6-SFT, a Protein from Leymus mollis, Positively Regulates Salinity Tolerance and Enhances Fructan Levels in Arabidopsis thaliana.
ABSTRACT: Fructans play vital roles in abiotic stress tolerance in plants. In this study, we isolated the sucrose:6-fructosyltransferase gene, which is involved in the synthesis of fructans, from Leymus mollis by rapid amplification of cDNA ends. The Lm-6-SFT gene was introduced into Arabidopsis thaliana cv. Columbia by Agrobacterium-mediated transformation. The transgenic plants were evaluated under salt stress conditions. The results showed that the expression of Lm-6-SFT was significantly induced by light, abscisic acid (ABA), salicylic acid (SA), and salt treatment in L. mollis plants. Overexpression of Lm-6-SFT in Arabidopsis promoted seed germination and primary root growth during the early vegetative growth stage under salt stress. We also found that the transgenic plants expressing Lm-6-SFT had increased proline and fructan levels. β-Glucuronidase staining and promoter analysis indicated that the promoter of Lm-6-SFT was regulated by light, ABA, and salt stress. Quantitative PCR suggested that overexpression of Lm-6-SFT could improve salt tolerance by interacting with the expression of some salt stress tolerance genes. Thus, we demonstrated that the Lm-6-SFT gene is a candidate gene that potentially confers salt stress tolerance to plants. Our study will aid the elucidation of the regulatory mechanism of 6-SFT genes in herb plants.
Project description:The main storage compounds in Lolium perenne are fructans with prevailing β(2-6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
Project description:As a class of water-soluble, fructose-based oligo- and polysaccharides, fructans are major nonstructural carbohydrates and an important carbon source for grain filling in wheat (Triticum aestivum L.). Four enzymes are involved in fructan synthesis in higher plants, and 6-SFT is a key enzyme in fructan biosynthesis. In this study, thirteen single nucleotide polymorphisms were detected in 6-SFT-A2 in 24 wheat accessions, forming three haplotypes. Two cleaved amplified polymorphic sequence markers developed based on polymorphisms at sites 1870(A-G) and 1951(A-G) distinguished the three haplotypes. 6-SFT-A2 was located on chromosome 4A, between markers P2454.3 and P3465.1 in a doubled haploid (DH) population derived from the cross Hanxuan 10 × Lumai 14. The DH population comprising 150 lines and a historical population consisting of 154 accessions were used in a 6-SFT-A2 marker-trait association analysis. The three haplotypes were significantly associated with thousand-grain weight (TGW) under rainfed conditions. HapIII had a significant positive effect on TGW. There were significant differences between the Hanxuan 10 and Lumai 14 genotypes in both rainfed and irrigated environments. The average TGW of Lumai 14 (HapIII) was higher than that of Hanxuan 10 (HapI). The frequencies of 6-SFT-A2 HapIII in cultivars released at different periods showed that it had been strongly positively selected in breeding programs. The preferred HapIII for TGW occurred at higher frequencies in Gansu, Beijing, Shanxi, and Hebei than other regions in northern China.
Project description:Plants, as sessile organisms experience various abiotic stresses, which pose serious threat to crop production. Plants adapt to environmental stress by modulating their growth and development along with the various physiological and biochemical changes. This phenotypic plasticity is driven by the activation of specific genes encoding signal transduction, transcriptional regulation, ion transporters and metabolic pathways. Rice is an important staple food crop of nearly half of the world population and is well known to be a salt sensitive crop. The completion and enhanced annotations of rice genome sequence has provided the opportunity to study functional genomics of rice. Functional genomics aids in understanding the molecular and physiological basis to improve the salinity tolerance for sustainable rice production. Salt tolerant transgenic rice plants have been produced by incorporating various genes into rice. In this review we present the findings and investigations in the field of rice functional genomics that includes supporting genes and networks (ABA dependent and independent), osmoprotectants (proline, glycine betaine, trehalose, myo-inositol, and fructans), signaling molecules (Ca2+, abscisic acid, jasmonic acid, brassinosteroids) and transporters, regulating salt stress response in rice.
Project description:Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.
Project description:Although allelic diversity of genes has been shown to contribute to many phenotypic variations associated with different physiological processes in plants, information on allelic diversity of abiotic stress-responsive genes is limited. Here it is shown that the alleles OsWRKY45-1 and OsWRKY45-2 play different roles in abscisic acid (ABA) signalling and salt stress adaptation in rice. The two alleles had different transcriptional responses to ABA and salt stresses. OsWRKY45-1-overexpressing lines showed reduced ABA sensitivity, whereas OsWRKY45-1-knockout lines showed increased ABA sensitivity. OsWRKY45-1 transgenic plants showed no obvious difference from negative controls in response to salt stress. In contrast, OsWRKY45-2-overexpressing lines showed increased ABA sensitivity and reduced salt stress tolerance, and OsWRKY45-2-suppressing lines showed reduced ABA sensitivity and increased salt stress tolerance. OsWRKY45-1 and OsWRKY45-2 transgenic plants showed differential expression of a set of ABA- and abiotic stress-responsive genes, but they showed similar responses to cold and drought stresses. These results suggest that OsWRKY45-1 negatively and OsWRKY45-2 positively regulates ABA signalling and, in addition, OsWRKY45-2 but not OsWRKY45-1 negatively regulates rice response to salt stress. The different roles of the two alleles in ABA signalling and salt stress may be due to their transcriptional mediation of different signalling pathways.
Project description:BACKGROUND:Strigolactones (SLs) are considered to be a novel class of phytohormone involved in plant defense responses. Currently, their relationships with other plant hormones, such as abscisic acid (ABA), during responses to salinity stress are largely unknown. RESULTS:In this study, the relationship between SL and ABA during the induction of H2O2 - mediated tolerance to salt stress were studied in arbuscular mycorrhizal (AM) Sesbania cannabina seedlings. The SL levels increased after ABA treatments and decreased when ABA biosynthesis was inhibited in AM plants. Additionally, the expression levels of SL-biosynthesis genes in AM plants increased following treatments with exogenous ABA and H2O2. Furthermore, ABA-induced SL production was blocked by a pre-treatment with dimethylthiourea, which scavenges H2O2. In contrast, ABA production was unaffected by dimethylthiourea. Abscisic acid induced only partial and transient increases in the salt tolerance of TIS108 (a SL synthesis inhibitor) treated AM plants, whereas SL induced considerable and prolonged increases in salt tolerance after a pre-treatment with tungstate. CONCLUSIONS:These results strongly suggest that ABA is regulating the induction of salt tolerance by SL in AM S. cannabina seedlings.
Project description:BACKGROUND:Basic region/leucine zipper (bZIP) transcription factors perform as crucial regulators in ABA-mediated stress response in plants. Nevertheless, the functions for most bZIP family members in tomato remain to be deciphered. RESULTS:Here we examined the functional characterization of SlbZIP1 under salt and drought stresses in tomato. Silencing of SlbZIP1 in tomato resulted in reduced expression of multiple ABA biosynthesis- and signal transduction-related genes in transgenic plants. In stress assays, SlbZIP1-RNAi transgenic plants exhibited reduced tolerance to salt and drought stresses compared with WT plants, as are evaluated by multiple physiological parameters associated with stress responses, such as decreased ABA, chlorophyll contents and CAT activity, and increased MDA content. In addition, RNA-seq analysis of transgenic plants revealed that the transcription levels of multiple genes encoding defense proteins related to responses to abiotic stress (e.g. endochitinase, peroxidases, and lipid transfer proteins) and biotic stress (e.g. pathogenesis-related proteins) were downregulated in SlbZIP1-RNAi plants, suggesting that SlbZIP1 plays a role in regulating the genes related to biotic and abiotic stress response. CONCLUSIONS:Collectively, the data suggest that SlbZIP1 exerts an essential role in salt and drought stress tolerance through modulating an ABA-mediated pathway, and SlbZIP1 may hold potential applications in the engineering of salt- and drought-tolerant tomato cultivars.
Project description:KEY MESSAGE:HbMBF1a was isolated and characterized in H. brevisubulatum, and overexpressed HbMBF1a could enhance the salt tolerance and ABA insensitivity in Arabidopsis thaliana. The transcript levels of stress-responsive genes were significantly increased in the transgenic lines under salt and ABA conditions. Salinity is an abiotic stress that considerably affects plant growth, yield, and distribution. Hordeum brevisubulatum is a halophyte that evolved to become highly tolerant to salinity. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator and an important regulator of stress tolerance. In this study, we isolated and characterized HbMBF1a based on the transcriptome data of H. brevisubulatum grown under saline conditions. We overexpressed HbMBF1a in Arabidopsis thaliana and compared the phenotypes of the transgenic lines and the wild-type in response to stresses. The results indicated that HbMBF1a expression was induced by salt and ABA treatments during the middle and late stages. The overexpression of HbMBF1a in A. thaliana resulted in enhanced salt tolerance and ABA insensitivity. More specifically, the enhanced salt tolerance manifested as the increased seed germination and seedling growth and development. Similarly, under ABA treatments, the cotyledon greening rate and seedling root length were higher in the HbMBF1a-overexpressing lines, suggesting the transgenic plants were better adapted to high exogenous ABA levels. Furthermore, the transcript levels of stress-responsive genes were significantly increased in the transgenic lines under salt and ABA conditions. Thus, HbMBF1a is a positive regulator of salt and ABA responses, and the corresponding gene may be useful for producing transgenic plants that are salt tolerant and/or ABA insensitive, with few adverse effects. This study involved a comprehensive analysis of HbMBF1a. The results may provide the basis and insight for the application of MBF1 family genes for developing stress-tolerant crops.
Project description:The abscisic acid (ABA)-responsive element binding factors (ABFs) play important regulatory roles in multiple abiotic stresses responses. However, information on the stress tolerance functions of ABF genes in sweetpotato (Ipomoea batatas [L.] Lam) remains limited. In the present study, we isolated and functionally characterized the sweetpotato IbABF4 gene, which encodes an abiotic stress-inducible basic leucine zipper (bZIP) transcription factor. Sequence analysis showed that the IbABF4 protein contains a typical bZIP domain and five conserved Ser/Thr kinase phosphorylation sites (RXXS/T). The IbABF4 gene was constitutively expressed in leaf, petiole, stem, and root, with the highest expression in storage root body. Expression of IbABF4 was induced by ABA and several environmental stresses including drought, salt, and heat shock. The IbABF4 protein localized to the nucleus, exhibited transcriptional activation activity, and showed binding to the cis-acting ABA-responsive element (ABRE) in vitro. Overexpression of IbABF4 in Arabidopsis thaliana not only increased ABA sensitivity but also enhanced drought and salt stress tolerance. Furthermore, transgenic sweetpotato plants (hereafter referred to as SA plants) overexpressing IbABF4, generated in this study, exhibited increased tolerance to drought, salt, and oxidative stresses on the whole plant level. This phenotype was associated with higher photosynthetic efficiency and lower malondialdehyde and hydrogen peroxide content. Levels of endogenous ABA content and ABA/stress-responsive gene expression were significantly upregulated in transgenic Arabidopsis and sweetpotato plants compared with wild-type plants under drought stress. Our results suggest that the expression of IbABF4 in Arabidopsis and sweetpotato enhances tolerance to multiple abiotic stresses through the ABA signaling pathway.
Project description:Abscisic acid (ABA) is a vital cellular signal in plants, and effective ABA signalling is pivotal for stress tolerance. AtLOS5 encoding molybdenum cofactor sulphurase is a key regulator of ABA biosynthesis. Here, transgenic AtLOS5 plants were generated to explore the role of AtLOS5 in salt tolerance in maize. AtLOS5 overexpression significantly up-regulated the expression of ZmVp14-2, ZmAO, and ZmMOCO, and increased aldehyde oxidase activities, which enhanced ABA accumulation in transgenic plants under salt stress. Concurrently, AtLOS5 overexpression induced the expression of ZmNHX1, ZmCBL4, and ZmCIPK16, and enhanced the root net Na(+) efflux and H(+) influx, but decreased net K(+) efflux, which maintained a high cytosolic K(+)/Na(+) ratio in transgenic plants under salt stress. However, amiloride or sodium orthovanadate could significantly elevate K(+) effluxes and decrease Na(+) efflux and H(+) influx in salt-treated transgenic roots, but the K(+) effluxes were inhibited by TEA, suggesting that ion fluxes regulated by AtLOS5 overexpression were possibly due to activation of Na(+)/H(+) antiport and K(+) channels across the plasma membrane. Moreover, AtLOS5 overexpression could up-regulate the transcripts of ZmPIP1:1, ZmPIP1:5, and ZmPIP2:4, and enhance root hydraulic conductivity. Thus transgenic plants had higher leaf water potential and turgor, which was correlated with greater biomass accumulation under salt stress. Thus AtLOS5 overexpression induced the expression of ABA biosynthetic genes to promote ABA accumulation, which activated ion transporter and PIP aquaporin gene expression to regulate root ion fluxes and water uptake, thus maintaining high cytosolic K(+) and Na(+) homeostasis and better water status in maize exposed to salt stress.