RESUMO
A promoter is a crucial component in driving the expression of a transgene of interest for biotechnological applications in crop improvement and thus characterization of varied regulatory regions is essential. Here, we identified the promoter of COR2-like (codeinone reductase-like) from banana and characterized its tissue specific and stress inducible nature. MusaCOR2-like of banana is closely related to COR2 and CHR (chalcone reductase) sequences from different plant species and contains signature sequences including a catalytic tetrad typical of proteins with aldo-keto reductase activity. Transcript level of MusaCOR2-like was strongly induced in response to drought, salinity and exposure of signaling molecules such as abscisic acid, methyl-jasmonate and salicylic acid. Induction of MusaCOR2-like under stress strongly correlated with the presence of multiple cis-elements associated with stress responses in the PMusaCOR2-like sequence isolated from Musa cultivar Rasthali. Transgenic tobacco lines harbouring PMusaCOR2-like-GUS displayed visible GUS expression in vascular tissue of leaves and stem while its expression was undetectable in roots under control conditions. Exposure to drought, salinity and cold strongly induced GUS expression from PMusaCOR2-like-GUS in transgenic tobacco shoots in a window period of 3H to 12H. Applications of salicylic acid, methyl-jasmonate, abscisic acid and ethephon also activate GUS in transgenic shoots at different period, with salicylic acid and abscisic acid being the stronger stimulants of PMusaCOR2-like. Using PMusaCOR2-like-GUS fusion and expression profiling, the current study sheds insights into a complex regulation of COR2-like, one of the least studied genes of secondary metabolite pathway in plants.
RESUMO
NAC transcription factors are known for their diverse role in plants. In this study, we have demonstrated the role of MusaATAF2, a banana NAC transcription factor, in leaf senescence. Its expression gets strongly up-regulated during the early stress responses of drought and high salinity exposure and down-regulated under ABA application, which suggests MusaATAF2 is a stress-related NAC transcription factor. To study the role of MusaATAF2 in banana, we have transformed the banana embryogenic cells with MusaATAF2 coding region and generated transgenic banana plants. Overexpression of MusaATAF2 in banana plants caused yellow leaf phenotype under control condition, suggesting its role as a senescence-associated transcription factor. Transgenic banana leaves exhibited low chlorophyll content and high H2 O2 accumulation. Hormone analysis of the leaves demonstrated a higher accumulation of ABA in the transgenic plants than the controls. Transgenic plants overexpressing MusaATAF2 have a higher transcript abundance of two chlorophyll catabolic pathway genes (PAO and HCAR) and lower transcript abundance of ROS scavenging enzymes (TDP, THIO, CAT, APX, and PRXDN) than control. Together, all these analyses indicate that MusaATAF2 induces senescence by inducing chlorophyll degradation and H2 O2 accumulation in banana plants and controls its own expression using an ABA-dependent feedback loop.
Assuntos
Regulação da Expressão Gênica de Plantas , Fatores de Transcrição , Ácido Abscísico/metabolismo , Clorofila/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Senescência Vegetal , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
KEY MESSAGE: Senescence-associated transcription factor ATAF2 regulates cytokinin signalling and in vitro shoot multiplication in banana plants. MusaATAF2-like protein is a stress-related NAC transcription factor of banana. It regulates senescence in rooted banana plants. During the early stages of plant development under in vitro conditions, the presence of 6-benzylaminopurine leads to vigorous shoot multiplication. The major contributor to plant shoot multiplication is auxin to cytokinin ratio and their signalling components. The LC-MS analysis of transgenic banana plants overexpressing MusaATAF2 indicated significantly higher cytokinin content and remarkably lower auxin content. Auxin transport has been reported to be inhibited by flavonoids. Their significantly higher abundance in the shoot tissues in transgenic lines suggested potential negative regulation of auxin signalling in transgenic plants. Enhanced shoot multiplication in transgenic lines was further corroborated by reduced transcript abundance of type-A Arabidopsis response regulator-like genes (inhibitors of cytokinin signalling pathway) and higher expression of Arabidopsis histidine kinase-like genes and type-B Arabidopsis response regulator-like genes (positive regulators of cytokinin signalling pathway) in transgenic lines. Altogether, the data concludes that MusaATAF2 induces cytokinin hypersensitivity in banana shoots by modulating/regulating the cytokinin signalling components and flavonoids content.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Musa , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citocininas/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Musa/genética , Musa/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Micro RNAs (miRNAs) are 20-24 nucleotides long non-coding RNA sequences identified and characterized in multiple plant and animal systems. miRNAs play multifarious roles ranging from plant development to stress tolerance by synchronizing physiological processes at the level of transcription and translation. Banana is a major horticultural crop with colossal production worldwide. Despite the recent encouraging developments, the information on functions of miRNAs in banana plants is still in its infancy. The available literature pertaining to miRNAs in banana plants hints towards their contribution as master regulators in crucial physiological processes for instance abiotic stress responses, pathogenic defence response, fruit ripening and so on. This review is focused on biogenesis of miRNAs, their identification and deciphering their respective roles in banana plants with special emphasis on abiotic stress responses, plant immune responses, fruit ripening and storage. Based on the prior reports, we identified a few miRNAs with prospective roles in stress tolerance and illustrated the potential applications of miRNAs in banana crop improvement utilizing recent biotechnological tools such as CRISPR cas9, RNAi and the nano particle based foliar spray of miRNAs. The review briefly explained the future directions in banana research with a special emphasis on miRNA regulatory networks and agronomic traits improvement. Finally, future domains in miRNA research in plants and their possible applications towards crop improvement in agriculture are described briefly.
Assuntos
MicroRNAs , Musa , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Musa/genética , Interferência de RNA , RNA de Plantas/genética , Estresse FisiológicoRESUMO
Increasing crop productivity in an ever-changing environmental scenario is a major challenge for maintaining the food supply worldwide. Generation of crops having broad-spectrum pathogen resistance with the ability to cope with water scarcity is the only solution to feed the expanding world population. Stomatal closure has implications on pathogen colonization and drought tolerance. Recent studies have provided novel insights into networks involved in stomatal closure which is being used in biotechnological applications for improving crop endurance. Despite that genetic engineering of stomata requires guard cell preferred or specific regulatory regions to avoid undesirable side effects. In the present study, we describe the 5'-upstream regulatory region of the WRKY18 transcription factor of banana and functionally analyzed its stress meditated activation and strong guard cell preferred activity. Expression of MusaWRKY18 is augmented in leaves of banana cultivars Karibale Monthan, Rasthali and Grand Nain under multiple stress conditions suggesting its role in stress responses of banana plants. Transgenic tobacco lines harboring PMusaWRKY18 -ß-D-glucuronidase (GUS) were regenerated and GUS staining demonstrated substantial GUS expression in guard cells which corroborates with multiple Dof1 binding cis-elements in PMusaWRKY18 . Fluorescent ß-galactosidase assay demonstrated the stress-mediated strong induction profiles of PMusaWRKY18 at different time points in transgenic tobacco lines exposed to drought, high-salinity, cold, and applications of abscisic acid, salicylic acid, methyl jasmonate, and ethephon. This study sheds novel insights into guard cell preferred expression of WRKY genes under stress and confirm the utility of PMusaWRKY18 for exploring guard cell functions and guard cell engineering.
Assuntos
Musa , Ácido Abscísico , Secas , Regulação da Expressão Gênica de Plantas , Musa/genética , Musa/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
KEY MESSAGE: MusaSNAC1 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. Drought 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 (ß-glucuronidase) under P MusaSNAC1 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.
Assuntos
Adaptação Fisiológica , Secas , Peróxido de Hidrogênio/metabolismo , Musa/fisiologia , Proteínas de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Musa/genética , Estômatos de Plantas/citologia , Plantas Geneticamente Modificadas , Ligação Proteica , Estresse Fisiológico/genéticaRESUMO
Vascular related NAC (NAM, ATAF and CUC) domain-containing genes regulate secondary wall deposition and differentiation of xylem vessel elements. MusaVND1 is an ortholog of Arabidopsis VND1 and contains the highly conserved NAC domain. The expression of MusaVND1 is highest in developing corm and during lignification conditions, the increase in expression of MusaVND1 coincides with the expression of PAL, COMT and C4H genes. MusaVND1 encodes a nuclear localized protein as MusaVND1-GFP fusion protein gets localized to nucleus. Transient overexpression of MusaVND1 converts banana embryogenic cells to xylem vessel elements, with a final differentiation frequency of 33.54% at the end of tenth day. Transgenic banana plants overexpressing MusaVND1 showed stunted growth and were characterized by PCR and Southern blot analysis. Transgenic banana plants showed transdifferentiation of various types of cells into xylem vessel elements and ectopic deposition of lignin in cells of various plant organs such as leaf and corm. Tracheary element formation was seen in the cortical region of transgenic corm as well as in epidermal cells of leaves. Biochemical analysis indicates significantly higher levels of lignin and cellulose content in transgenic banana lines than control plants. MusaVND1 overexpressing transgenic banana plants showed elevated expression levels of genes involved in lignin and cellulose biosynthesis pathway. Further expression of different MYB transcription factors positively regulating secondary wall deposition was also up regulated in MusaVND1 transgenic lines.
Assuntos
Musa/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Sequência de Aminoácidos , Transdiferenciação Celular , Parede Celular/genética , Celulose/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Dados de Sequência Molecular , Musa/citologia , Musa/metabolismo , Proteínas de Plantas/metabolismo , Xilema/genética , Xilema/fisiologiaRESUMO
The DNA double strand break repair in plants is preferentially by non homologous end joining (NHEJ) pathway. A key protein of NHEJ pathway is Ku70. We have identified Ku70 homolog (VvKu70) from grapevine genome database. In this report we characterize a Ku70 homologue from Vitis vinifera cv. Mango. The VvKu70 expression was found to increase strongly in response to gamma radiation. The transcript level of VvKu70 was found to increase up to 36 h in gamma irradiated shoots of grapevine. The expression of VvKu70 was found in many organs like stem, leaves and roots. A GFP fused VvKu70 protein was found to be nuclear localized which indicates that the VvKu70 is a nuclear localized protein. The VvKu70 identified by in silico approaches is present as a single copy number in V. vinifera cv. Mango genome. The VvKu70-GFP fused protein possesses ATPase activity and fails to bind dsDNA but binds ssDNA.
Assuntos
Antígenos Nucleares/isolamento & purificação , Reparo do DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Nicotiana/genética , Plantas Geneticamente Modificadas/efeitos da radiação , Animais , Antígenos Nucleares/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Quebras de DNA de Cadeia Simples/efeitos da radiação , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Proteínas de Ligação a DNA/genética , Raios gama , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Autoantígeno Ku , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Raízes de Plantas/genética , Raízes de Plantas/efeitos da radiação , Vitis/genética , Vitis/efeitos da radiaçãoRESUMO
Understanding the molecular mechanisms governed by genes and cross-talks among stress signaling pathways is vital for generating a broad view on stress responses in plants. Here, we analysed the effects of MusaNAC29-like transcription factor of banana on stress responses and report the quantitative modulation of phytohormone and flavonoid content and analysed the growth parameters and yield trait in transgenic banana plants. Expression of MusaNAC29-like transcription factor was strongly altered in responses to stress conditions and application of signaling molecules. Under control conditions, PMusaNAC29-like-GUS is activated in cells bordering xylem vessel elements and is strongly triggered in other cells types after influence of salicylic acid and abscisic acid. Transgenic banana plants of cultivar Rasthali and Grand Naine overexpressing MusaNAC29-like transcription factor displayed superior tolerance towards drought and salinity stress. LC-MS analysis indicated elevated levels of jasmonic acid and salicylic acid while content of zeatin was significantly reduced in leaves of transgenic banana lines. Transgenic banana lines displayed increased levels of gallic acid, coumaric acid, naringenin, chlorogenic acid while levels of vanillic acid and piperine were significantly reduced. Expression of stress related genes coding for antioxidants, thiol peptidase proteins, cold-regulated proteins, late embryogenesis abundant proteins, ethylene-responsive transcription factors, bHLH proteins, jasmonate-zim-domain proteins and WRKY transcription factors were significantly induced in transgenic banana lines. Though MusaNAC29-like transcription factor improved stress tolerance, its overexpression resulted in retarded growth of transgenic lines resulting in reduced yield of banana fruits.
Assuntos
Musa , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Musa/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Secas , Ácido Salicílico/metabolismo , Estresse Fisiológico/genéticaRESUMO
Generation of crops with broad-spectrum tolerance to biotic and abiotic stress conditions depends upon availability of genetic elements suitable for varied situations and diverse genotypes. Here, we characterize the 5'-upstream regulatory region of flavonoid 3'5'-hydroxylase-1 (F3'5'H-1) gene from banana and analyzed its tissue-specific and stress-mediated activation in genetic background of tobacco plants. MusaF3'5'H-1 is a stress-responsive gene as its expression is induced in banana after application of salicylic acid and methyl jasmonate while its transcript levels were drastically reduced in response to drought, high salinity and abscisic acid. PMusaF3'5'H-1 harbours cis-elements associated with stress conditions and those responsible for tissue-specific expression. Transgenic lines harbouring PMusaF3'5'H-1-GUS displays strong GUS expression in guard cells of stomata indicating guard cell preferred activity of PMusaF3'5'H-1 while its activity was undetectable in roots. Drought and high salinity induce strong expression of GUS in transgenic tobacco lines and exposure to abscisic acid, salicylic acid and methyl jasmonate revealed distinct profiles of GUS expression in transgenic lines confirming involvement of F3'5'H-1 in plant stress responses. Fluorescent ß-galactosidase assay revealed induction profiles of PMusaF3'5'H-1 at different time points in transgenic lines exposed to salicylic acid and abscisic acid while strong suppression in GUS expression was observed after application of methyl jasmonate. The guard cell preferred activity of PMusaF3'5'H-1 and stress-mediated expression profiles of MusaF3'5'H-1 indicated the suitability of PMusaF3'5'H-1 for generating stress-enduring crops and analyzing guard cell functions.
Assuntos
Musa , Musa/genética , Musa/metabolismo , Ácido Abscísico/farmacologia , Sequências Reguladoras de Ácido Nucleico , Ácido Salicílico , Plantas Geneticamente Modificadas/genética , Regulação da Expressão Gênica de Plantas , Secas , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Augmenting shoot multiplication through genetic engineering is an emerging biotechnological application desirable in optimizing regeneration of genetically modified plants on selection medium and rapid clonal propagation of elite cultivars. Here, we report the improved shoot multiplication in transgenic banana lines with overexpression of MusaSNAC1, a drought-associated NAC transcription factor in banana. Overexpression of MusaSNAC1 induces hypersensitivity of transgenic banana lines toward 6-benzylaminopurine ensuing higher shoot number on different concentrations of 6-benzylaminopurine. Altered transcript levels of multiple genes involved in auxin signaling (Aux/IAA and ARFs) and cytokinin signaling pathways (ARRs) in banana plants overexpressing MusaSNAC1 corroborate the hypersensitivity of transgenic banana plants toward 6-benzylaminopurine. Modulation in expression of ARRs reported to be involved in ABA-hypersensitivity and closure of stomatal aperture correlates with the function of MusaSNAC1 as a drought-responsive NAC transcription factor. Present study suggests a prospective cross talk between shoot multiplication and drought responses coordinated by MusaSNAC1 in banana plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02744-5.
RESUMO
In the present study, the 5'-regulatory region of chalcone isomerase gene (MusaCHI-1) of banana was functionally analysed for its tissue specific, stress mediated and strong guard cell preferred activity. Expression of MusaCHI-1 was altered in leaves of banana plants exposed to various stress conditions and signalling molecules. Transgenic lines of tobacco harbouring PMusaCHI-1-GUS displays prominent GUS staining in vascular region and guard cells of leaves which corroborates with array of Dof1 binding cis-elements in PMusaCHI-1 region. Multiple cis-elements associated with various stress conditions were detected in PMusaCHI-1 which directly correlates with alteration of MusaCHI-1 transcript level in banana exposed to stress conditions. GUS staining of transgenic tobacco plants harbouring PMusaCHI-1-GUS and exposed to drought, salinity, and applications of methyl jasmonate and abscisic acid indicated activation of PMusaCHI-1 under these conditions while exposure of salicylic acid strongly suppresses GUS expression from PMusaCHI-1.
Assuntos
Musa , Ácido Abscísico , Secas , Regulação da Expressão Gênica de Plantas , Musa/genética , Regiões Promotoras GenéticasRESUMO
Mitogen activated protein kinases (MAPKs) are known to play important functions in stress responses of plants. We have functionally characterized a MAPK, MusaMPK5 from banana and demonstrated its function in cold tolerance response of banana plants. Expression of MusaMPK5 showed positive response to cold, methyl-jasmonate and salicylic acid treatment. Transgenic banana plants harbouring PMusaMPK5::GUS after exposure to cold stress (8⯰C) showed strong induction of GUS in cells surrounding central vascular cylinder of corm and cortical cells of pseudostem. Transgenic banana lines overexpressing MusaMPK5 were regenerated and four different transgenic lines were confirmed for T-DNA insertions by Southern blot and PCR analysis. In an in-vitro growth assay transgenic lines gained better shoot length and fresh weight during recovery from cold stress indicating improved cold tolerance ability of transgenic lines than control plants. Leaf discs of transgenic lines bleached less and retain lower MDA content than leaf discs of control plants after cold stress (4⯰C and 8⯰C). Cold stress tolerance analysis using two month old plants suggested that improved cold tolerance ability of transgenic lines might be associated with increased level of proline and reduced MDA content. MusaMPK5 gets localized in cytoplasm as observed in onion epidermal cells transiently overexpressing either MusaMPK5-GFP or MusaMPK5-GUS fusion protein. MusaMPK5 is a functional kinase as it autophosphorylate itself and phosphorylate myelin basic protein (MBP) in an in vitro reaction. Purified MusaMPK5 can phosphorylate NAC042 and SNAC67 transcription factors of banana which are important regulators of stress tolerance in banana plants.
Assuntos
Musa , Sequência de Aminoácidos , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno , Plantas Geneticamente Modificadas , Estresse FisiológicoRESUMO
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 (ß-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.
Assuntos
Musa/genética , Musa/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Xilema/metabolismoRESUMO
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.
Assuntos
Clorofila/metabolismo , Redes e Vias Metabólicas , Musa/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Sequência de Bases , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Cruzamentos Genéticos , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Musa/genética , Musa/fisiologia , Musa/ultraestrutura , Especificidade de Órgãos/genética , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Salinidade , Estresse Fisiológico/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Banana is an important fruit crop and its yield is hampered by multiple abiotic stress conditions encountered during its growth. The NAC (NAM, ATAF, and CUC) transcription factors are involved in plant response to biotic and abiotic stresses. In the present study, we studied the induction of banana NAC042 transcription factor in drought and high salinity conditions and its overexpression in transgenic banana to improve drought and salinity tolerance. MusaNAC042 expression was positively associated with stress conditions like salinity and drought and it encoded a nuclear localized protein. Transgenic lines of banana cultivar Rasthali overexpressing MusaNAC042 were generated by Agrobacterium-mediated transformation of banana embryogenic cells and T-DNA insertion was confirmed by PCR and Southern blot analysis. Our results using leaf disc assay indicated that transgenic banana lines were able to tolerate drought and high salinity stress better than the control plants and retained higher level of total chlorophyll and lower level of MDA content (malondialdehyde). Transgenic lines analyzed for salinity (250 mM NaCl) and drought (Soil gravimetric water content 0.15) tolerance showed higher proline content, better Fv/Fm ratio, and lower levels of MDA content than control suggesting that MusaNAC042 may be involved in responses to higher salinity and drought stresses in banana. Expression of several abiotic stress-related genes like those coding for CBF/DREB, LEA, and WRKY factors was altered in transgenic lines indicating that MusaNAC042 is an efficient modulator of abiotic stress response in banana.
Assuntos
Secas , Musa/fisiologia , Proteínas de Plantas/metabolismo , Salinidade , Tolerância ao Sal , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Musa/embriologia , Musa/genética , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regeneração , Reprodutibilidade dos Testes , Tolerância ao Sal/genética , Alinhamento de Sequência , Frações Subcelulares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional/genéticaRESUMO
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.
Assuntos
Parede Celular/genética , Regulação da Expressão Gênica de Plantas , Genes myb , Musa/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Lignina/biossíntese , Musa/metabolismo , Fenilpropionatos/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polifenóis/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismoRESUMO
Secondary-wall deposition in xylem vessel elements is regulated by vascular-related NAC transcription factors (VNDs). We show that three banana VNDs (MusaVND1, MusaVND2 and MusaVND3) directly regulate multiple secondary-wall associated genes by binding to their 5'-upstream regulatory region. Transgenic banana harboring either PMusaVND1:GUS, PMusaVND2:GUS or PMusaVND3:GUS showed specific GUS staining in lignified tissues. MusaVND1, MusaVND2 and MusaVND3 encodes transcriptional-activators as its C-terminal region drive expression of reporter genes in vivo in yeast. Purified MusaVND1, MusaVND2 and MusaVND3 proteins in gel shift assay bind to 19-bp secondary-wall NAC binding element (SNBE) while it fails to bind mutated SNBE. Putative SNBE sites in the 5'-upstream regulatory region of important secondary-wall associated genes related to programmed cell death (XCP1), cell-wall modification (IRX1/CesA8, IRX3/CesA7,IRX5/CesA4, IRX8, IRX10 and IRX12) and transcriptional regulation (MYB52, MYB48/59, MYB85, MYB58/72, MYB46, and MYB83) in banana was identified and mobility of these regulatory regions got retarded by MusaVND1, MusaVND2 and MusaVND3. Transcript level of these important secondary wall associated genes were elevated in transgenic banana overexpressing either MusaVND1, MusaVND2 or MusaVND3. Present study suggested promoters with prospective utilization in wall modification in banana (a potential biofuel crop) and suggest a complex transcriptional regulation of secondary wall deposition in plants.
Assuntos
Regulação da Expressão Gênica de Plantas , Musa/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Parede Celular/genética , Musa/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
NAM, ATAF, and CUC (NAC) domain-containing proteins are plant-specific transcription factors involved in stress responses and developmental regulation. MusaVND2 and MusaVND3 are vascular-related NAC domain-containing genes encoding for nuclear-localized proteins. The transcript level of MusaVND2 and MusaVND3 are gradually induced after induction of lignification conditions in banana embryogenic cells. Banana embryogenic cells differentiated to tracheary element-like cells after overexpression of MusaVND2 and MusaVND3 with a differentiation frequency of 63.5 and 23.4 %, respectively, after ninth day. Transgenic banana plants overexpressing either of MusaVND2 or MusaVND3 showed ectopic secondary wall deposition as well as transdifferentiation of cells into tracheary elements. Transdifferentiation to tracheary element-like cells was observed in cortical cells of corm and in epidermal and mesophyll cells of leaves of transgenic plants. Elevated levels of lignin and crystalline cellulose were detected in the transgenic banana lines than control plants. The results obtained are useful for understanding the molecular regulation of secondary wall development in banana.
Assuntos
Parede Celular/metabolismo , Musa/genética , Folhas de Planta/genética , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Transdiferenciação Celular , Celulose/metabolismo , Celulose/ultraestrutura , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Lignina/ultraestrutura , Musa/citologia , Musa/metabolismo , Filogenia , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
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.