The Re-Localization of Proteins to or Away from Membranes as an Effective Strategy for Regulating Stress Tolerance in Plants.

The membranes of plant cells are dynamic structures composed of phospholipids and proteins. Proteins harboring phospholipid-binding domains or lipid ligands can localize to membranes. Stress perception can alter the subcellular localization of these proteins dynamically, causing them to either associate with or detach from membranes. The mechanisms behind the re-localization involve changes in the lipidation state of the proteins and interactions with membrane-associated biomolecules. The functional significance of such re-localization includes the regulation of molecular transport, cell integrity, protein folding, signaling, and gene expression. In this review, proteins that re-localize to or away from membranes upon abiotic and biotic stresses will be discussed in terms of the mechanisms involved and the functional significance of their re-localization. Knowledge of the re-localization mechanisms will facilitate research on increasing plant stress adaptability, while the study on re-localization of proteins upon stresses will further our understanding of stress adaptation strategies in plants.

Identification of stably expressed reference genes for expression studies in Arabidopsis thaliana using mass spectrometry-based label-free quantification.

Arabidopsis thaliana has been used regularly as a model plant in gene expression studies on transcriptional reprogramming upon pathogen infection, such as that by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), or when subjected to stress hormone treatments including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been extensively employed to quantitate these gene expression changes. However, the accuracy of the quantitation is largely dependent on the stability of the expressions of reference genes used for normalization. Recently, RNA sequencing (RNA-seq) has been widely used to mine stably expressed genes for use as references in RT-qPCR. However, the amplification step in RNA-seq creates an intrinsic bias against those genes with relatively low expression levels, and therefore does not provide an accurate quantification of all expressed genes. In this study, we employed mass spectrometry-based label-free quantification (LFQ) in proteomic analyses to identify those proteins with abundances unaffected by Pst DC3000 infection. We verified, using RT-qPCR, that the levels of their corresponding mRNAs were also unaffected by Pst DC3000 infection. Compared to commonly used reference genes for expression studies in A. thaliana upon Pst DC3000 infection, the candidate reference genes reported in this study generally have a higher expression stability. In addition, using RT-qPCR, we verified that the mRNAs of the candidate reference genes were stably expressed upon stress hormone treatments including JA, SA, and ABA. Results indicated that the candidate genes identified here had stable expressions upon these stresses and are suitable to be used as reference genes for RT-qPCR. Among the 18 candidate reference genes reported in this study, many of them had greater expression stability than the commonly used reference genes, such as ACT7, in previous studies. Here, besides proposing more appropriate reference genes for Arabidopsis expression studies, we also demonstrated the capacity of mass spectrometry-based LFQ to quantify protein abundance and the possibility to extend protein expression studies to the transcript level.

AtGAP1 Promotes the Resistance to Pseudomonas syringae pv. tomato DC3000 by Regulating Cell-Wall Thickness and Stomatal Aperture in Arabidopsis.

GTP is an important signaling molecule involved in the growth, development, and stress adaptability of plants. The functions are mediated via binding to GTPases which are in turn regulated by GTPase-activating proteins (GAPs). Satellite reports have suggested the positive roles of GAPs in regulating ABA signaling and pathogen resistance in plants. However, the molecular mechanisms that bring forth the pathogen resistance have remained unclear. In this study, we demonstrated that the expression of AtGAP1 was inducible by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The overexpression of AtGAP1 in Arabidopsis promoted the expression of PR1 and the resistance to Pst DC3000. Proteomic analyses revealed the enhanced accumulation of cell-wall-modifying proteins as a result of AtGAP1 overexpression. By microscopic analyses, we showed that the overexpression of AtGAP1 resulted in increased thickness of the mesophyll cell wall and reduced stomatal aperture, which are effective strategies for restricting the entry of foliar pathogens. Altogether, we demonstrated that AtGAP1 increases the resistance to Pst DC3000 in Arabidopsis by promoting cellular strategies that restrict the entry of pathogens into the cells. These results point to a future direction for studying the modes of action of GAPs in regulating plant cell structures and disease resistance.

Using the Knowledge of Post-transcriptional Regulations to Guide Gene Selections for Molecular Breeding in Soybean.

The omics approaches allow the scientific community to successfully identify genomic regions associated with traits of interest for marker-assisted breeding. Agronomic traits such as seed color, yield, growth habit, and stress tolerance have been the targets for soybean molecular breeding. Genes governing these traits often undergo post-transcriptional modifications, which should be taken into consideration when choosing elite genes for molecular breeding. Post-transcriptional regulations of genes include transcript regulations, protein modifications, and even the regulation of the translational machinery. Transcript regulations involve elements such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) for the maintenance of transcript stability or regulation of translation efficiency. Protein modifications involve molecular modifications of target proteins and the alterations of their interacting partners. Regulations of the translational machinery include those on translation factors and the ribosomal protein complex. Post-transcriptional regulations usually involve a set of genes instead of a single gene. Such a property may facilitate molecular breeding. In this review, we will discuss the post-transcriptional modifications of genes related to favorable agronomic traits such as stress tolerance, growth, and nutrient uptake, using examples from soybean as well as other crops. The examples from other crops may guide the selection of genes for marker-assisted breeding in soybean.

The Tiny Companion Matters: The Important Role of Protons in Active Transports in Plants.

In plants, the translocation of molecules, such as ions, metabolites, and hormones, between different subcellular compartments or different cells is achieved by transmembrane transporters, which play important roles in growth, development, and adaptation to the environment. To facilitate transport in a specific direction, active transporters that can translocate their substrates against the concentration gradient are needed. Examples of major active transporters in plants include ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion (MATE) transporters, monosaccharide transporters (MSTs), sucrose transporters (SUTs), and amino acid transporters. Transport via ABC transporters is driven by ATP. The electrochemical gradient across the membrane energizes these secondary transporters. The pH in each cell and subcellular compartment is tightly regulated and yet highly dynamic, especially when under stress. Here, the effects of cellular and subcellular pH on the activities of ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters will be discussed to enhance our understanding of their mechanics. The relation of the altered transporter activities to various biological processes of plants will also be addressed. Although most molecular transport research has focused on the substrate, the role of protons, the tiny counterparts of the substrate, should also not be ignored.

The Identification of MATE Antisense Transcripts in Soybean Using Strand-Specific RNA-Seq Datasets.

Natural antisense transcripts (NATs) have been generally reported as negative regulators of their sense counterparts. Multidrug and toxic compound extrusion (MATE) proteins mediate the transport of various substrates. Although MATEs have been identified genome-wide in various plant species, their transcript regulators remain unclear. Here, using the publicly available strand-specific RNA-seq datasets of Glycine soja (wild soybean) which have the data from various tissues including developing pods, developing seeds, embryos, cotyledons and hypocotyls, roots, apical buds, stems, and flowers, we identified 35 antisense transcripts of MATEs from 28 gene loci after transcriptome assembly. Spearman correlation coefficients suggested the positive expression correlations of eight MATE antisense and sense transcript pairs. By aligning the identified transcripts with the reference genome of Glycine max (cultivated soybean), the MATE antisense and sense transcript pairs were identified. Using soybean C08 (Glycine max), in developing pods and seeds, the positive correlations between MATE antisense and sense transcript pairs were shown by RT-qPCR. These findings suggest that soybean antisense transcripts are not necessarily negative transcription regulators of their sense counterparts. This study enhances the existing knowledge on the transcription regulation of MATE transporters by uncovering the previously unknown MATE antisense transcripts and their potential synergetic effects on sense transcripts.

The Poly-Glutamate Motif of GmMATE4 Regulates Its Isoflavone Transport Activity.

Multidrug and toxic compound extrusion (MATE) transporters in eukaryotes have been characterized to be antiporters that mediate the transport of substrates in exchange for protons. In plants, alkaloids, phytohormones, ion chelators, and flavonoids have been reported to be the substrates of MATE transporters. Structural analyses have been conducted to dissect the functional significance of various motifs of MATE proteins. However, an understanding of the functions of the N- and C-termini has been inadequate. Here, by performing phylogenetic analyses and protein sequence alignment of 14 representative plant species, we identified a distinctive N-terminal poly-glutamate motif among a cluster of MATE proteins in soybean. Amongst them, GmMATE4 has the most consecutive glutamate residues at the N-terminus. A subcellular localization study showed that GmMATE4 was localized at the vacuolar membrane-like structure. Protein charge prediction showed that the mutation of the glutamate residues to alanine would reduce the negative charge at the N-terminus. Using yeast as the model, we showed that GmMATE4 mediated the transport of daidzein, genistein, glycitein, and glycitin. In addition, the glutamate-to-alanine mutation reduced the isoflavone transport capacity of GmMATE4. Altogether, we demonstrated GmMATE4 as an isoflavone transporter and the functional significance of the N-terminal poly-glutamate motif of GmMATE4 for regulating the isoflavone transport activity.

MATE-Type Proteins Are Responsible for Isoflavone Transportation and Accumulation in Soybean Seeds.

Soybeans are nutritionally important as human food and animal feed. Apart from the macronutrients such as proteins and oils, soybeans are also high in health-beneficial secondary metabolites and are uniquely enriched in isoflavones among food crops. Isoflavone biosynthesis has been relatively well characterized, but the mechanism of their transportation in soybean cells is largely unknown. Using the yeast model, we showed that GmMATE1 and GmMATE2 promoted the accumulation of isoflavones, mainly in the aglycone forms. Using the tobacco BrightYellow-2 (BY-2) cell model, GmMATE1 and GmMATE2 were found to be localized in the vacuolar membrane. Such subcellular localization supports the notion that GmMATE1 and GmMATE2 function by compartmentalizing isoflavones in the vacuole. Expression analyses showed that GmMATE1 was mainly expressed in the developing soybean pod. Soybean mutants defective in GmMATE1 had significantly reduced total seed isoflavone contents, whereas the overexpression of GmMATE1 in transgenic soybean promoted the accumulation of seed isoflavones. Our results showed that GmMATE1, and possibly also GmMATE2, are bona fide isoflavone transporters that promote the accumulation of isoflavones in soybean seeds.

GmDNJ1, a type-I heat shock protein 40 (HSP40), is responsible for both Growth and heat tolerance in soybean.

Global warming poses severe threats to agricultural production, including soybean. One of the major mechanisms for organisms to combat heat stress is through heat shock proteins (HSPs) that stabilize protein structures at above-optimum temperatures, by assisting in the folding of nascent, misfolded, or unfolded proteins. The HSP40 subgroups, or the J-domain proteins, functions as co-chaperones. They capture proteins that require folding or refolding and pass them on to HSP70 for processing. In this study, we have identified a type-I HSP40 gene in soybean, GmDNJ1, with high basal expression under normal growth conditions and also highly inducible under abiotic stresses, especially heat. Gmdnj1-knockout mutants had diminished growth in normal conditions, and when under heat stress, exhibited more severe browning, reduced chlorophyll contents, higher reactive oxygen species (ROS) contents, and higher induction of heat stress-responsive transcription factors and ROS-scavenging enzyme-encoding genes. Under both normal and heat-stress conditions, the mutant lines accumulated more aggregated proteins involved in protein catabolism, sugar metabolism, and membrane transportation, in both roots and leaves. In summary, GmDNJ1 plays crucial roles in the overall plant growth and heat tolerance in soybean, probably through the surveillance of misfolded proteins for refolding to maintain the full capacity of cellular functions.

Secretory Peptides as Bullets: Effector Peptides from Pathogens against Antimicrobial Peptides from Soybean.

Soybean is an important crop as both human food and animal feed. However, the yield of soybean is heavily impacted by biotic stresses including insect attack and pathogen infection. Insect bites usually make the plants vulnerable to pathogen infection, which causes diseases. Fungi, oomycetes, bacteria, viruses, and nematodes are major soybean pathogens. The infection by pathogens and the defenses mounted by soybean are an interactive and dynamic process. Using fungi, oomycetes, and bacteria as examples, we will discuss the recognition of pathogens by soybean at the molecular level. In this review, we will discuss both the secretory peptides for soybean plant infection and those for pathogen inhibition. Pathogenic secretory peptides and peptides secreted by soybean and its associated microbes will be included. We will also explore the possible use of externally applied antimicrobial peptides identical to those secreted by soybean and its associated microbes as biopesticides.

Understanding the Composition, Biosynthesis, Accumulation and Transport of Flavonoids in Crops for the Promotion of Crops as Healthy Sources of Flavonoids for Human Consumption.

Flavonoids are a class of polyphenolic compounds that naturally occur in plants. Sub-groups of flavonoids include flavone, flavonol, flavanone, flavanonol, anthocyanidin, flavanol and isoflavone. The various modifications on flavonoid molecules further increase the diversity of flavonoids. Certain crops are famous for being enriched in specific flavonoids. For example, anthocyanins, which give rise to a purplish color, are the characteristic compounds in berries; flavanols are enriched in teas; and isoflavones are uniquely found in several legumes. It is widely accepted that the antioxidative properties of flavonoids are beneficial for human health. In this review, we summarize the classification of the different sub-groups of flavonoids based on their molecular structures. The health benefits of flavonoids are addressed from the perspective of their molecular structures. The flavonoid biosynthesis pathways are compared among different crops to highlight the mechanisms that lead to the differential accumulation of different sub-groups of flavonoids. In addition, the mechanisms and genes involved in the transport and accumulation of flavonoids in crops are discussed. We hope the understanding of flavonoid accumulation in crops will guide the proper balance in their consumption to improve human health.