Transgenic Seedlings Research Articles

The Recretohalophyte Tamarix TrSOS1 Gene Confers Enhanced Salt Tolerance to Transgenic Hairy Root Composite Cotton Seedlings Exhibiting Virus-Induced Gene Silencing of GhSOS1.

The salt overly sensitive 1 (SOS1) gene encodes the plasma membrane Na+/H+ antiporter, SOS1, that is mainly responsible for extruding Na+ from the cytoplasm and reducing the Na+ content in plants under salt stress and is considered a vital determinant in conferring salt tolerance to the plant. However, studies on the salt tolerance function of the TrSOS1 gene of recretohalophytes, such as Tamarix, are limited. In this work, the effects of salt stress on cotton seedlings transformed with tobacco-rattle-virus-based virus-induced gene silencing (VIGS) of the endogenous GhSOS1 gene, or Agrobacterium rhizogenes strain K599-mediated TrSOS1-transgenic hairy root composite cotton plants exhibiting VIGS of GhSOS1 were first investigated. Then, with Arabidopsis thaliana AtSOS1 as a reference, differences in the complementation effect of TrSOS1 or GhSOS1 in a yeast mutant were compared under salt treatment. Results showed that compared to empty-vector-transformed plants, GhSOS1-VIGS-transformed cotton plants were more sensitive to salt stress and had reduced growth, insufficient root vigor, and increased Na+ content and Na+/K+ ratio in roots, stems, and leaves. Overexpression of TrSOS1 enhanced the salt tolerance of hairy root composite cotton seedlings exhibiting GhSOS1-VIGS by maintaining higher root vigor and leaf relative water content (RWC), and lower Na+ content and Na+/K+ ratio in roots, stems, and leaves. Transformations of TrSOS1, GhSOS1, or AtSOS1 into yeast NHA1 (Na+/H+ antiporter 1) mutant reduced cellular Na+ content and Na+/K+ ratio, increased K+ level under salt stress, and had good growth complementation in saline conditions. In particular, the ability of TrSOS1 or GhSOS1 to complement the yeast mutant was better than that of AtSOS1. This may indicate that TrSOS1 is an effective substitute and confers enhanced salt tolerance to transgenic hairy root composite cotton seedlings, and even the SOS1 gene from salt-tolerant Tamarix or cotton may have higher efficiency than salt-sensitive Arabidopsis in regulating Na+ efflux, maintaining Na+ and K+ homeostasis, and therefore contributing to stronger salt tolerance.

The constitutive expression of alfalfa MsMYB2L enhances salinity and drought tolerance of Arabidopsis thaliana

MYB-type transcription factors are known to participate in the response of plants to a number of stress agents. MsMYB2L is an alfalfa member of this large gene family. Its transcription in alfalfa seedlings was found to be rapidly and strongly induced by salinity, moisture deficiency and exogenously supplied abscisic acid. An analysis based on a yeast one hybrid assay indicated that its product is able to activate transcription, consistent with its function as a transcription factor. When the gene was constitutively expressed in Arabidopsis thaliana, both germination and seedling growth were more sensitive to ABA treatment than wild type, and growth was less strongly compromised by salinity and moisture deficiency stress, presumably as a result of the induction of certain stress-related genes active in ABA-dependent pathways. The transgenic seedlings’ enhanced the synthesis of many osmotic regulatory substances such as proline and soluble sugar, and decreased the lipid peroxidation. In all, MsMYB2L represents a potential candidate gene for manipulating the salinity and drought tolerance of alfalfa.

Chemical enhancers of posttranscriptional gene silencing in Arabidopsis.

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.

Overproduction of fungal endo-β-1,4-glucanase leads to characteristic lignocellulose modification for considerably enhanced biomass enzymatic saccharification and bioethanol production in transgenic rice straw

Genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance for enhanced biomass enzymatic saccharification and biofuel production in bioenergy crops. Although endo-β-1,4-glucanase (EG II) secreted by fungi has been broadly applied for enzymatic hydrolysis of cellulose, it remains to explore its role in cellulose modification when the EG II gene is overexpressed in plant. In this study, we selected transgenic rice plants that overproduced Trichoderma reesei EG II enzyme specifically deposited into plant cell walls, and then examined much higher enzymatic activities by fourfold to fivefold in transgenic young seedlings than those of wild type in vitro. Notably, despite slightly altered cell wall compositions and polymer interlinkages relative to the wild type, the transgenic mature rice straw exhibited significantly reduced cellulose DP and CrI values and hemicellulosic Xyl/Ara ratio, leading to much increased biomass porosity. These should play integrated impact for enhanced biomass enzymatic saccharification and bioethanol production even under mild alkali pretreatment. Therefore, the results suggested that the EG II deposition should have enzymatic activity specific for minor-modification of cellulose microfibrils in transgenic rice plants. It has also provided a potential strategy for mild cell wall modification and optimal biomass process in rice and other bioenergy crops.

The high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and early seedling growth of Brassica napus

BackgroundSeed germination and seedling establishment are two of the most critical phases in plant development. However, the molecular mechanisms underlying the effect of phosphorus on seed germination and post-germinated growth of oilseed rape are unclear so far. Here, we report the role of BnPHT1;4 in seed germination and early seedling development of Brassica napus.ResultsOur results show that BnPHT1;4 is preferentially expressed in cotyledons of early developing seedlings. Overexpression of BnPHT1;4 in oilseed rape promoted seed germination and seedling growth. Expression levels of the genes related to ABA and GA biosynthesis and signaling were significantly altered in BnPHT1;4 transgenic seedlings. Consequently, active GA level was up-regulated, whereas ABA content was down-regulated in BnPHT1;4 transgenic seedlings. Furthermore, exogenous GA could promote seed germination of wild type, while exogenous ABA could partially recover the advanced-germination phenotype of BnPHT1;4 transgenic seeds. Total phosphorus content in cotyledons of the transgenic seedlings was decreased more rapidly than that in wild type when Pi was supplied or deficient, and Pi contents in shoots and roots of the BnPHT1;4 transgenic plants were higher than those in wild type under high and low Pi conditions.ConclusionsOur data suggest that the high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and seedling growth of Brassica napus by modulating ABA and GA biosynthesis.

Accumulation of cadmium (Cd) in T1 transgenic tobacco seedlings expressing metallothionein gene from Eleusine indica

Cadmium (Cd) contamination of urban and agricultural soils is toxic to humans, animals and may cause negative effects on plant growth and crop production. The existing conventional methods are found to be not efficient to remove Cd from contaminated soil. The present experiment reports the analysis of nine T1 lines of transgenic tobacco carrying metallothionein gene (eiMT1) from Eleusine indica, with potential for high efficiency to remediate Cd in contaminated soils. Thirty-days old tobacco plants were treated with different concentrations of CdNO3 (0, 50, 100 and, 150 µmol) for seven days and the accumulation of Cd in the whole seedling was quantitatively determined by using atomic absorption spectrometer (AAS). All transgenic tobacco lines showed greater tolerance and accumulated higher level of Cd than the wild type with lines 18D, 20D1 and, 18C were among the highest (678.7, 623.0 and 611.9 mgkg-1 Cd, respectively). Meanwhile, transgenic tobacco lines 18B1 and 20D1 showed higher expression of eiMT1 gene. These results suggest that the cadmium accumulation in transgenic tobacco did not strictly associate with the expression level of eiMT1 gene. However, expression of eiMT1 greatly required for higher accumulation of Cd in transgenic tobacco seedling.

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice.

A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice. A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides - 351 and - 121, which contains the sugar response complex (- 266 to - 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.

Hydrogen sulfide: A novel component in Arabidopsis peroxisomes which triggers catalase inhibition.

Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as H2 O2 , superoxide radical (O2 · - ), nitric oxide and peroxynitrite (ONOO- ). These organelles have an active nitro-oxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (H2 S) is a new signaling gasotransmitter which can mediate the posttranslational modification (PTM) persulfidation. We used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) fused to a canonical peroxisome targeting signal 1 (PTS1) to visualize peroxisomes in living cells, as well as a specific fluorescent probe which showed that peroxisomes contain H2 S. H2 S was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory H2 O2 -generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a H2 S donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the H2 S donor. To corroborate the inhibitory effect of H2 S on catalase activity, we also assayed pure catalase from bovine liver and pepper fruit-enriched samples, in which catalase activity was inhibited. Taken together, these data provide evidence of the presence of H2 S in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal H2 O2 metabolism.

Evidence for the Accumulation of 3‐Hydroxypropionate and β‐Alanine in Arabidopsis thaliana Seedlings via Isoleucine Degradation

Amino acids serve several cellular processes outside of proteinogenesis that influence the growth and development of the plant, as well as the generation of metabolic energy. Here, we examine the contribution of branched‐chain amino acid degradation to the production of β‐alanine in the model organism, Arabidopsis thaliana. β‐alanine is a key intermediate in the synthesis of the essential molecules vitamin B5 and coenzyme A (CoA). Despite the importance, the current knowledge surrounding β‐alanine lacks strong evidence for the primary biosynthetic pathways in higher plants. Previous studies found that polyamines, uracil, and propionate can all serve as sources for β‐alanine. Given that the catabolism of branched‐chain amino acids (BCAAs) valine and isoleucine result in the production of propionyl‐CoA (a derivative of propionate), we hypothesized that these BCAAs could also serve as a source of β‐alanine. Propionyl‐CoA is typically converted to acetyl‐CoA via β‐oxidation, however, one of the intermediates, malonate semialdehyde, can react to form β‐alanine. To trace the production of β‐alanine from propionyl‐CoA and the BCAA isoleucine, we treated wild‐type and transgenic seedlings with isotopically‐labeled precursors and analyzed extracts using 13C‐NMR and GC‐MS. We observed the production of both β‐alanine and a key intermediate, 3‐hydroxypropionate (3HP) in samples treated with propionate and isoleucine. Our data shows that the fed 13C‐isotopes were efficiently incorporated into and recycled through propionyl‐CoA metabolism, resulting in the production of 13C‐labeled β‐alanine. These findings suggest that while there is evidence for the production of β‐alanine via polyamines, uracil, and propionate in other plants; isoleucine, and possibly valine, can also serve as a precursor to this metabolically important molecule.Support or Funding InformationClare Boothe Luce Undergraduate Scholars program funded by the Henry Luce Foundation (Grant #9601)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A LEA Gene from a Vietnamese Maize Landrace Can Enhance the Drought Tolerance of Transgenic Maize and Tobacco

Maize (Zea mays) is a major cereal crop worldwide, and there is increasing demand for maize cultivars with enhanced tolerance to desiccation. Late embryogenesis abundant (LEA) proteins group 5C is involved in plants’ responses to various osmotic stresses such as drought and salt. A putative group 5C LEA gene from Z. mays cv. Tevang 1 was isolated, named ZmLEA14tv, and cloned into a T-DNA for expression in plants. The deduced amino acid of ZmLEA14tv showed a conserved Pfam LEA_2 domain and a high proportion of hydrophobic residues, characteristic of group 5C LEA proteins. Transgenic tobacco and maize plants expressing ZmLEA14tv were generated. During drought simulation conditions, the ZmLEA14tv-expressing plants of tobacco showed improved recovery ability, while those of maize enhanced the seed germination in comparison with the non-transgenic control plants. In addition, the survival rate of ZmLEA14tv transgenic maize seedlings was twice as high as the control. These results indicated that ZmLEA14tv might be involved in the drought tolerance of plants and could be a candidate gene for developing enhanced drought-tolerant crops.

Loss of Arabidopsis Halotolerance 2-like (AHL), a 3'-phosphoadenosine-5'-phosphate phosphatase, suppresses insensitive response of Arabidopsis thaliana ring zinc finger 1 (atrzf1) mutant to abiotic stress.

Destruction of PAP phosphatase AHL suppresses atrzf1 phenotype in abiotic stress responses. AHL plays an intermediate role in the regulation of proline accumulation by PAP nucleotidase. Proline (Pro) metabolism is important for environmental responses, plant development, and growth. However, the role of Pro in abiotic stress process is unclear. Using atrzf1 (Arabidopsis thaliana ring zinc finger 1) mutant as a parental line for T-DNA tagging mutagenesis, we identified a suppressor mutant designated as proline content alterative 17 (pca17) that suppressed insensitivity of atrzf1 to abiotic stresses during early seedling growth. Pro content of pca17 was lower than that in both wild type (WT) and atrzf1 while complementary lines were less sensitive to abscisic acid (ABA) and abiotic stresses compared to WT. Thermal Asymmetric Interlaced (TAIL)-PCR of pca17 showed that T-DNA was inserted at site of At5g54390 (AHL for Arabidopsis Halotolerance 2-like) encoding 3'-phosphoadenosine-5'-phosphate (PAP) phosphatase. Under drought stress condition, products of sulfate metabolism such as PAP and adenosine monophosphate were significantly lower in pca17 than those in WT and atrzf1. Furthermore, pca17 showed significantly higher levels of several important drought parameters including malondialdehyde, ion leakage, and water loss than WT and atrzf1. Fluorescence signal of green fluorescent protein (GFP)-tagged AHL was quite strong in nuclei of the root and guard cells of transgenic seedlings. Additionally, AHL promoter-β-glucuronidase (GUS) construct revealed substantial gene expression in vasculature tissues and pollen. Collectively, these findings demonstrate that pca17 acts as a dominant suppressor mutant of atrzf1 in abiotic stress response by modulating proline and sulfate metabolism.

Maize annexin genes ZmANN33 and ZmANN35 encode proteins that function in cell membrane recovery during seed germination.

Plasma membrane (PM) recovery from the impaired dry state is essential for seed germination, but its underlying mechanism remains unclear. In this study, we found that ZmANN33 and ZmANN35, two annexin genes in maize, encode proteins that participate in PM recovery during seed germination. The expression of both genes was up-regulated during seed germination and strongly repressed by chilling (either 15 or 5 °C) as compared with the normal temperature (25 °C). In addition, the increased membrane damage caused by chilling imbibition was correlated with suppressed expression of ZmANN33 and ZmANN35, while rapid recovery of their expression levels accompanied the rescue of the damaged membrane. Arabidopsis seedlings ectopically expressing ZmANN33 or ZmANN35 had longer seedling length than wild-type (WT) plants during the recovery period after 3 d of chilling stress, indicating the positive roles of these two gene products in the plant's recovery from chilling injury. Moreover, these transgenic seedlings had lower lipid peroxidation and higher peroxidase activities than WT during the recovery period. Consistently, root cells of these transgenic seedlings had more intact PM after chilling stress, supporting the proposition that ZmANN33 and ZmANN35 contribute to the maintenance of PM integrity. The enhanced PM integrity is likely due to the accelerated exocytotic process after chilling stress. We also showed that both ZmANN33 and ZmANN35 localized in the cytosol near the plasma membrane. Thus, we conclude that ZmANN33 and ZmANN35 play essential roles in membrane recovery during maize seed germination.

The WRKY Transcription Factor GmWRKY12 Confers Drought and Salt Tolerance in Soybean.

WRKYs are important regulators in plant development and stress responses. However, knowledge of this superfamily in soybean is limited. In this study, we characterized the drought- and salt-induced gene GmWRKY12 based on RNA-Seq and qRT-PCR. GmWRKY12, which is 714 bp in length, encoded 237 amino acids and grouped into WRKY II. The promoter region of GmWRKY12 included ABER4, MYB, MYC, GT-1, W-box and DPBF cis-elements, which possibly participate in abscisic acid (ABA), drought and salt stress responses. GmWRKY12 was minimally expressed in different tissues under normal conditions but highly expressed under drought and salt treatments. As a nucleus protein, GmWRKY12 was responsive to drought, salt, ABA and salicylic acid (SA) stresses. Using a transgenic hairy root assay, we further characterized the roles of GmWRKY12 in abiotic stress tolerance. Compared with control (Williams 82), overexpression of GmWRKY12 enhanced drought and salt tolerance, increased proline (Pro) content and decreased malondialdehyde (MDA) content under drought and salt treatment in transgenic soybean seedlings. These results may provide a basis to understand the functions of GmWRKY12 in abiotic stress responses in soybean.

The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression

BackgroundGRAS transcription factor (TF) family is unique and numerous in higher plants with diverse functions that involving in plant growth and development processes, such as gibberellin (GA) signal transduction, root development, root nodule formation, and mycorrhiza formation. Walnut tree is exposed to various environmental stimulus that causing concern about its resistance mechanism. In order to understand the molecular mechanism of walnut to adversity response, a GRAS TF (JrGRAS2) was cloned and characterized from Juglans regia in this study.ResultsA 1500 bp promoter fragment of JrGRAS2 was identified from the genome of J. regia, in which the cis-elements were screened. This JrGRAS2 promoter displayed expression activity that was enhanced significantly by high temperature (HT) stress. Yeast one-hybrid assay, transient expression and chromatin immunoprecipitation (Chip)-PCR analysis revealed that JrDof3 could specifically bind to the DOFCOREZM motif and share similar expression patterns with JrGRAS2 under HT stress. The transcription of JrGRAS2 was induced by HT stress and up-regulated to 6.73-~11.96-fold in the leaf and 2.53-~4.50-fold in the root to control, respectively. JrGRAS2 was overexpressed in Arabidopsis, three lines with much high expression level of JrGRAS2 (S3, S7, and S8) were selected for HT stress tolerance analysis. Compared to the wild type (WT) Arabidopsis, S3, S7, and S8 exhibited enhanced seed germination rate, fresh weight accumulation, and activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione-S-transferase (GST) under HT stress. In contrast, the Evans blue staining, electrolyte leakage (EL) rates, hydrogen dioxide (H2O2) and malondialdehyde (MDA) content of transgenic seedlings were all lower than those of WT exposed to HT stress. Furthermore, the expression of heat shock proteins (HSPs) in S3, S7, and S8 was significant higher than those in WT plants. The similar results were obtained in JrGRAS2 transient overexpression walnut lines under normal and HT stress conditions.ConclusionsOur results suggested that JrDof3 TF contributes to improve the HT stress response of JrGRAS2, which could effectively control the expression of HSPs to enhance HT stress tolerance. JrGRAS2 is an useful candidate gene for heat response in plant molecular breeding.

Salt-inducible expression of OsJAZ8 improves resilience against salt-stress

BackgroundProductivity of important crop rice is greatly affected by salinity. The plant hormone jasmonate plays a vital role in salt stress adaptation, but also evokes detrimental side effects if not timely shut down again. As novel strategy to avoid such side effects, OsJAZ8, a negative regulator of jasmonate signalling, is expressed under control of the salt-inducible promoter of the transcription factor ZOS3–11, to obtain a transient jasmonate signature in response to salt stress. To modulate the time course of jasmonate signalling, either a full-length or a dominant negative C-terminally truncated version of OsJAZ8 driven by the ZOS3–11 promoter were expressed in a stable manner either in tobacco BY-2 cells, or in japonica rice.ResultsThe transgenic tobacco cells showed reduced mortality and efficient cycling under salt stress adaptation. This was accompanied by reduced sensitivity to Methyl jasmonate and increased responsiveness to auxin. In the case of transgenic rice, the steady-state levels of OsJAZ8 transcripts were more efficiently induced under salt stress compared to the wild type, this induction was more pronounced in the dominant-negative OsJAZ8 variant.ConclusionsThe result concluded that, more efficient activation of OsJAZ8 was accompanied by improved salt tolerance of the transgenic seedlings and demonstrates the impact of temporal signatures of jasmonate signalling for stress tolerance.

Enhanced tolerance to heat stress in transgenic tomato seeds and seedlings overexpressing a trehalose-6-phosphate synthase/phosphatase fusion gene

A fusion gene coding for trehalose-6-phosphate synthase/phosphatase (TPSP) derived from Escherichia coli was introduced into tomato plants (Solanum lycopersicum cv. Joyful) to generate overexpressing lines and enhance the level of trehalose in the seeds. Trehalose has been implicated in tolerance to abiotic stress in plants, so the response to heat stress of seeds from the transgenic lines and seeds from wild-type plants were compared. Wild-type heat-treated seeds had a germination rate of 17%, while four transgenic lines had germination rates over 50% following heat stress. In one heat-treated transgenic line, 100% of the seeds germinated, and therefore had a germination rate six times that of seeds from wild-type plants. Moreover, quantitative PCR revealed that the expression of diverse genes that respond to heat stress was enhanced in TPSP transgenic seeds compared to wild-type seeds 150 min after the onset of heat stress. The enhanced germination rate and expression of these genes in the transgenic seeds were essentially mimicked in wild-type seeds treated with 1 mM exogenous trehalose. Therefore, accumulated trehalose and associated metabolites may act as signaling molecules that enhance the expression of heat stress-responsive genes and confer heat-stress tolerance to seeds.

Promoter of the wheat lipid transfer protein, TdLTP4, drives leaf-preferential expression in transgenic Arabidopsis plants.

In a previous report, a gene encoding a durum wheat lipid transfer protein, TdLTP4, was characterised as induced by abiotic and biotic stresses. In the present work, we investigated the regulation of the gene TdLTP4. A TdLTP4 promoter (PrTdLTP4) region of around 868-bp was isolated and sequenced. Its analysis revealed the presence of several DNA boxes known to be important mainly in the regulation of genes expressed under abiotic stress (salt and dehydration), abscisic acid (ABA) and pathogen responsiveness. The whole PrTdLTP4 fragment was fused to the reporter gene β-glucuronidase (gusA) and analysed in transgenic Arabidopsis plants. Histochemical assays of transgenic Arabidopsis plants showed that the 868-bp fragment of TdLTP4 gene promoter was found to be sufficient for both spatial and temporal patterns of its expression. Under control conditions, GUS histochemical staining was observed significantly only in young leaves of 8- and 12-day-old plants. Whereas after stress challenge especially with NaCl and mannitol, GUS transcripts expression increased substantially in leaves of 30-day-old transgenic seedlings. Real-time qPCR expression analysis of the gusA gene, confirmed the results of histochemical assays. Taken together these data provide evidence that PrTdLTP4 functions as abiotic-stress-inducible promoter in a heterologous dicot system and could be an excellent tool for future crop improvement.

In Vivo DNA Affinity Purification and Histone Deacetylase Inhibitor Treatment Proves the Role of Histone Acetylation in the Expression Regulation of High-Molecular-Weight Glutenin Genes

High-molecular-weight glutenin subunit (HMW GS) proteins are major components of the gluten matrix, which is the physical basis of bread-making in wheat. Epigenetic and transcriptional regulations of HMW GS genes were studied both in silico and in wet lab to understand their tissue (endosperm) specific expression. Our co-expressional network analysis identified key transcription factor (TF) genes that regulate HMW GS genes. We also show here that HMW GS genes are inhibited in vegetative tissues by histone deacetylation as revealed by strong GUS expression in vascular tissues of transgenic barley seedlings harbouring HMW GS gene promoter::uidA-reporter gene fusions upon treatment with a histone deacetylase inhibitor. A novel method termed in vivo DNA affinity purification (IP) has been developed here for the isolation of histones and transcription factors binding to target DNA regions. The technique is based on the biolistic introduction of biotinylated PCR probes amplified from HMW GS gene promoters into wheat leaves. Twenty-four hours later, the probe is cross-linked with interacting factors and subsequently re-purified from plant nuclear extracts. Many proteins, ribosomal proteins and histones have so far been isolated. No lysine-acetylated histone protein fragments were found which further highlight the inhibiting effect of histone deacetylation on HMW GS gene expression.

The interaction between AtMT2b and AtVDAC3 affects the mitochondrial membrane potential and reactive oxygen species generation under NaCl stress in Arabidopsis.

AtMT2b interacts with AtVDAC3 in mitochondria in Arabidopsis. The overexpression of the AtMT2b and AtVDAC3 T-DNA insertion mutant confers tolerance to NaCl stress in Arabidopsis. Both AtMT2b and AtVDAC3 are involved in the regulation of the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) under NaCl stress. Metallothioneins (MTs) are small, cysteine rich, metal-binding proteins that perform multiple functions, such as heavy metal detoxification and reactive oxygen species (ROS) scavenging. MTs have been reported to be involved in mitochondrial function in mammals. However, whether a direct relationship exists between MTs and mitochondrial proteins remains unclear. In the present study, we used yeast two-hybrid and bimolecular fluorescence complementation assays to demonstrate that AtMT2b, which is a type 2 MT in Arabidopsis, interacts with the outer mitochondrial membrane voltage-dependent anion channel AtVDAC3. AtMT2b bound AtVDAC3, leading to its co-localization in mitochondria. AtMT2b transgenic seedlings exhibited increased tolerance to salt stress, and the atvdac3 mutant showed a similar phenotype. The mitochondrial membrane potential (MMP) was maintained, and ROS generation was reduced following AtMT2b overexpression and AtVDAC3 knockout under NaCl stress. Both AtMT2b and AtVDAC3 were shown to be involved in MMP regulation and ROS production under NaCl stress but showed opposite effects. We conclude that AtMT2b might negatively interact with AtVDAC3 in mitochondria, and both proteins are involved in the regulation of MMP and ROS under NaCl stress.

Overexpression of a WRKY Transcription Factor TaWRKY2 Enhances Drought Stress Tolerance in Transgenic Wheat.

Drought is a major environmental stress that severely restricts plant growth and crop productivity. A previous study showed that TaWRKY2 from wheat (Triticum aestivum) plays an important role in drought stress tolerance. In the present study, we isolated the promoter of TaWRKY2 and identified multiple regulatory cis-elements in the promoter region. The activity of the TaWRKY2 promoter was induced by drought, salt, heat, and abscisic acid (ABA). We also generated TaWRKY2-overexpressing transgenic wheat, and found that the transgenic seedlings exhibited significantly enhanced tolerance to drought stress, as evidenced by a higher survival rate and lower water loss rate of detached leaves compared with wild type (WT) plants. In addition, the transgenic lines had higher contents of free proline, soluble sugar, and chlorophyll. During a prolonged period of drought stress before the heading stage, the growth of WT plants was inhibited, whereas the TaWRKY2-overexpressing lines progressed to the heading stage. The increased grain yield of the transgenic wheat lines reflected the cumulative effects of longer panicle length, more kernels per spike, and greater aboveground biomass. Our findings show that TaWRKY2 can enhance drought tolerance and increase grain yield in wheat, thus providing a promising candidate target for improving the drought tolerance of wheat cultivars through genetic engineering.