Transgenic Tobacco Plants Research Articles

Effect of ipt Gene Induction in Transgenic Tobacco Plants on Hydraulic Conductance, Formation of Apoplastic Barriers and Aquaporin Activity under Heat Shock.

Cytokinins are known to keep stomata open, which supports gas exchange and correlates with increased photosynthesis. However, keeping the stomata open can be detrimental if the increased transpiration is not compensated for by water supply to the shoots. In this study, we traced the effect of ipt (isopentenyl transferase) gene induction, which increases the concentration of cytokinins in transgenic tobacco plants, on transpiration and hydraulic conductivity. Since water flow depends on the conductivity of the apoplast, the deposition of lignin and suberin in the apoplast was studied by staining with berberine. The effect of an increased concentration of cytokinins on the flow of water through aquaporins (AQPs) was revealed by inhibition of AQPs with HgCl2. It was shown that an elevated concentration of cytokinins in ipt-transgenic plants increases hydraulic conductivity by enhancing the activity of aquaporins and reducing the formation of apoplastic barriers. The simultaneous effect of cytokinins on both stomatal and hydraulic conductivity makes it possible to coordinate the evaporation of water from leaves and its flow from roots to leaves, thereby maintaining the water balance and leaf hydration.

Genomic-Wide Analysis Identifies the PI-PLC Gene Family and Expression of Its Member PsnPI-PLC6 Confers Cadmium Tolerance in Transgenic Tobacco Plants

Genomic-Wide Analysis Identifies the PI-PLC Gene Family and Expression of Its Member PsnPI-PLC6 Confers Cadmium Tolerance in Transgenic Tobacco Plants

NPR1 Translocation from Chloroplast to Nucleus Activates Plant Tolerance to Salt Stress.

Chloroplasts play crucial roles in biotic and abiotic stress responses, regulated by nuclear gene expression through changes in the cellular redox state. Despite lacking the N-terminal chloroplast transit peptide (cTP), nonexpressor of pathogenesis-related genes 1 (NPR1), a redox-sensitive transcriptional coactivator was consistently found in the tobacco chloroplasts. Under salt stress and after exogenous application of H2O2 or aminocyclopropane-1-carboxylic acid, an ethylene precursor, transgenic tobacco plants expressing green fluorescent protein (GFP)-tagged NPR1 (NPR1-GFP) showed significant accumulation of monomeric nuclear NPR1, irrespective of the presence of cTP. Immunoblotting and fluorescence image analyses indicated that NPR1-GFP, with and without cTP, had similar molecular weights, suggesting that the chloroplast-targeted NPR1-GFP is likely translocated from the chloroplasts to the nucleus after processing in the stroma. Translation in the chloroplast is essential for nuclear NPR1 accumulation and stress-related expression of nuclear genes. An overexpression of chloroplast-targeted NPR1 enhanced stress tolerance and photosynthetic capacity. In addition, compared to the wild-type lines, several genes encoding retrograde signaling-related proteins were severely impaired in the Arabidopsis npr1-1 mutant, but were enhanced in NPR1 overexpression (NPR1-Ox) transgenic tobacco line. Taken together, chloroplast NPR1 acts as a retrograding signal that enhances the adaptability of plants to adverse environments.

A novel soybean transcription factor, DREB7, regulates RD29A and SODFe gene expression in transgenic tobacco plants

A novel soybean transcription factor, DREB7, regulates RD29A and SODFe gene expression in transgenic tobacco plants

Transcriptomic and functional analysis reveals that VvSAUR43 may be involved the elongation of grape berries

The overall shape of grape (Vitis vinifera) berries is an important quality trait. The application of gibberellin (GA3) will elongate fruit, however, the molecular mechanisms involved in this response are unknown. Results of the present study indicated that the application of GA3 promoted the levels of endogenous IAA and IAA-AsP. In regard to cell development, compared with the control group, GA3 treatment contributing to lengthen the longitudinal elongation of cell and the fruit shape index. Gene Ontology (GO) enrichment analysis show that DEGs were mainly enriched in the cell periphery, external encapsulating structure, and cell wall of cellular components. KEGG analysis revealed that the GA3 treatment positively regulated the signal transduction pathways for auxin, especially IAA, and brassinosteroid (BR). Notably, the IAA signal transduction pathway accounted for the largest proportion of the assigned genes, being 50%, 60%, and 61% of the total number of DEGs in the T6h vs CK6h, T24h vs CK24h, and T24h vs T6h comparison, respectively. Among the responding genes, Small Auxin-Upregulated RNA (SAUR) genes responded rapidly and were significantly enriched. Phylogenetic analysis of the SAUR gene family indicated that SAUR genes in grape and Arabidopsis have high homology. Furthermore, VvSAUR43 gene overexpressed in tomato and tobacco produce elongated fruit in tomato, leaf and the shape index of silique are also significantly up-regulated in tobacco transgenic plants. The study revealed that IAA plays a major role in the response to the GA3 treatment, and that increased IAA levels enhance VvSAUR expression, ultimately resulting in an elongated fruit shape. Our study provides new insights into the molecular mechanism responsible for the effect of GA3 on fruit shape.

Identification of cucumber S-adenosylmethionine decarboxylase genes and functional analysis of CsSAMDC3 in salt tolerance.

As one of the key enzymes in the biosynthesis of polyamines, S-adenosylmethionine decarboxylase (SAMDC) plays an important role in plant stress resistance. In this study, four SAMDC genes (CsSAMDC1-4) were identified in cucumber (Cucumis sativus L.) and divided into three groups (I, II, and III) by phylogenetic analysis. Motif analysis suggested the existence of many conserved motifs, which is compatible with SAMDC protein classification. Gene structure analysis revealed that CsSAMDC2 and CsSAMDC3 in group I have no intron, which showed a similar response to salt stress by gene expression analysis. CsSAMDC3 responded differently to hormone and stress treatments, and was more susceptible to salt stress. Compared with wild-type (WT) tobacco, the activities of superoxide dismutase, peroxidase, and catalase were increased in CsSAMDC3-overexpressing tobacco under salt stress, but the content of electrolyte leakage, malondialdehyde, and hydrogen peroxide were decreased, which alleviated the inhibition of growth induced by salt stress. Under salt stress, overexpression of CsSAMDC3 in transgenic tobacco plants exhibited salt tolerance, mainly in the form of a significant increase in dry and fresh weight, the maximal quantum yield of PSII photochemistry, the net photosynthetic rate and the content of spermidine and spermine, while the content of putrescine was reduced. In addition, the expression levels of antioxidase-related coding genes (NtSOD, NtPOD, NtCAT) and PAs metabolism-related coding genes (NtSAMS, NtSPDS, NtSPMS, NtPAO) in transgentic plants was lower than WT under salt stress, which suggested that overexpression of CsSAMDC3 affected the expression of these genes. In summary, our results showed that CsSAMDC3 could be used as a potential candidate gene to improve salt tolerance of cucumber by regulating polyamine and antioxidant metabolism.

Plant-derived PAP proteins fused to immunoglobulin A and M Fc domains induce anti-prostate cancer immune response in mice

In this study, recombinant Fc-fused Prostate acid phosphatase (PAP) proteins were produced in transgenic plants. PAP was fused to immunoglobulin (Ig) A and M Fc domain (PAP-IgA Fc and PAP-IgM Fc), which were tagged to the ER retention sequence KDEL to generate PAP-IgA FcK and PAP-IgM FcK. Agrobacterium-meditated transformation was performed to produce transgenic tobacco plants expressing four recombinant proteins. Genomic PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK in tobacco plant leaves. Western blot confirmed the expression of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK proteins. SEC-HPLC and Bio-TEM analyses were performed to confirm the size and shape of the plant-derived recombinant PAP-Fc fusion proteins. In mice experiments, the plant-derived IgA and IgM Fc fused proteins induced production of total IgGs including IgG1 against PAP. This result suggests that IgA and IgM Fc fusion can be applied to produce recombinant PAP proteins as a prostate cancer vaccine in plant expression system.

Ectopic expression of DnaJ type-I protein homolog of Vigna aconitifolia (VaDJI) confers ABA insensitivity and multiple stress tolerance in transgenic tobacco plants.

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants.

Phytomelatonin interferes with flavonols biosynthesis to regulate ROS production and stomatal closure in tobacco

Flavonols are well-known antioxidants that prevent stomatal closure via interfering with ROS signaling. Phytomelatonin regulates stomatal closure, but the signaling pathways are still largely unknown. Here, we investigated the role of flavonols in phytomelatonin-mediated stomatal closure in tobacco plants. The application of melatonin induced stomatal closure through NADPH oxidase-mediated ROS production. Transgenic tobacco plants overexpressing soybean GmSNAT1 (coding for serotonin N-acetyltransferase that catalyzes the penultimate step in phytomelatonin biosynthesis) had higher phytomelatonin concentration, accumulated more ROS in guard cells and were more sensitive to melatonin-induced stomatal closure than the wild-type plants, which was associated with the higher expression of PMTR1-homologous genes. Exogenous melatonin decreased flavonol concentrations in guard cells and the expression of flavonoid-related genes in wild-type and transgenic tobacco plants, and these inhibitory effects were more obvious in GmSNAT1-overexpressing plants than the wild type. However, the melatonin-mediated stomatal closure and ROS production were diminished by the application of kaempferol (a type of flavonol). Additionally, transgenic tobacco plants with increased expression of NtFLS (encoding flavonol synthase) were less sensitive to melatonin-induced stomatal closure. In conclusion, phytomelatonin hampers the biosynthesis of flavonols in guard cells, which results in high concentration of ROS and induces stomatal closure in tobacco plants.

Overexpression of NtGCN2 improves drought tolerance in tobacco by regulating proline accumulation, ROS scavenging ability, and stomatal closure

Drought stress is a severe threat to plants. Genes that respond to drought stress are essential for plant growth and development. General control nonderepressible 2 (GCN2) encodes a protein kinase that responds to various biotic and abiotic stresses. However, the mechanism of GCN2 in plant drought tolerance remains unclear. In the present study, the promoters of NtGCN2 from Nicotiana tabacum K326, which contained a drought-responsive Cis-acting element MYB that can be activated by drought stress, were cloned. Furthermore, the drought tolerance function of NtGCN2 was investigated using NtGCN2-overexpressed transgenic tobacco plants. NtGCN2-overexpressed transgenic plants were more tolerant to drought stress than wild-type (WT) plants. The transgenic tobacco plants exhibited higher proline and abscisic acid (ABA) contents, antioxidant enzyme activities, leaf relative water content, and expression levels of genes encoding key antioxidant enzymes and proline synthase, but lower levels of malondialdehyde and reactive oxygen species, and reduced stomatal apertures, stomatal densities, and stomatal opening rates compared to WT plants under drought stress. These results indicated that overexpression of NtGCN2 conferred drought tolerance in transgenic tobacco plants. RNA-seq analysis showed that overexpression of NtGCN2 responded to drought stress by regulating the expression of genes related to proline synthesis and catabolism, abscisic acid synthesis and catabolism, antioxidant enzymes, and ion channels in guard cells. These results showed that NtGCN2 might regulate drought tolerance by regulating proline accumulation, reactive oxygen species (ROS) scavenging, and stomatal closure in tobacco and may have the potential for application in the genetic modification of crop drought tolerance.

Manipulation of CBTS1 Expression Alters Tobacco Resistance to Spodoptera frugiperda and Phytophthora nicotianae

Cembranoids produced by tobacco glandular trichomes have bioactivities in resistance to insect pests and pathogens. Cembratrien-ol synthase (CBTS) plays a key role in the biosynthesis of cembranoids and directly determines the cembranoid content in tobacco. This study examined the effect of changing CBTS1 expression on tobacco resistance to the insect pest Spodoptera frugiperda and oomycete pathogen Phytophthora nicotianae. The CDS sequence of CBTS1 was cloned into gene overexpression and silencing vectors and introduced into tobacco (Nicotiana tabacum L. cv. TN90) to obtain CBTS1-overexpression plants (CBTS1-OE) and CBTS1-silenced plants (CBTS1-RI). Compared with control plants, the content of cembratrien-ol (CBT-ol) was increased 4.48 times in the CBTS1-OE plants but decreased by 68% in the CBTS1-RI plants, while that of cembratrien-diol (CBT-diol) was increased 3.17 times in the CBTS1-OE plants but decreased by 76% in the CBTS1-RI plants. The S. frugiperda resistance of transgenic tobacco plants was evaluated by in vitro toxicity test, and the results showed that the resistance of CBTS1-OE plants to S. frugiperda was significantly improved but that of CBTS1-RI plants was reduced. The P. nicotianae resistance of transgenic tobacco plants was assessed by the detached leaf assay, and the results showed that the resistance of CBTS1-OE plants to P. nicotianae was enhanced, while that of CBTS1-RI plants was attenuated. Further gene expression analysis showed that overexpression of CBTS1 increased the expression of the pathogen-related gene PR-1a, while silencing of CBTS1 decreased its expression. This study demonstrated that manipulating the expression of CBTS1 could change the cembranoid content in tobacco plants and alter their resistance to both insect pests and oomycete pathogens.

Proline metabolism as a mechanism for the energy dissipation in VaP5CSF129A transgenic tobacco plants under water deficit

In plants, proline accumulation in cells is a common response to alleviate the stress caused by water deficits. It has been shown that foliar proline spraying, as well as its overaccumulation in transgenic plants can increase drought tolerance, as proline metabolism plays important roles in cell redox balance and on energy dissipation pathways. The aim of this work was to evaluate the role of exogenous proline application or its endogenous overproduction as a potential mechanism for energy dissipation. For this, wild-type and VaP5CSF129A transgenic tobacco plants were sprayed with proline (10 mM) and submitted to water deficit. Changes in plant physiology and biochemistry were evaluated. Transcriptional changes in the relative expression of genes involved in proline synthesis and catabolism, NAD (P)-dependent malate dehydrogenase (NAD(P)-MDH), alternative oxidase (AOX), and VaP5CSF129A transgene were measured. Exogenous proline reduced the negative effects of water deficit on photosynthetic activity in both genotypes; with the transgenic plants even less affected. Water deficit caused an increase in the relative expression of proline biosynthesis genes. On the other hand, the expression of catabolism genes decreased, primarily in transgenic plants. Exogenous proline reduced activity of the NADP-MDH enzyme and decreased expression of the AOX and NADP-MDH genes, mainly in transgenic plants under water stress. Finally, our results suggest that proline metabolism could act as a complementary/compensatory mechanism for the energy dissipation pathways in plants under water deficit.

Transcriptional Plasticity and Cell Wall Characterization in High-Methanol-Producing Transgenic Tobacco Plants

Volatile organic compounds emanating from plant surfaces serve as a sustainable natural solution to combat biotic stresses in plants. Leaf methanol is the simplest and second major volatile organic compound after isoprene emitted through the leaf surface. Methanol has been neglected as a by-product of other secondary metabolites for a long time, but recent studies have suggested its importance in development and stress responses. In our previous findings, we had revealed that transgenic plants over-expressing PME, enhanced methanol production providing resistance against a broad spectrum of insects. In the current study, we extended our previous work to provide new insights by performing differential transcriptomics of high-methanol-producing insect-resistant transgenic lines. We found that 2262 genes were differentially expressed in the transgenics plants, including transcription factors, cell wall modulating, phytohormones signaling and development-related genes. Our results demonstrated that the expression levels of transcription factors associated with development and biotic stress were altered in the transgenic lines. In addition, phytohormones ABA and gibberellin signalling genes were upregulated, whereas ethylene and auxin signalling genes were downregulated. Moreover, biochemical characteristics of cell walls in both transgenic tobacco plants were comparable to the control plants.

Overexpression of TgERF1, a Transcription Factor from Tectona grandis, Increases Tolerance to Drought and Salt Stress in Tobacco

Teak (Tectona grandis) is one of the most important wood sources, and it is cultivated in tropical regions with a significant market around the world. Abiotic stresses are an increasingly common and worrying environmental phenomenon because it causes production losses in both agriculture and forestry. Plants adapt to these stress conditions by activation or repression of specific genes, and they synthesize numerous stress proteins to maintain their cellular function. For example, APETALA2/ethylene response factor (AP2/ERF) was found to be involved in stress signal transduction. A search in the teak transcriptome database identified an AP2/ERF gene named TgERF1 with a key AP2/ERF domain. We then verified that the TgERF1 expression is rapidly induced by Polyethylene Glycol (PEG), NaCl, and exogenous phytohormone treatments, suggesting a potential role in drought and salt stress tolerance in teak. The full-length coding sequence of TgERF1 gene was isolated from teak young stems, characterized, cloned, and constitutively overexpressed in tobacco plants. In transgenic tobacco plants, the overexpressed TgERF1 protein was localized exclusively in the cell nucleus, as expected for a transcription factor. Furthermore, functional characterization of TgERF1 provided evidence that TgERF1 is a promising candidate gene to be used as selective marker on plant breeding intending to improve plant stress tolerance.

Genome-Wide Characterization of the PIFs Family in Sweet Potato and Functional Identification of IbPIF3.1 under Drought and Fusarium Wilt Stresses.

Phytochrome-interacting factors (PIFs) are essential for plant growth, development, and defense responses. However, research on the PIFs in sweet potato has been insufficient to date. In this study, we identified PIF genes in the cultivated hexaploid sweet potato (Ipomoea batatas) and its two wild relatives, Ipomoea triloba, and Ipomoea trifida. Phylogenetic analysis revealed that IbPIFs could be divided into four groups, showing the closest relationship with tomato and potato. Subsequently, the PIFs protein properties, chromosome location, gene structure, and protein interaction network were systematically analyzed. RNA-Seq and qRT-PCR analyses showed that IbPIFs were mainly expressed in stem, as well as had different gene expression patterns in response to various stresses. Among them, the expression of IbPIF3.1 was strongly induced by salt, drought, H2O2, cold, heat, Fusarium oxysporum f. sp. batatas (Fob), and stem nematodes, indicating that IbPIF3.1 might play an important role in response to abiotic and biotic stresses in sweet potato. Further research revealed that overexpression of IbPIF3.1 significantly enhanced drought and Fusarium wilt tolerance in transgenic tobacco plants. This study provides new insights for understanding PIF-mediated stress responses and lays a foundation for future investigation of sweet potato PIFs.

Overexpression of tomato glutathione reductase (SlGR) in transgenic tobacco enhances salt tolerance involving the S-nitrosylation of GR

S-nitrosylation, a post-translational modification (PTM) dependent on nitric oxide, is essential for plant development and environmental responsiveness. However, the function of S-nitrosylation of glutathione reductase (GR) in tomato (SlGR) under NaCl stress is yet uncertain. In this study, sodium nitroprusside (SNP), an exogenous NO donor, alleviated the growth inhibition of tomato under NaCl treatment, particularly at 100 μM. Following NaCl treatment, the transcripts, enzyme activity, and S-nitrosylated level of GR were increased. In vitro, the SlGR protein was able to be S-nitrosylated by S-nitrosoglutathione (GSNO), significantly increasing the activity of GR. SlGR overexpression transgenic tobacco plants exhibited enhanced germination rate, fresh weight, and increased root length in comparison to wild-type (WT) seedlings. The accumulation of reactive oxygen species (ROS) was lower, whereas the expression and activities of GR, ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT); the ratio of ascorbic acid/dehydroascorbic acid (AsA/DHA), reduced glutathione/oxidized glutathione (GSH/GSSG), total soluble sugar and proline contents; and the expression of stress-related genes were higher in SlGR overexpression transgenic plants in comparison to the WT plants following NaCl treatment. The accumulation of NO and S-nitrosylated levels of GR in transgenic plants was higher in comparison to WT plants following NaCl treatment. These results indicated that S-nitrosylation of GR played a significant role in salt tolerance by regulating the oxidative state.

Ectopic expression of Jatropha curcas JcTAW1 improves the vegetative growth, yield, and drought resistance of tobacco

BackgroundJatropha curcas is a promising alternative bio-energy resource. However, underrun limited its broad application in the industry. Luckily, TAW1 is a high-productivity promoting gene that increases the lateral branches by prolonging the identification of inflorescence meristems to generate more spikes and flowers.ResultsIn the current study, we introduced the Jatropha JcTAW1 gene into tobacco to depict its functional profile. Ectopically expressed JcTAW1 increased the lateral branches and ultimate yield of the transgenic tobacco plants. Moreover, the JcTAW1 lines had significantly higher plant height, longer roots, and better drought resistance than those of wild-type (W.T.). We performed RNA sequencing and weighted gene co-expression network analysis to determine which biological processes were affected by JcTAW1. The results showed that biological processes such as carbon metabolism, cell wall biosynthesis, and ionization transport were extensively promoted by the ectopic expression of JcTAW1. Seven hub genes were identified. Therein, two up-regulated genes affect glucose metabolism and cell wall biosynthesis, five down-regulated genes are involved in DNA repair and negative regulation of TOR (target-of-rapamycin) signaling which was identified as a central regulator to promote cell proliferation and growth.ConclusionsOur study verified a new promising candidate for Jatropha productive breeding and discovered several new features of JcTAW1. Except for boosting flowering, JcTAW1 was found to promote stem and root growth. Additionally, transcriptome analysis indicated that JcTAW1 might promote glucose metabolism while suppressing the DNA repair system.

Heterologous expression of HsPstS gene reducing arsenic accumulation and improving As-tolerance in transgenic tobaccos by enhancing CAT activity

Arsenic (As) is a toxic metalloid element that affects plant growth and development. Reducing the uptake of arsenic by plants via genetic engineering strategy can effectively improve the tolerance and safety of economic crops in As-contaminated soil. In this paper, the HsPstS gene coded ABC-type periplasmic phosphate-binding protein (PBP) of Halomonas strain GFAJ-1 was introduced into tobacco K326 by Agrobacterium-mediated genetic transformation to create transgenic tobaccos. Under As stress, NBT and DAB staining of tobacco leaves showed significant accumulation of H2O2 in wild-type and CK plants, and the further determination showed that the H2O2 content in CK plants was higher than that in transgenic plants except for L35S-2 and LREL-4 at 3 d after stress. Generally, the activity of antioxidant enzymes (CAT and POD) in tobaccos increased first and then decreased under As stress, and the CAT activity in most transgenic tobacco plants was significantly higher than that in wild-type and CK plants at 5 d after stress. By contrast, POD activity in CK and wild-type plants was significantly higher than that in transgenic tobaccos except for L35S-2. Additionally, As content determination showed that all transgenic tobacco plants except for CK showed the characteristic of low As-accumulation, especially in transgenic tobaccos L35S-2 and LREL-4, which suggested that the introduction of HsPstS could significantly reduce the As absorption in HsPstS-contained transgenic tobaccos, while there was no significant influence on agronomic traits and photosynthetic characteristics of transgenic tobaccos compared with wild-type ones. Interestingly, the introduction of HsPstS gene also reduced the content of nicotine and nornicotine in transgenic tobacco plants, while there was no significant difference on K content between transgenic and non-transgenic tobaccos. These results above provided ideal parental materials for cultivating tobacco germplasm with the characteristic of low As-accumulation.

Functional analysis of soybean miR156 and miR172 in tobacco highlights their role in plant morphology and floral transition

Soybean (Glycine max), a significant oilseed and protein source for humans and livestock feed, needs short day photoperiod for floral induction. Further, soybean has a paleopolyploid genome with multiple copies of flowering genes adding to the complexity of genetic regulation of flowering, and seed set, especially in investigating the role of the noncoding genome. microRNAs, a class of noncoding RNA, play a regulatory role in plant development. miR156 and miR172 are major components of the essential regulatory hub controlling juvenile and vegetative developments and initiation of reproductive phase change leading to flowering. These microRNAs have been originally isolated and studied from model plant, Arabidopsis. However, a study on soybean microRNAs is lacking. We investigated the temporal expression patterns of gma-miR156a and gma-miR172a and found inversely related - gma-miR156a expression was higher in the vegetative stage, and gma-miR172a expression was elevated under inductive flowering conditions. The functions of gma-miR156a and gma-miR172a were evaluated via heterologous expressions in transgenic tobacco plants (Nicotiana tabacum L.). The analysis of overexpression transgenic lines highlighted that gma-miR156a plays a role in juvenile development via repression of the SPL transcription factor family. In contrast, gma-miR172a plays a pivotal role in the reproductive development phase by down-regulating its target genes, AP2. In addition, ectopic expression of gma-miR156a and gma-miR172a affected plant morphology and physiology during plant growth. Collectively, our results suggest that gma-miR156a and gma-miR172a regulate multiple morpho-physiological traits that could be used to enhance crop yield under changing climate conditions.

Synthetic sub-genomic transcript promoter from Horseradish Latent Virus (HRLV).

We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; -340 to + 10; 351bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology.