Late Embryogenesis Abundant (<Emphasis Type="Italic">LEA</Emphasis>) Gene Family in Maize: Identification,Evolution, and Expression Profiles

Late embryogenesis abundant (LEA) proteins are identified as a large and highly diverse group of polypeptides accumulating in response to cellular dehydration in many organisms. However, there are only very limited reports of this protein family in maize until this study. In the present paper, we identified 32 LEA genes in maize. A total of 83 LEA proteins including 51 members in Arabidopsis and 32 putative members in maize were classified into nine groups. Gene organization and motif compositions of the LEA members are highly conserved in each of the groups, indicative of their functional conservation. The predicted ZmLEA genes were non-random distributed across chromosomes, and transposition event and segmental duplication contributed to the expansion of the LEA gene family in maize. Some abiotic stress-responsive cis-elements were also found in the promoters of ZmLEA genes. Microarray expression analyses revealed different accumulation patterns of ZmLEA family members. Moreover, some members of ZmLEAs were regulated under IAA and some abiotic stresses. This study will provide comprehensive information for maize LEA gene family and may pave the way for deciphering their functions in further studies.     

The <Emphasis Type="Italic">Populus trichocarpa PtHSP17.8</Emphasis> involved in heat and salt stress tolerances

Key message

PtHSP17.8 was regulated by various abiotic stresses. Overexpression of PtHSP17.8 enhanced the tolerance to heat and salt stresses through maintain ROS homeostasis and cooperate with stress-related genes in Arabidopsis.

Abstract

Small heat shock proteins (sHSPs) play important roles in response to diverse biotic and abiotic stresses, especially in heat tolerance. However, limited information is available on the stress tolerance roles of sHSPs in woody species. To explore the function of sHSPs in poplar, we isolated and characterized PtHSP17.8 from Populus trichocarpa. Phylogenetic analysis and subcellular localization revealed that PtHSP17.8 was a cytosolic class I sHSP. The gene expression profile of PtHSP17.8 in various tissues showed that it was significantly accumulated in stem and root, which was consistent with the GUS expression pattern driven by promoter of PtHSP17.8. The expression of PtHSP17.8 could be induced by various abiotic stresses and significantly activated by heat stress. Overexpression of PtHSP17.8 enhanced the tolerance to heat and salt stresses in Arabidopsis. The seedling survival rate, root length, relative water content, antioxidative enzyme activities, proline, and soluble sugar content were increased in transgenic Arabidopsis under heat and salt stresses, but not in normal condition. The co-expression network of PtHSP17.8 were constructed and demonstrated many stress responsive genes included. The stress-related genes in the co-expression network were up-regulated in the PtHSP17.8 overexpression seedlings. These results suggest that PtHSP17.8 confers heat and salt tolerances in plants.

     

<Emphasis Type="Italic">ZmFKBP20-1</Emphasis> improves the drought and salt tolerance of transformed Arabidopsis

FK506-binding proteins (FKBPs), which belong to the peptidyl-prolyl cis/trans isomerase superfamily, are involved in plant response to abiotic stresses. A number of FKBP family genes have been isolated in plants, but little has been reported of FKBP genes in maize. In this study, a drought-induced FKBP gene, ZmFKBP20-1, was isolated from maize and was characterized for its role in stress responses using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes, and physiological parameter analysis. During drought and salt stresses, ZmFKBP20-1 transgenic Arabidopsis plants exhibited enhanced tolerance, which was concomitant with the altered expression of stress/ABA-responsive genes, such as COR15a, COR47, ERD10, RD22, KIN1, ABI1, and ABI2. The resistance characteristics of ZmFKBP20-1 overexpression were associated with a significant increase in survival rate. These results suggested that ZmFKBP20-1 plays a positive role in drought and salt stress responses in Arabidopsis and provided new insights into the mechanisms of FKBP in response to abiotic stresses in plants.     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

Genome-wide identification and expression analysis of the GRAS family proteins in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The GRAS gene family performs a variety of functions in plant growth and development processes, and they also play essential roles in plant response to environmental stresses. Medicago truncatula is a diploid plant with a small genome used as a model organism. Despite the vital role of GRAS genes in plant growth regulation, few studies on these genes in M. truncatula have been conducted to date. Using the M. truncatula reference genome data, we identified 68 MtGRAS genes, which were classified into 16 groups by phylogenetic analysis, located on eight chromosomes. The structure analysis indicated that MtGRAS genes retained a relatively constant exon–intron composition during the evolution of the M. truncatula genome. Most of the closely related members in the phylogenetic tree had similar motif compositions. Different motifs distributed in different groups of the MtGRAS genes were the sources of their functional divergence. Twenty-eight MtGRAS genes were expressed in six tissues, namely root, bud, blade, seedpod, nodule, and flower tissues, suggesting their putative function in many aspects of plant growth and development. Nine MtGRAS genes were upregulated under cold, freezing, drought, ABA, and salt stress treatments, indicating that they play vital roles in the response to abiotic stress in M. truncatula. Our study provides valuable information that can be utilized to improve the quality and agronomic benefits of M. truncatula and other plants.     

Identification and expression analysis of cytokinin response regulators in <Emphasis Type="Italic">Fragaria vesca</Emphasis>

Cytokinin response regulators (RRs) are important components of the two component signal systems, which are involved in the regulation of plant growth and development, and in the response to abiotic stress. In this study, 18 cytokinin RR genes were identified in Fragaria vesca through the genome-wide search. They were further classified into three types: type-A (FvRR1–7), type-B (FvRR8–14) and type-C (FvRR15–18) according to the domain architecture and the phylogeny. Phylogenetic analysis demonstrated that most cytokinin response regulators of F. vesca and Arabidopsis formed clear orthologous pairs. Expression patterns of the cytokinin FvRR genes in various tissues and organs at reproductive stages were detected in this study. Additionally, gene expression response patterns to ABA and abiotic stresses including high temperature and osmotic stress were investigated. The results showed that different types of cytokinin FvRRs have different expression patterns, suggesting the functional differentiation of cytokinin FvRRs during the evolution. This systematic study provides insights into possible functions of the cytokinin FvRR genes and a basis for further functional analysis.     

Metabolic evolution and a comparative omics analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for putrescine production

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor l-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.     

Molecular genetics and functional genomics of abiotic stress-responsive genes in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.): a review of recent advances and future

Abiotic stresses are the key factors which negatively influence plant development and productivity and are the main cause of extensive agricultural production losses worldwide. Brassica napus is an oilseed crop of global economic significance and major contributor to the total oilseed production, quite often encounters abiotic stresses, resulting in reduced agricultural productivity. Hence, there is an immediate need being felt to raise B. napus cultivars which would be more suitable for various abiotic stress conditions presently and in the years to come. Biotechnology and molecular plant breeding has emerged as an important tool for molecular understanding of plant response to various abiotic stresses. Currently, various stress-responsive genes and mechanisms have been identified and functionally characterized in model plant Arabidopsis and other major crop plants such as Oryza sativa and Zea mays. However, very inadequate success has been achieved in this direction in a major oilseed crop such as B. napus. In this review, we present the latest methods and approaches of studying abiotic stress in B. napus. In this review, we describe the genes functioning as markers for crop breeding and discuss the recent progress and advances in genome editing by break through CRISPR/Cas9 multigene–multiplex approaches for developing multiple abiotic stress tolerance with our on-going research as a scheme. We also throw some light on molecular genetics, plant breeding and abiotic stress biotechnology of B. napus which offer a new prospective on the research directions for the practical plant breeding and functional genomics of B. napus in response to different abiotic stress conditions.     

Genome-wide survey and expression analysis of the stress-associated protein gene family in desert poplar, <Emphasis Type="Italic">Populus euphratica</Emphasis>

Stress-associated proteins (SAPs) are a novel class of zinc finger proteins that extensively participate in abiotic stress responses. To date, no overall analysis and expression profiling of SAP genes in woody plants have been reported. Populus euphratica is distributed in desert regions and is extraordinarily adaptable to abiotic stresses. Thus, it is regarded as a promising candidate for studying abiotic stress resistance mechanisms of woody plants. In this study, 18 non-redundant SAP genes were identified from the genome of P. euphratica using basic local alignment search tool algorithms and functional domain verification. Among these 18 PeuSAP genes, 15 were intronless. To investigate the evolutionary relationships of SAP genes in P. euphratica and other Salicaceae plants, phylogenetic analyses were performed. Subsequently, the expression profiles of the 18 PeuSAP genes were analyzed in different tissues and under various stresses (drought, salt, heat, cold, and abscisic acid (ABA) treatment) using quantitative real-time PCR. Tissue expression analysis indicated that PeuSAPs showed no tissue specificity. PeuSAPs were induced by multiple abiotic stresses, especially drought, salt, and heat stresses, perhaps because of abundant cis-acting heat shock elements and drought-inducible elements in the promoter regions of the PeuSAPs. Moreover, single nucleotide polymorphisms (SNPs) variant analysis revealed many synonymous and non-synonymous SNPs in PeuSAP genes, but the zinc finger structure was conserved during evolution. These results provide an overview of the SAP gene family in P. euphratica and a reference for further functional research on PeuSAP genes.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Molecular cloning and expression analysis of two <Emphasis Type="Italic">FAD2</Emphasis> genes from chia (<Emphasis Type="Italic">Salvia hispanica</Emphasis>)

FATTY ACID DESATURASE 2 (FAD2, EC 1.3.1.35), also known as delta-12 oleate desaturase, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. Chia (Salvia hispanica) seeds contain the highest known proportion of α-linolenic acid in any plant sources. In this study, two full-length FAD2 genes, named as ShFAD2-1 and ShFAD2-2, were isolated from S. hispanica based on RACE method. Both ShFAD2-1 and ShFAD2-2 proteins possess strong transmembrane helices, three histidine motifs and a C-terminal ER-located signal (YNNKL). Phylogenetic analysis showed that both ShFAD2-1 and ShFAD2-2 are grouped with constitutive plant FAD2s. Heterologous expression in Saccharomyces cerevisiae indicated that ShFAD2-1 and ShFAD2-2 genes both encode a bio-functional delta-12 oleate desaturase. ShFAD2-2 was mainly expressed in flowers and early-stage seeds while ShFAD2-1 expression was almost constitutive in different organs. qRT-PCR results demonstrated that ShFAD2-1 and ShFAD2-2 show a cold-induced and heat-repressed expression pattern, whereas they also were differentially up-regulated or repressed by other abiotic stresses. This is the first cloning and function characterization of FAD2 from S. hispanica, which can provide insights into molecular mechanism of high ALA traits of S. hispanica and enrich our understanding of the roles of FAD2 genes in various abiotic stresses.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.