Himanshu Tak

Cloning and molecular characterization of a putative bZIP transcription factor VvbZIP23 from Vitis vinifera

The proteins harboring bZIP domains comprise a large family and play key roles in many cellular processes, one of them being tolerance to biotic and abiotic stresses in plants. In the present study, we characterize a putative bZIP transcription factor from Vitis vinifera namely VvbZIP23. Our studies revealed that a GFP fusion of VvbZIP23 is localized in the nucleus showing VvbZIP23 codes for a nuclear localized protein. VvbZIP23 identified by in silico approaches from grapevine DNA databases available in the public domain NCBI is present in a single copy in the grapevine genome as shown by Southern blot analysis. Expression of VvbZIP23 is induced by a wide spectrum of abiotic stresses, including drought, salt, and cold. Exogenous application of signaling chemicals like abscisic acid, methyl viologen, salicylic acid, jasmonic acid, and ethephon also induced expression of VvbZIP23. This shows that VvbZIP23 is involved in regulating a number of stress responses in V. vinifera. The 5′ proximal region of VvbZIP23 contains many cis-acting elements, which show induction of VvbZIP23 expression in multiple stress responses. Transcripts of VvbZIP23 were found in many parts of the grapevine plant with the highest expression detected in leaves. Further in silico analysis shows that the open reading frame of VvbZIP23 is 822 bp long and codes for a 273 amino acid long protein having a characteristic bZIP domain in its N-terminal end. Overexpression of VvbZIP23-GFP fusion protein in grapevine callus leads to enhanced transcript levels of genes, homologues of which are reported to be important in regulating many stress conditions.

Expression analysis of MusaNAC68 transcription factor and its functional analysis by overexpression in transgenic banana plants

NAC (NAM, ATAF and CUC) proteins are plant-specific transcription factors regulating development and responses to stress. One of NAC proteins from banana is NAC68, and expression analysis indicated its positive association to stress conditions. The 5′-proximal region of MusaNAC68 was isolated and sequence analysis indicated presence of stress related cis-elements and cis-elements involved in auxin-signaling. Expression of MusaNAC68 was maximum in roots and positively correlated with application of α-naphthaleneacetic acid. Nuclear localization of MusaNAC68 was determined by fusion of green-fluorescent protein with MusaNAC68 and transiently overexpressing in banana embryogenic cells. Transgenic lines were marginally taller and displayed more abundant roots than control along with altered expression of auxin-responsive genes like auxin-responsive factors and IAA/Aux (indoleacetic acid-induced protein) genes. Transgenic plants overexpressing MusaNAC68 displayed higher activity of polyphenol oxidase and rapid browning of plant extract. Transgenic lines showed better tolerance to stress induced by NaCl and mannitol and produced more shoot biomass. Leaf disc assay showed that transgenic lines retain more chlorophyll and lower malondialdehyde than control under salinity and drought. Transgenic line constitutively overexpressing MusaNAC68 showed elevated expression of many stress-responsive genes indicating its involvement in salinity and drought tolerance in banana.

Molecular characterization of VvSDIR1 from Vitis vinifera and its functional analysis by heterologous expression in Nicotiana tabacum

The proteins harboring really interesting new gene (RING) finger domains comprise a large family and play key roles in a variety of cellular processes. One among them is the tolerance to biotic and abiotic stresses in plants. In the present study, we characterize Vitis vinifera salt- and drought-induced RING finger 1 (VvSDIR1) a homologue of the Arabidopsis SDIR1 gene obtained from V. vinifera. The VvSDIR1 gene was identified using in silico approaches and encodes a membrane-localized protein. This was evident as expression of VvSDIR1 fused with green fluorescent protein was detected in cell membrane. Southern blot analysis indicates that VvSDIR1 is present in single copy number in grape genome. The expression of VvSDIR1 gene is elevated by multiple abiotic stresses like salt, drought, cold, and heat as well as upon exogenous application of methyl jasmonate, salicylic acid, methyl viologen, abscisic acid, and ethephon. In silico analysis shows that the VvSDIR1 cDNA is 831-bp long and codes for a 276-amino acid-long protein containing a characteristic RING finger domain in its C-terminal end. Overexpression of VvSDIR1 in tobacco leads to enhanced transcript levels of many genes, homologues of which are reported to be important in regulating many stress conditions. The heterologous expression of VvSDIR1 in tobacco was found to enhance the oxidative stress tolerance in tobacco. Tobacco lines transgenic for VvSDIR1 showed enhanced tolerance to treatment with methyl viologen, NaCl, and polyethylene glycol. To the best of our knowledge, this is the first report of the heterologous expression of VvSDIR1 in oxidative stress tolerance in transgenic tobacco.

A banana NAC transcription factor ( MusaSNAC1 ) impart drought tolerance by modulating stomatal closure and H 2 O 2 content

Key messageMusaSNAC1 function in H2O2 mediated stomatal closure and promote drought tolerance by directly binding to CGT[A/G] motif in regulatory region of multiple stress-related genes.AbstractDrought is a abiotic stress-condition, causing reduced plant growth and diminished crop yield. Guard cells of the stomata control photosynthesis and transpiration by regulating CO2 exchange and water loss, thus affecting growth and crop yield. Roles of NAC (NAM, ATAF1/2 and CUC2) protein in regulation of stress-conditions has been well documented however, their control over stomatal aperture is largely unknown. In this study we report a banana NAC protein, MusaSNAC1 which induced stomatal closure by elevating H2O2 content in guard cells during drought stress. Overexpression of MusaSNAC1 in banana resulted in higher number of stomata closure causing reduced water loss and thus elevated drought-tolerance. During drought, expression of GUS (β-glucuronidase) under PMusaSNAC1 was remarkably elevated in guard cells of stomata which correlated with its function as a transcription factor regulating stomatal aperture closing. MusaSNAC1 is a transcriptional activator belonging to SNAC subgroup and its 5′-upstream region contain multiple Dof1 elements as well as stress-associated cis-elements. Moreover, MusaSNAC1 also regulate multiple stress-related genes by binding to core site of NAC-proteins CGT[A/G] in their 5′-upstream region. Results indicated an interesting mechanism of drought tolerance through stomatal closure by H2O2 generation in guard cells, regulated by a NAC-protein in banana.

Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate its role as a negative regulator of lignin biosynthesis in banana

Lignin and polyphenols are important cellular components biosynthesized through phenylpropanoid pathway. Phenylpropanoid pathway in plants is regulated by some important transcription factors including R2R3 MYB transcription factors. In this study, we report the cloning and functional characterization of a banana R2R3-MYB transcription factor (MusaMYB31) by overexpression in transgenic banana plants and evaluated its potential role in regulating biosynthesis of lignin and polyphenols. Sequence analysis of MusaMYB31 indicated its clustering with members of subgroup 4 (Sg4) of R2R3MYB family which are well known for their role as repressors of lignin biosynthesis. Expression analysis indicated higher expression of MusaMYB31 in corm and root tissue, known for presence of highly lignified tissue than other organs of banana. Overexpression of MusaMYB31 in banana cultivar Rasthali was carried out and four transgenic lines were confirmed by GUS histochemical staining, PCR analysis and Southern blot. Histological and biochemical analysis suggested reduction of cell wall lignin in vascular elements of banana. Transgenic lines showed alteration in transcript levels of general phenylpropanoid pathway genes including lignin biosynthesis pathway genes. Reduction of total polyphenols content in transgenic lines was in line with the observation related to repression of general phenylpropanoid pathway genes. This study suggested the potential role of MusaMYB31 as repressor of lignin and polyphenols biosynthesis in banana.

A stress associated NAC transcription factor MpSNAC67 from banana (Musa x paradisiaca) is involved in regulation of chlorophyll catabolic pathway

Process of senescence includes multiple steps involving break-down of chlorophyll to degrade photosynthetic machinery. In this study, we showed that a stress-associated NAC transcription factor MpSNAC67 regulates senescence by promoting chlorophyll-catabolic genes. MpSNAC67 encodes a transcriptional activator and its promoter activity is restricted to vascular tissue of banana. Expression of MpSNAC67 showed positive responses to multiple abiotic stress conditions suggesting that MpSNAC67 is a stress associated NAC transcription factor. Transgenic banana lines overexpressing MpSNAC67 showed highly senesced phenotype including yellowing and de-greening of leaves similar to etiolated leaves. Transgenic leaves possessed low chlorophyll content and failed to retain normal chloroplast morphology including loss of granum thylakoid, non-uniform chloroplast membrane and increased number as well as size of plastoglobulins. In a gel shift assay MpSNAC67 could retard the mobility of chlorophyll catabolic genes such as PAO-like (Pheophorbide-a-oxygenase), HCAR-like (hydroxymethyl chlorophyll-a-reductase), NYC/NOL-like (Chlorophyll-b-reductase) as well as ORS1-like (a SenNAC). Expression of these genes were highly elevated in transgenic lines which indicate that MpSNAC67 is a positive regulator of senescence in banana and exercise its effect by regulating the expression of chlorophyll catabolic genes and ORS1.

Xylem specific activation of 5’ upstream regulatory region of two NAC transcription factors (MusaVND6 and MusaVND7) in banana is regulated by SNBE-like sites

Deposition of secondary cell wall in the xylem elements is controlled by a subgroup of NAC (NAM, ATAF, CUC) family, known as vascular-related NAC transcription factors (VNDs). In the present study, we analyzed the 5’ upstream regulatory region of two banana NAC transcription factors (MusaVND6 and MusaVND7) for tissue specific expression and presence of 19-bp secondary-wall NAC binding element (SNBE)-like motifs. Transgenic banana plants of Musa cultivar Rasthali harboring either PMusaVND7::GUS or PMusaVND6::GUS showed specific GUS (β-D-Glucuronidase) activity in cells of the xylem tissue. Approximately 1.2kb promoter region of either MusaVND6 or MusaVND7 showed presence of at least two SNBE-like motifs. This 1.2kb promoter region was retarded in a gel shift assay by three banana VND protein (VND1,VND2 and VND3). The banana VND1-VND3 could also retard the mobility of isolated SNBE-like motifs of MusaVND6 or MusaVND7 in a gel shift assay. Transcript levels of MusaVND6 and MusaVND7 were elevated in transgenic banana overexpressing either banana VND1, VND2 or VND3. Present study suggested a probable regulation of banana VND6 and VND7 expression through direct interaction of banana VND1- VND3 with SNBE-like motifs. Our study also indicated two promoter elements for possible utilization in cell wall modifications in plants especially banana, which is being recently considered as a potential biofuel crop.

Molecular Farming: Prospects and Limitation

Plant molecular farming is the production of recombinant pharmaceutical and nonpharmaceutical proteins of commercial importance utilizing plants as bioreactors. Research and development on plant-derived recombinant proteins have gained momentum in recent years. Advantages of employing plants as bioreactors for recombinant protein generation are many including low cost of production, easier scale-up, cost-effective storage, and absence of animal pathogens in protein preparations. This article reviews the various technologies developed for employing plants as bioreactors, different plant systems being used as expression host, and limitations and research advances to overcome these limitations. An overview of different plant-derived products whether currently in market or are in different stages of development, including phases of clinical trials, is described. Special emphasis has been given on banana being used as an expression host, advantages and limitations of using banana in plant molecular farming, and different approaches which can be utilized to overcome those limitations have been described.