Transgenic Tobacco Seedlings Research Articles

McWRKY43 Confers Cold Stress Tolerance in Michelia crassipes via Regulation of Flavonoid Biosynthesis.

WRKY transcription factor (TF) plays a crucial role in plant abiotic stress response, but it is rarely reported in Michelia crassipes. Our studies have found that the transcription factor McWRKY43, a member of the IIc subgroup, is strongly upregulated under cold stress. In this study, we cloned the full length of McWRKY43 to further investigate the function of McWRKY43 in resistance to cold stress and its possible regulatory pathways in M. crassipes. Under cold stress, the seed-germination rate of transgenic tobacco was significantly higher than that of the wild type, and the flavonoid content, antioxidant enzyme activities, and proline content of transgenic tobacco seedlings were significantly increased, which promoted the expression of flavonoid pathway structural genes. In addition, the transient transformation of McWRKY43 in the M. crassipes leaves also found the accumulation of flavonoid content and the transcription level of flavonoid structural genes, especially McLDOX, were significantly increased under cold stress. Yeast one-hybrid (Y1H) assay showed that McWRKY43 could bind to McLDOX promoter, and the transcription expression of McLDOX was promoted by McWRKY43 during cold stress treatment. Overall, our results indicated that McWRKY43 is involved in flavonoid biosynthetic pathway to regulate cold stress tolerance of M. crassipes, providing a basis for molecular mechanism of stress resistance in Michelia.

AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

Cloning and overexpression of the DaSOD1 gene from Dioscorea alata improves cadmium resistance in transgenic tobacco plants

ABSTRACT Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar characterized by its strong resistance to Cd stress. This study aims to investigate the enzymatic antioxidant system of MH1 under Cd stress. Under Cd treatment, MH1 exhibited an elevation in superoxide dismutase (SOD) activity, while the activities of ascorbate peroxidase, catalase, and peroxidase remained unchanged. A Cu/Zn-SOD gene, DaSOD1, was cloned and found to be up-regulated following Cd treatment. The DaSOD1 gene was further introduced into tobacco plants for functional analysis. Transgenic tobacco seedlings overexpressing DaSOD1 under Cd treatments exhibited increased chlorophyll contents and seed germination rate, while the levels of superoxide anion and malondialdehyde decreased compared to the wild-type plants. This suggests that DaSOD1 overexpression mitigated the negative effects of Cd stress by reducing oxidative damage in plants. Characterization of the SOD activity and its corresponding DaSOD1 gene is expected to improve our understanding of Cd-resistance mechanism in yam plants.

Functional Verification of the Four Splice Variants from Ajania purpurea NST1 in Transgenic Tobacco

Ajania purpurea is a small semi-shrub in the Asteraceae family. Its corolla is purplish red from the middle to the top, and its leaves and flowers are all fragrant. It can be introduced and cultivated as ornamental plants. In order to survive adversity, plants actively regulate the expression of stress response genes and transcripts. Alternative splicing is a common phenomenon and an important regulation mode of eukaryotic gene transcription, which plays an important role in various biological processes. In this study, four splice variants of the NST1 gene were identified from A. purpurea, and the molecular mechanism of NST1 alternative splice variants involved in abiotic stress was explored through bioinformatics, transgenics and paraffin sectionalization. The analysis of amino acid sequences showed that ApNST1.1 had alternative 5′splicing, ApNST1.2 had alternative 3′splicing and ApNST1 had the two splicing types. The main conclusions from studying transgenic tobacco seedlings and adult seedlings under abiotic stress were as follows: ApNST1, ApNST1.1 and ApNST1.3 showed salt tolerance at seedling stage, especially ApNST1.3. At the mature seedling stage, the stem height of ApNST1.1 increased significantly, and ApNST1.1 showed obvious salt tolerance, while ApNST1.2 showed obvious cold resistance. Compared to Super35S::GFP, the xylem of ApNST1 thickened by 94 μm, and the cell wall thickened by 0.215 μm. These results are of great significance to the breeding and application of ApNST1 to select splice variants with more resistance to abiotic stress, and to future study in this area. At the same time, they provide a new direction for A. purpurea breeding, and increase the possibility of garden applications.

Genome-wide characterization of the U-box gene in Camellia sinensis and functional analysis in transgenic tobacco under abiotic stresses

Plants U-box genes are crucial for plant survival, and they extensively regulate plant growth, reproduction and development as well as coping with stress and other processes. In this study, we identified 92 CsU-box genes through genome-wide analysis in the tea plant (Camellia sinensis), all of them contained the conserved U-box domain and were divided into 5 groups, which supported by the further genes structure analysis. The expression profiles in eight tea plant tissues and under abiotic and hormone stresses were analyzed using the TPIA database. 7 CsU-box genes (CsU-box27/28/39/46/63/70/91) were selected to verify and analyze expression patterns under PEG-induced drought and heat stress in tea plant respectively, the qRT-PCR results showed consistent with transcriptome datasets; and the CsU-box39 were further heterologous expressed in tobacco to perform gene function analysis. Phenotypic analyses of overexpression transgenic tobacco seedlings and physiological experiments revealed that CsU-box39 positively regulated the plant response to drought stress. These results lay a solid foundation for studying the biological function of CsU-box, and will provide breeding strategy basis for tea plant breeders.

Transgenic tobacco plant overexpressing ginkgo dihydroflavonol 4-reductase gene GbDFR6 exhibits multiple developmental defects.

Dihydroflavonol Q 4-reductase (DFR), a key enzyme in the flavonoid biosynthetic pathway in plants, significantly influences plant survival. However, the roles of DFR in the regulation of plant development are largely unknown. In the present study, phenotypes of transgenic tobacco plants overexpressing the Ginkgo biloba DFR gene, GbDFR6, were investigated. Transgenic tobacco seedlings exhibited relatively low fresh weights, long primary roots, decreased lateral root numbers, and impaired root gravitropic responses when compared to wild-type tobacco plants. Adult transgenic tobacco plants exhibited a considerably high percentage of wrinkled leaves when compared to the wild-type tobacco plants. In addition to the auxin-related phenotypic changes, transgenic tobacco plants exhibited delayed flowering phenotypes under short-day conditions. Gene expression analysis revealed that the delayed flowering in transgenic tobacco plants was caused by the low expression levels of NtFT4. Finally, variations in anthocyanin and flavonoid contents in transgenic tobacco plants were evaluated. The results revealed that the levels of most anthocyanins identified in transgenic tobacco leaves increased. Specifically, cyanidin-3,5-O-diglucoside content increased by 9.8-fold in transgenic tobacco plants when compared to the wild-type tobacco plants. Pelargonidin-3-O-(coumaryl)-glucoside was only detected in transgenic tobacco plants. Regarding flavonoid compounds, one flavonoid compound (epicatechin gallate) was upregulated, whereas seven flavonoid compounds (Tamarixetin-3-O-rutinoside; Sexangularetin-3-O-glucoside-7-O-rhamnoside; Kaempferol-3-O-neohesperidoside; Engeletin; 2'-Hydoxy,5-methoxyGenistein-O-rhamnosyl-glucoside; Diosmetin; Hispidulin) were downregulated in both transgenic tobacco leaves and roots. The results indicate novel and multiple roles of GbDFR6 in ginkgo and provide a valuable method to produce a late flowering tobacco variety in tobacco industry.

Ectopic overexpression of mulberry MnT5H2 enhances melatonin production and salt tolerance in tobacco.

Soil salinization severely inhibits plant growth and has become one of the major limiting factors for global agricultural production. Melatonin (N-acetyl-5-methoxytryptamine) plays an important role in regulating plant growth and development and in responding to abiotic stresses. Tryptamine-5-hydroxylase (T5H) is an enzyme essential for the biosynthesis of melatonin in plants. Previous studies have identified the gene MnT5H for melatonin synthesis in mulberry (Morus notabilis), but the role of this gene in response to salinity stress in mulberry is remain unclear. In this study, we ectopically overexpressed MnT5H2 in tobacco (Nicotiana tabacum L.) and treated it with NaCl solutions. Compared to wild-type (WT), melatonin content was significantly increased in the overexpression-MnT5H2 tobacco. Under salt stress, the expression of NtCAT, NtSOD, and NtERD10C and activity of catalase (CAT), peroxidase (POD), and the content of proline (Pro) in the transgenic lines were significantly higher than that in WT. The Malondialdehyde (MDA) content in transgenic tobacco was significantly lower than that of WT. Furthermore, transgenic tobacco seedlings exhibited faster growth in media with NaCl. This study reveals the changes of melatonin and related substance content in MnT5H2-overexpressing tobacco ultimately lead to improve the salt tolerance of transgenic tobacco, and also provides a new target gene for breeding plant resistance to salt.

AcdS gene of Bacillus cereus enhances salt tolerance of seedlings in tobacco (Nicotiana tabacum L.)

Previous studies show that 1-aminocyclopropane-1-carboxylate (ACC) deaminase can facilitate the growth and stress tolerance of hosts by reducing ethylene levels. In this study, the acdS gene encoding ACC deaminase from Bacillus cereus (HK012) was cloned and transformed into tobacco (Nicotiana tabacum L.) by the leaf disc method using Agrobacterium. Molecular detection and physiological analysis of the transgenic tobacco plants were performed. Our results showed the acdS gene was integrated into the tobacco genome and fluorescence microscopy showed that the fusion protein was located on the cell membrane of tobacco root. Compared with control, the transgenic plants showed increases in plant height, root length, dry weight, fresh weight and chlorophyll content; and significant increases in the concentration of proline of 55.15% and 42.7% under salt stress conditions (150 mmol L−1 and 300 mmol L−1 NaCl, respectively). The superoxide dismutase, peroxidase, catalase and ACC deaminase activities of transgenic tobacco were higher than those of control tobacco at 150 and 300 mmol L−1 salt concentrations. Transgenic tobacco seedlings expressing the acdS gene of B. cereus HK012 showed higher salt tolerance than the control plants. The obtained results suggest that the acdS gene of B. cereus can be used to promote salt tolerance in glycophytes by using biotechnology strategies.

Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis).

Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

Cytological and physiological tolerance of transgenic tobacco to Cd stress is enhanced by the ectopic expression of SmZIP8

Zrt and Irt-like proteins (ZIPs) are responsible for transporting various divalent metal cations. However, information about the characteristics of the cellular and physiological tolerance of plant ZIPs to Cd stress is still limited. The expression levels of SmZIP8 in Salix matsudana Koidz were upregulated by Cd stress. The complete length of SmZIP8 from S. matsudana was cloned, and transgenic tobacco was obtained by Agrobacterium-mediated transformation. Then, the tolerance to Cd stress of wild-type (WT) and transgenic tobacco seedlings was analyzed and compared by studying the cytotoxicity of the root tip cells, photosynthetic parameters, histochemical staining of O2− and H2O2, the activities of antioxidant enzymes, and malondialdehyde content under Cd stress. In comparison with WT tobacco, the ectopic expression of SmZIP8 in tobacco promoted the cytological tolerance of the transgenic tobacco to Cd stress by reducing cell damage, raising the mitotic indexes, and reducing the rate of chromosome aberration of the root cells. Meanwhile, the results of increased photosynthetic capacity, decreased oxidative damage, and activated antioxidant enzymes showed that the physiological tolerance of transgenic tobacco to Cd was enhanced. The principal component analysis for the above physiological parameters explained 96.08% of the total variance (PC1, 77.77%; PC2, 18.31%), indicating a significant difference in Cd tolerance abilities between the tobacco expressing SmZIP8 and WT tobacco. Therefore, SmZIP8 may be considered as an important genetic resource for the phytoremediation of Cd or other heavy metal pollution via the use of transgenic plants obtained through genetic transformation.

Cloning and characterization of the DIR1 promoter from Eucommia ulmoides Oliv and its response to hormonal and abiotic stress

The lignans of Eucommia ulmoides have been extensively studied and shown to have a dual mechanism of regulating blood pressure. Studies have shown that DIR1 is a key gene in the biosynthetic pathway of lignans in Eucommia ulmoides Oliv. In this study, a 2000 bp upstream promoter sequence was cloned, and part of the sequence (1495 bp) and its 5′-end truncated segment were constructed into the pCAMBIA1391Z expression plasmid upstream of s-glucuronidase (GUS). Agrobacterium-mediated genetic transformation produced stable transgenic tobacco lines. The results showed that although both full-length and truncated promoters could initiate GUS expression, their levels were influenced by the degree of deletion at the 5′ end. GUS histochemical staining showed that the core promoter region was located in the region containing the transcription initiation site (TIS) within 212 bp. In addition, the DIR1 promoter responded to environmental and hormonal stressors, such as jasmonic acid (MeJA), abscisic acid (ABA), d-mannitol (drought mimic), and high concentrations of NaCl. In transgenic tobacco seedlings, MeJA, d-mannitol, and ABA could activate the DIR1 promoter, whereas high concentrations of NaCl could inhibit it. In E. ulmoides Oliv seedlings, MeJA, NaCl, and d-mannitol activated the DIR1 promoter, whereas ABA had an inhibitory effect. In summary, our findings provide a theoretical basis for the use of the DIR1 promoter in plant genetic engineering, indicating its potential. Our study also presents novel insights for lignan biosynthesis and sheds light on the mechanisms of E. ulmoides Oliv in response to stress. We successfully cloned the DIR1 gene promoter and studied its function in regulating the expression of the DIR1 gene.

Peanut (Arachis hypogaea L.) S-adenosylmethionine decarboxylase confers transgenic tobacco with elevated tolerance to salt stress.

Polyamines play an important role in stress response. In the pathway of polyamines synthesis, S-adenosylmethionine decarboxylase (SAMDC) is one of the key enzymes. In this study, a full length cDNA of SAMDC (AhSAMDC) was isolated from peanut (Arachis hypogaea L.). Phylogenetic analysis revealed high sequence similarity between AhSAMDC and SAMDC from other plants. In peanut seedlings exposed to sodium chloride (NaCl), the transcript level of AhSAMDC in roots was the highest at 24 h that decreased sharply at 72 and 96 h after 150 mM NaCl treatment. However, the expression of AhSAMDC in peanut leaves was significantly inhibited, and the transcript levels in leaves were not different compared with control These results implied the tissue-specific and time-specific expression of AhSAMDC. The physiological effects and functional mechanism of AhSAMDC were further evaluated by overexpressing AhSAMDC in tobaccos. The transgenic tobacco lines exhibited higher germination rate and longer root length under salt stress. Reduced membrane damage, higher antioxidant enzyme activity, and higher proline content were also observed in the transgenic tobacco seedlings. What's more, AhSAMDC also led to higher contents of spermidine and spermine, which can help to scavenge reactive oxygen species. Together, this study suggests that AhSAMDC enhances plant resistance to salt stress by improving polyamine content and alleviating membrane damage.

Overexpression of NtDOG1L-T Improves Heat Stress Tolerance by Modulation of Antioxidant Capability and Defense-, Heat-, and ABA-Related Gene Expression in Tobacco.

Drought and heat stresses are two major environmental stress factors that severely threaten crop growth and productivity. Plant delay of germination 1-like (DOG1L) family genes play important roles in various developmental processes and stress responses. In our previous study, a tobacco DOG1L gene (NtDOG1L-T) was found to regulate seedling growth and drought response. Unfortunately, the role of DOG1L genes in heat stress response is yet to be studied. Here, we present data supporting the role of DOG1L genes in heat stress and possible underlying molecular mechanisms. Transcript levels of NtDOG1L-T were rapidly induced by heat or abscisic acid (ABA) treatment. Furthermore, NtDOG1L-T promoter activity was markedly activated by ABA or heat stress, as revealed by histochemical staining in transgenic tobacco seedlings. Overexpression of NtDOG1L-T in transgenic lines improved heat stress tolerance. The NtDOG1L-T-transgenic plants exhibited lower levels of reactive oxygen species (ROS) and lipid peroxidation but higher antioxidant enzyme activities in response to heat stress. Furthermore, transcript abundance of some defense-, heat-, and ABA-responsive marker genes was significantly upregulated, as shown by reverse transcription quantitative PCR (qPCR) in these transgenic plants. In conclusion, NtDOG1L-T positively regulates heat stress tolerance possibly by modulation of antioxidant capability and defense-, heat-, and ABA-related gene expression in tobacco. This study may provide valuable resource for the potential exploitation of DOG1Ls in genetic improvement of heat stress tolerance in crops.

Overexpression of the spinach S-nitrosoglutathione reductase (SoGSNOR) in tobacco resulted in enhanced nitrate stress tolerance

S-nitrosylation, the addition of nitric oxide (NO) moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. S-nitrosoglutathione reductase (GSNOR) is thought to be the major regulator of total cellular SNO levels in plants. However, its role in excess nitrate stress has not been investigated in spinach. In this study, a spinach GSNOR gene (GenBank Accession No. KR381778) was amplified and designated as SoGSNOR. The transcript and protein level of SoGSNOR were reduced after excess nitrate treatment for 24 h. Addition of NO donor to the nitrate solution decreased the SoGSNOR expression, while supplementation inhibitor of nitrate reductase and nitric oxide synthase increased its expression. Overexpression of SoGSNOR in tobacco increased the germination rate of transgenic seeds, compared to the wild type (WT) under nitrate stress. Higher photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency and expression level of some stress-related genes were detected in the transgenic seedlings than the WT under nitrate stress. The transgenic tobacco seedlings have lower malondialdehyde content, reactive oxygen species (ROS) fluorescence, and higher activities and transcript level of superoxide dismutase, catalase, peroxidase under nitrate stress. SoGSNOR transgenic tobacco plants have lower NR activity and protein level, higher GSNOR and non-symbiotic class 1 hemoglobin (nsHb) protein level than the WT plants, leading to lower NO accumulation and SNOs contents under nitrate stress. These results suggested that overexpression of SoGSNOR increased nitrate stress tolerance of tobacco by regulating ROS and RNS metabolism. A spinach S-nitrosoglutathione reductase (SoGSNOR) gene was isolated from spinach root. The expression level and activities of SoGSNOR were reduced by excess nitrate treatment. Overexpression of SoGSNOR in tobacco increased nitrate stress tolerance by regulating ROS and RNS metabolism.

Heat stress inducible cytoplasmic isoform of ClpB1 from Z. nummularia exhibits enhanced thermotolerance in transgenic tobacco.

Previously, we isolated CDS of Ziziphus nummularia isoform ZnJClpB1-C from heat stress-tolerant genotype Jaisalmer. To further functionally validate ZnJClpB1-C assumed function in tobacco and to generate novel germplasm for heat stress tolerance, this gene was transformed in theNicotiana tabacum. ClpB proteins are the major key player required for basal and induced heat stress tolerance in plant cells under heat stress. In Ziziphus nummularia ClpB1-C transcript from genotype Jaisalmer was highly upregulated under heat stress conditions, as reported earlier. Nine transgenic lines (T1) from three transgenic tobacco events with single-copy integration (T0 stage) were taken for heat stress analysis at seedling stage. Mature tobacco transgenic plants did not show any deformity as compared to wild plants when grown under normal conditions. Overexpression of ZnJClpB1-C in tobacco significantly increased the tolerance to heat stress. Under heat stress conditions (42°C), T1 transgenic tobacco seedlings showed higher photosynthetic rate, relative water content, membrane stability index and lower levels of MDA, compared to the wild type untransformed plants. The qRT-PCR analysis revealed different level of transgene expression (1.08 to 3.89 folds) in 9 T1 transgenic lines. In vitro roles of ZnJClpB1-C regulating thermotolerance is not reported so far. These results demonstrated the positive roles of ZnJClpB1-C in enhancing thermotolerance and its use as a genomic resource in the near future for developing heat stress-tolerant germplasm.

Asparagus racemosus bZIP transcription factor-regulated squalene epoxidase (ArSQE) promotes germination and abiotic stress tolerance in transgenic tobacco

A. racemosus is a rich source of pharmacologically active steroidal saponins. Most of the studies are related to its chemistry and pharmacology, but the pathway involved in the biosynthesis of steroidal saponin is not much emphasized. Squalene epoxidase acts as a rate-limiting enzyme in this biosynthesis. In this study, we have selected root specific squalene epoxidase ArSQE from A. racemosus for its characterization. ArSQE was able to complement ergosterol auxotrophy in erg1 yeast mutants. Mutants were sensitive to the antifungal drug terbinafine, whereas ArSQE complementation made them tolerant to the same drug. ArSQE plays a significant role in early germination in transgenic tobacco. The transgenic tobacco seedlings overexpressing ArSQE were tolerant to terbinafine and abiotic stress. Expression analysis of transcripts in ArSQE transgenic lines suggests that it mostly affects ABA, GA, stress, and sterol related functions in transgenic tobacco. Further, root specific MeJA responsive A. racemosus bZIP transcription factors (TFs), ArTGA1 and ArTGA2, were identified that bind to MeJA responsive cis-element present in the promoter region of ArSQE. Characterization of ArSQE of A. racemosus provides new information about its regulation through MeJA responsive bZIP TF along with its role in the development and abiotic stress response in transgenic tobacco.

Overexpression of the alfalfa zeaxanthin epoxidase gene delaysseed germination in transgenic tobacco

Zeaxanthin epoxidase (ZEP) plays important roles in plant growth and development due to its functions in abscisic acid (ABA) biosynthesis and in the xanthophyll cycle. Many studies have been exploring the roles of ZEP in seed germination and response to various environmental stresses. In this study, we reported that heterologously overexpressing the ZEP gene from Medicago sativa (MsZEP) in tobacco increased ABA content in tobacco mature seeds and negatively regulated seed germination. Enhanced ABA synthesis in seed embryo and delayed germination might also be related to the increased 9-cis-epoxycarotenoid dioxygenase (NCED6) expression. Moreover, we found that overexpression of MsZEP resulted in an increased expression of the SOMNUS gene but a decreased expression of the DNA ligase 6 gene (Lig6) suggesting that MsZEP might affect seed proteome and DNA integrity. Furthermore, enhanced chlorophyll content in transgenic tobacco seedlings overexpressing MsZEP might be due to its function in the xanthophyll cycle and ABA 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.

Heterologous expression of Brassica juncea annexin, AnnBj2 confers salt tolerance and ABA insensitivity in transgenic tobacco seedlings.

Annexins are multifunctional proteins with roles in plant development and alleviation of stress tolerance. In the present communication, we report on the effect of heterologous expression of Brassica juncea annexin, AnnBj2 in tobacco. Transgenic tobacco plants expressing AnnBj2 exhibited salt-tolerant and abscisic acid (ABA)-insensitive phenotype at the seedling stage. Biochemical analysis showed that AnnBj2 transgenic plants retained higher chlorophyll and proline content, and lower malondialdehyde (MDA)levels compared to the null line under salt stress. They exhibited better water retention capacity compared to the null segregant (NS) line. AnnBj2 overexpression altered the transcript levels of several stress-related marker genes involved in reactive oxygen species (ROS) scavenging and abiotic stress signaling. Taken together, these results suggest a positive role for AnnBj2 in salt stress response upon heterologous expression in tobacco.

OsCBSCBSPB4 is a Two Cystathionine-β-Synthase Domain-containing Protein from Rice that Functions in Abiotic Stress Tolerance.

Cystathionine β-synthase (CBS) domains have been identified in a wide range of proteins of unrelated functions such as, metabolic enzymes, kinases and channels, and usually occur as tandem re-peats, often in combination with other domains. In plants, CBS Domain-Containing Proteins (CDCPs) form a multi-gene family and only a few are so far been reported to have a role in development via regu-lation of thioredoxin system as well as in abiotic and biotic stress response. However, the function of majority of CDCPs still remains to be elucidated in plants. Here, we report the cloning, characterization and functional validation of a CBS domain containing protein, OsCBSCBSPB4 from rice, which pos-sesses two CBS domains and one PB1 domain. We show that OsCBSCBSPB4 encodes a nucleo-cytoplasmic protein whose expression is induced in response to various abiotic stress conditions in salt-sensitive IR64 and salt-tolerant Pokkali rice cultivars. Further, heterologous expression of OsCBSCB-SPB4 in E. coli and tobacco confers marked tolerance against various abiotic stresses. Transgenic tobac-co seedlings over-expressing OsCBSCBSPB4 were found to exhibit better growth in terms of delayed leaf senescence, profuse root growth and increased biomass in contrast to the wild-type seedlings when subjected to salinity, dehydration, oxidative and extreme temperature treatments. Yeast-two hybrid stud-ies revealed that OsCBSCBSPB4 interacts with various proteins. Of these, some are known to be in-volved in abiotic stress tolerance. Our results suggest that OsCBSCBSPB4 is involved in abiotic stress response and is a potential candidate for raising multiple abiotic stress tolerant plants.