPs synthesis process in the presence of NADPH or NADH as electron donors. Finally, using Fourier-transform infrared spectrometry, protein and lipid residues were detected on the surface of the biogenic SeNPs. Based on these observations, A. faecalis Se03 has the potential to be an eco-friendly candidate for the bioremediation of selenium-contaminated soil/water and a bacterial catalyst for the biogenesis of SeNPs.

Whole genome bisulfite sequencing methylome analysis of mulberry (Morus alba) reveals epigenome modifications in response to drought stress.

DNA methylation plays a significant role in many biological processes. Although some studies of DNA methylation have been performed in woody plant, none is known about the methylation patterns of mulberry (Morus alba). In this study, we performed whole genome bisulfite sequencing under drought stress to generate a methylated cytosines map and assessed the effects of the changes on gene expression combined with transcriptomics. We found that the percentage of methylated cytosines varied depending on the local sequence context (CG, CHG and CHH) and external treatment (control, CK; drought stress, DS). The methylation levels under DS were 8.64% higher than that of CK, and differences that were mainly due to the contribution of mCG (6.24%). Additionally, there were 3,243 different methylation and expression associated genes. In addition, methylated genes were enriched within GO subcategories including catalytic activity, cellular process, metabolic process, response to stimulus and regulation of biological process. This is the first study to comprehensively present methylation patterns in mulberry and reveal widespread DNA methylation changes in response to drought stress, which has the potential to enhance our understanding of links between DNA methylation and the modulation of gene expression in plants subjected to abiotic stresses.

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori.

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified. However, molecular mechanisms of most of these mutants remain to be explored. Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing (mw) mutation which exhibits exceedingly small wings during pupal and adult stages. Compared with the wild type silkworm, relative messenger RNA expression of Bm-app is significantly decreased in the u11 mutant strain which shows mw phenotype. A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity. Furthermore, clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant, demonstrating that Bm-app controls wing development in B. mori. Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins, indicating that it is required for cell mitosis and growth during wing development. We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B. mori.

Mulberry (Morus alba) MmSK gene enhances tolerance to drought stress in transgenic mulberry.

Shaggy-like protein kinase (SK) plays important roles in the plant growth development, signal transduction, abiotic stress and biotic stress and substance metabolism regulation. However, the exact function of the response to drought stress in mulberry with SK remains unclear. In this study, a new SK gene that was designated as MmSK (GenBank accession NO: KY348867) was isolated and cloned from mulberry (Morus alba). MmSK contains two SK conservation domains of ATP domain and Serine/Threonine protein kinases active-site signature, and belonged to GSK3/shaggy protein kinase family. The expression of MmSK in mulberry was up-regulated under various abiotic stress treatments. Meanwhile, we observed higher expression levels in the phloem contrasted with other tissues. Mulberry MmSK gene was successfully silenced by virus induced gene silencing (VIGS), and after MmSK was silenced, the expression of MmSK in pTRV2-MmSK-VIGS plant (transgenic mulberry) dropped to 34.02% compared with the negative control inoculated with empty vector pTRV2-00 (CK). Under drought stress, the soluble protein content, proline content, superoxide dismutase (SOD) and peroxidase (POD) activities in transgenic mulberry decreased in different degree compared with the CK. In contrast, the accumulation of malondialdehyde (MDA) increased significantly in transgenic mulberry. With the extension of drought stress treatment time, the soluble protein content, proline content and MDA content gradually increased. The SOD activity and POD activity under drought stress gradually rose to the maximum on the fifth day and then decreased, which consistent with the change trend of MmSK gene expression. These results suggested that MmSK gene could function as a positive regulator of drought stress in mulberry.

High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba).

MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding the molecular mechanisms of drought resistance in mulberry.