GmHXK2 promotes the salt tolerance of soybean seedlings by mediating AsA synthesis, and auxin synthesis and distribution.

BACKGROUND: Salt is an important factor that affects crop productivity. Plant hexokinases (HXKs) are key enzymes in the glycolytic pathway and sugar signaling transduction pathways of plants. In previous studies, we identified and confirmed the roles of GmHXK2 in salt tolerance. RESULTS: In this study, we analyzed the tissue-specific expression of GmHXK2 at different growth stages throughout the plant's life cycle. The results showed that GmHXK2 was expressed significantly in all tissues at vegetative stages, including germination and seedling. However, no expression was detected in the pods, and there was little expression in flowers during the later mature period. Arabidopsis plants overexpressing the GmHXK2 (OE) had more lateral roots. The OE seedlings also produced higher levels of auxin and ascorbic acid (AsA). Additionally, the expression levels of genes PMM, YUC4/YUC6/YUC8, and PIN/LAX1,LAX3, which are involved respectively in the synthesis of AsA and auxin, as well as polar auxin transport, were upregulated in OE plants. This upregulation occurred specifically under exogenous glucose treatment. AtHKT1, AtSOS1, and AtNHX1 were up-regulated in OE plants under salt stress, suggesting that GmHXK2 may modulate salt tolerance by maintaining ion balance within the cells and alleviating damage caused by salt stress. Additionally, we further confirmed the interaction between GmHXK2 and the protein GmPMM through yeast two-hybridization and bimolecular fluorescence complementation assays, respectively. CONCLUSION: The expression of GmHXK2 gene in plants is organ-specific and developmental stage specific. GmHXK2 not only regulates the synthesis of AsA and the synthesis and distribution of auxin, but also promotes root elongation and induces lateral root formation, potentially enhancing soil water absorption. This study reveals the crosstalk between sugar signaling and hormone signaling in plants, where GmHXK2 acts as a glucose sensor through its interaction with GmPMM, and sheds light on the molecular mechanism by which GmHXK2 gene is involved in salt tolerance in plants.

Co-inoculation of Soybean Seedling with Trichoderma asperellum and Irpex laceratus Promotes the Absorption of Nitrogen and Phosphorus.

Soybean are one of the main oil crops in the world. The study demonstrated that co-inoculation with Trichoderma asperellum (Sordariomycetes, Hypocreomycetidae) and Irpex laceratus (Basidiomycota, Polyporales) isolated from Kosteletzkya virginica can promote the growth of soybean seedlings. The two fungi were found to produce various enzymes, including cellulase, amylase, laccase, protease, and urease. Upon inoculation, T. asperellum mainly colonized within the phloem of the roots in soybean seedlings, while I. laceratus mainly in the xylem and phloem of the roots. Physiological parameters, such as plant height, root length, and fresh weight, were significantly increased in soybean seedlings co-inoculated with T. asperellum and I. laceratus. Moreover, the expression of key genes related to N and P absorption and metabolism was also increased, leading to improved N and P utilization efficiency in soybean seedlings. These results indicate that the two fungi may have complementary roles in promoting plant growth, co-inoculation with T. asperellum and I. laceratus can enhance the growth and nutrient uptake of soybean. These findings suggest that T. asperellum and I. laceratus have the potential to be used as bio-fertilizers to improve soybean growth and yield.

Overexpression of KvCHX Enhances Salt Tolerance in Arabidopsis thaliana Seedlings.

The CHX (cation/H+ exchanger) family plays an important role in the transmembrane transport of cation/H+ in plants. The aim of this study was to identify and functionally analyze the KvCHX gene in the halophyte Kosteletzkya virginica to investigate its role in regulating the K+/Na+ ratio under salinity tolerance. Based on a partial gene sequence of EST from K. virginica, the full-length DNA sequence of the KvCHX gene was obtained using genome walking technology. Structural analysis and phylogenetic relationship analysis showed that the KvCHX gene was closely related to the AtCHX17 gene. The KvCHX overexpression vector was successfully constructed and transformed into Arabidopsis via floral dipping. Arabidopsis seedlings overexpressing KvCHX showed an enhanced tolerance to salt stress compared with wild-type plants. Transgenic Arabidopsis seedlings grew better under K+ deficiency than WT. The results showed that KvCHX could promote the uptake of K+, increase the ratio of K+/Na+, and promote the growth of plants under K+ deficiency and treatment with NaCl solution. KvCHX is involved in K+ transport and improves plant salt tolerance by coordinating K+ acquisition and homeostasis.

GhCNGC13 and 32 Act as Critical Links between Growth and Immunity in Cotton.

Cyclic nucleotide-gated ion channels (CNGCs) remain poorly studied in crop plants, most of which are polyploid. In allotetraploid Upland cotton (Gossypium hirsutum), silencing GhCNGC13 and 32 impaired plant growth and shoot apical meristem (SAM) development, while triggering plant autoimmunity. Both growth hormones (indole-3-acetic acid and gibberellin) and stress hormones (abscisic acid, salicylic acid, and jasmonate) increased, while leaf photosynthesis decreased. The silenced plants exhibited an enhanced resistance to Botrytis cinerea; however, Verticillium wilt resistance was weakened, which was associated with LIPOXYGENASE2 (LOX2) downregulation. Transcriptomic analysis of silenced plants revealed 4835 differentially expressed genes (DEGs) with functional enrichment in immunity and photosynthesis. These DEGs included a set of transcription factors with significant over-representation in the HSF, NAC, and WRKY families. Moreover, numerous members of the GhCNGC family were identified among the DEGs, which may indicate a coordinated action. Collectively, our results suggested that GhCNGC13 and 32 functionally link to photosynthesis, plant growth, and plant immunity. We proposed that GhCNGC13 and 32 play a critical role in the "growth-defense tradeoff" widely observed in crops.

Dynamic Expression, Differential Regulation and Functional Diversity of the CNGC Family Genes in Cotton.

Cyclic nucleotide-gated channels (CNGCs) constitute a family of non-selective cation channels that are primarily permeable to Ca2+ and activated by the direct binding of cyclic nucleotides (i.e., cAMP and cGMP) to mediate cellular signaling, both in animals and plants. Until now, our understanding of CNGCs in cotton (Gossypium spp.) remains poorly addressed. In the present study, we have identified 40, 41, 20, 20, and 20 CNGC genes in G. hirsutum, G. barbadense, G. herbaceum, G. arboreum, and G. raimondii, respectively, and demonstrated characteristics of the phylogenetic relationships, gene structures, chromosomal localization, gene duplication, and synteny. Further investigation of CNGC genes in G. hirsutum, named GhCNGC1-40, indicated that they are not only extensively expressed in various tissues and at different developmental stages, but also display diverse expression patterns in response to hormones (abscisic acid, salicylic acid, methyl jasmonate, ethylene), abiotic (salt stress) and biotic (Verticillium dahlia infection) stimuli, which conform with a variety of cis-acting regulatory elements residing in the promoter regions; moreover, a set of GhCNGCs are responsive to cAMP signaling during cotton fiber development. Protein-protein interactions supported the functional aspects of GhCNGCs in plant growth, development, and stress responses. Accordingly, the silencing of the homoeologous gene pair GhCNGC1&18 and GhCNGC12&31 impaired plant growth and development; however, GhCNGC1&18-silenced plants enhanced Verticillium wilt resistance and salt tolerance, whereas GhCNGC12&31-silenced plants had opposite effects. Together, these results unveiled the dynamic expression, differential regulation, and functional diversity of the CNGC family genes in cotton. The present work has laid the foundation for further studies and the utilization of CNGCs in cotton genetic improvement.

CYP3A4*1G regulates CYP3A4 intron 10 enhancer and promoter activity in an allelic-dependent manner.

OBJECTIVE: CYP3A4*1G (G > A) in human CYP3A4 intron 10 is associated with therapeutic effects of CYP3A4-metabolized drugs. The aim of this study was to predict its function in the regulation of CYP3A4 expression. METHODS: Functional analysis of the CYP3A4*1G allele was performed using bioinformatic methods and enhancer or promoter reporter assays in HepG2 cells. RESULTS: Transcription regulatory elements like CAATboxes, TATA-boxes, Sp1, SMARCA3.01, and Box II-like sequence were present in the intron 10 of CYP3A4. SMARCA3.01 and Box II-like sequence were responsible for differential binding of transcription factors on the CYP3A4*1G allele. In CYP3A4*1G, the G allele enhanced expression of the CYP3A4 promoter in a position-dependent and orientation-dependent manner, however, the A allele enhanced expression of the CYP3A4 promoter in a position-independent and orientation-independent manner. In addition, the G allele and the A allele both displayed strong transcriptional activation, but the latter showed higher promoter activity than the former. Also, the A allele showed greater activity than the CYP3A4 promoter. CONCLUSION: These results in vitro suggest that CYP3A4*1G regulates CYP3A4 intron 10 enhancer and promoter activity in an allelic-dependent manner.

Response of maize serine/arginine-rich protein gene family in seedlings to drought stress.

Alternative splicing (AS) in eukaryotic organisms is closely related to the gene regulation in plant abiotic stress responses, in which serine/arginine-rich proteins (SR proteins) act as key regulators. The genome sequence of maize inbred line B73 was analyzed, showing that the promoter regions of SR genes possess about three to eight kinds of cis-acting regulatory elements. Twenty-seven SR genes encode alkaline proteins, and 23 of which are divided into five subgroups in terms of the first RNA recognition motif (RRM) at the amino terminal. The expression of SR genes showed tissue-specific and genotype-dependent features under drought stress in the hybrid Zhengdan-958 and its parents, Zheng-58 and Chang-7-2 via bidirectional hierarchical clustering. SR genes were down-regulated in roots while they were up-regulated in shoots under drought stress. However, SR genes were down-regulated in both roots and shoots in three different rehydration stages after severe drought stress. Additionally, a widespread alternative splicing exists in all SR genes although SR genes showed differential expression tendency under drought stress and/or during rehydration stages. Results above will deepen our understanding of the molecular mechanisms of plant response to abiotic stress from the perspective of AS-network.

Characterization of hexokinase gene family members in Glycine max and functional analysis of GmHXK2 under salt stress.

Hexokinase (HXK) is a bifunctional enzyme involved in carbohydrate metabolism and sugar signal sensing. HXK gene family has been extensively discussed in many species, while the detailed investigations of the family in Glycine max have yet to be reported. In this study, 17 GmHXK genes (GmHXKs) were identified in the G. max genome and the features of their encoded proteins, conserved domains, gene structures, and cis-acting elements were systematically characterized. The GmHXK2 gene isolated from G. max was firstly constructed into plant expression vector pMDC83 and then transformed with Agrobacterium tumefaciens into Arabidopsis thaliana. The expression of integrated protein was analyzed by Western Blotting. Subcellular localization analysis showed that the GmHXK2 was located on both vacuolar and cell membrane. Under salt stress, seedlings growth was significantly improved in Arabidopsis overexpressing GmHXK2 gene. Furthermore, physiological indicators and expression of salt stress responsive genes involved in K+ and Na+ homeostasis were significantly lower in GmHXK2-silenced soybean seedlings obtained by virus-induced gene silencing (VIGS) technique under salt stress compared with the control plants. Our study showed that GmHXK2 gene played an important role in resisting salt stress, which suggested potential value for the genetic improvement of abiotic resistant crops.

Characterization key genes of Arabidopsis seedlings in response to ß-caryophyllene, eugenol using combined transcriptome and WGCN analysis.

Weeds present a significant challenge to high crop yield and quality. In our study, we investigated the phytotoxic activity of ß-caryophyllene (BCP) and eugenol, which are natural allelopathic chemical compounds, on Arabidopsis seedlings. We found that these compounds inhibited the growth of Arabidopsis thaliana plants. When either BCP or eugenol was applied, it led to decrease in the content of cell wall components such as lignin, cellulose, hemicellulose, and pectin; and increase in the levels of endogenous hormones like ETH, ABA, SA, and JA in the seedlings. Through transcriptome profiling, we identified 7181 differentially expressed genes (DEGs) in the roots and shoots that were induced by BCP or eugenol. The genes involved in the synthesis of lignin, cellulose, hemicellulose, and pectin were down-regulated, whereas genes related to synthesis and signal transduction of ABA, ETH, SA, and JA were up-regulated. However, genes related to IAA synthesis and signal transduction were found to be down-regulated. Furthermore, we characterized 24 hub genes using Weighted Correlation Network Analysis (WGCNA). Among them, the identified 16 genes in response to BCP was primarily associated with hypoxia stress, while 8 genes induced by eugenol were linked to inhibition of cell division. Our results suggested that BCP and eugenol had ability to target multiple genes to inhibit growth and development of Arabidopsis plants. Therefore, they can serve as excellent candidates for natural biological herbicides.

Identification of endophytic fungi with ACC deaminase-producing isolated from halophyte Kosteletzkya Virginica.

Seashore mallow (Kosteletzkya virginica), as a noninvasive perennial halophytic oilseed-producing dicot, is native from the Gulf to the Atlantic coasts of the U.S. The purpose of our research was to investigate 1-aminocyclopropane-1carboxylic acid deaminase (ACCD) producing endophytic fungi from K.virginica. A total of 59 endophytic fungal strains, isolated from roots in K.virginica of seedlings, were grouped into 12 genera including in Penicillium, Aspergillus, Fusarium, Trichoderma, Rhizopycnis sp., Ceriporia Donk, Trametes sp., Schizophyllum commune sp., Alternaria, Cladosporium, Cylindrocarpon, and Scytalidium according to sequences of ITS. The ACD activity of 10 endophytic fungi isolated was detected. T.asperellum had the highest ACC deaminase activity among all 10 isolated genera of fungal strains, followed by T. viride. Dry weight and fresh weight of plant, plant height, root length, SOD activity, and chlorophyll content of wheat and soybean inoculated with T.asperellum or T. viride was increased compared with non-inoculated control plants under non salt or salt stress. Further analysis showed that T.asperellum or T.viride strains induced downregulation of the expression of ethylene synthesis-related genes such as ACC oxidase (ACO) and ACC synthase (ACS), thereby reducing ethylene synthesis and damage to plants under salt stress. These endophytic fungi can be used as alternative bioinoculants to increase crop yield in saline soil.

Gene expression of halophyte Kosteletzkya virginica seedlings under salt stress at early stage.

Gene differential expression of Kosteletzkya virginica seedlings under salt stress at two time points (2, 24 h) in roots and leaves was analyzed using the cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Polymorphic transcript-derived fragments (TDFs) among control plants and salt-treated plants were grouped into four main differential expression patterns: repression (A), de novo induction (B), up-regulation (C) and down-regulation (D). Among them, 34 differentially expressed gene fragments were homologous to known genes from other species and 4 were sequences with unknown functions. These differentially expressed genes can be classified into four groups according to their putative functions: (1) genes for re-establishing ion homeostasis and protecting the plant from stress damage; (2) genes involved in metabolism or energy and resuming plant growth and development under salt stress; (3) genes involved in regulation of gene expression; (4) genes for signal transduction. Changes of eight differentially expressed genes were confirmed by quantitative real time RT-PCR.

[Analysis of gene expression involved in the response to salt stress in the dicot halophyte Kosteletzkya virginica L. seedlings].

Kosteletzkya virginica L. Presl. is an obligate wetland species indigenous to southeastern US. Its niche in salt marsh foretells its high salinity tolerance. cDNA-AFLP technique was used to identify the gene transcriptional profiles of leaves and roots from K. virginica seedlings under salt stress in order to clarify the molecular architecture of stress tolerance in the dicot halophyte. Expression analysis over time intervals and under various salt stresses in leaves or roots showed that the quantitatively expressed pattern (in which genes were quantitatively up- or down-regulated under salt stress or fluctuate with different NaCl concentrations) was more prevalent than the qualitatively expressed pattern (in which genes were induced or silenced under salt stress) in K. virginica seedlings under salt stress. The qualitative pattern was appreciably more predominant than the quantitative one only in roots when exposed to salt stress for 2 h. Although each expression pattern was observed in leaves as well as in roots, the percentage of genes (i.e., up-/down-regulated or induced/silenced under salt stress) was dynamically changeable under salt stress at different time intervals. All these results indicated that there was no established formula of gene expression patterns in deciphering the sophisticated mechanism of plant salinity tolerance, considering that plants undergo a series of dynamically physiological and metabolic pathways in sensing and response to salt stress for different tissues and during different stages of stress. A number of Trivially distributed file system (TDFs) up-regulated or induced under salt stress from leaves and roots were sequenced, and the sequences were blasted against the NCBI non-redundant protein database using translated nucleotide query (Blastx). The TDFs from K. virginica seedlings involved in sensing and response to salt stress can be classified at least into three groups according to their putative functions: (1) genes for re-establishing ionic homeostasis or preventing from damage (specially genes for transporter); (2) genes for resuming plant growth and development under salt stress, such as key enzymes involved in energy synthesis or hormone regulatory pathway; (3) genes for signal transduction and so on. The relationship of expression patterns of these TDFs with the molecular mechanism of salt tolerance in K. virginica was discussed.

Relationship between differential gene expression patterns in functional leaves of maize inbreds & hybrids at spikelet differentiation stage and heterosis.

To understand the molecular mechanism of maize heterosis, differential gene expression patterns in functional leaves between 10 maize inbreds and 38 hybrids at spikelet differentiation stage were analyzed by using cDNA-AFLP. The correlation analysis of various differential gene expression patterns with the performance and heterosis of main maize agronomic traits was carried out. The main results are as follows: (1) There are differential gene expression patterns in quality and quantity between hybrids and their parents. The differential expression patterns in quality include: bands expressed only in one parent, bands expressed only in both parents, bands expressed only in one parent and F1, bands expressed only in F1. (2) At spikelet differentiation stage, there are large variations among different hybrids for the same differentially expressed patterns. In general from mean data, there were 25.22% bands expressed only in F1, 21.46% bands expressed in one parent and F1, 8.27% bands expressed only in both parents and 33.49% bands expressed only in one parent. (3) For bands expressed only in one parent, significant positive correlation was detected with the relationship to the performance of plant height. For bands expressed only in both parents, significant negative correlation was detected with the relationship to the heterosis of ear diameter. For bands expressed only in one parent and F1, significant negative correlations were detected with the relationships to the heterosis of rows per ear and seed weight per ear. However, for bands expressed only in F1, and for bands only in two parents or only in F1, no significant correlation was detected with the relationship to the performance and heterosis of all agronomic traits.