MusaATAF2 like protein, a stress-related transcription factor, induces leaf senescence by regulating chlorophyll catabolism and H2 O2 accumulation.

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.

MusaATAF2-like protein regulates shoot development and multiplication by inducing cytokinin hypersensitivity and flavonoid accumulation in banana plants.

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.

The 5'-upstream region of WRKY18 transcription factor from banana is a stress-inducible promoter with strong expression in guard cells.

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.

A banana NAC transcription factor (MusaSNAC1) impart drought tolerance by modulating stomatal closure and H2O2 content.

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.

Cloning and functional characterization of MusaVND1 using transgenic banana plants.

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.

MusaNAC29-like transcription factor improves stress tolerance through modulation of phytohormone content and expression of stress responsive genes.

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.

Functional characterization of 5'-regulatory region of flavonoid 3',5'-hydroxylase-1 gene of banana plants.

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.

Overexpression of MusaSNAC1 improves shoot proliferation in transgenic banana lines.

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.

Studies on the tissue specific nature and stress inducible activation of the CHI-1 gene from banana.

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.

MusaMPK5, a mitogen activated protein kinase is involved in regulation of cold tolerance in banana.

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.

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 (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.

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.

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.

Native vascular related NAC transcription factors are efficient regulator of multiple classes of secondary wall associated genes in banana.

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.

Banana NAC transcription factor MusaNAC042 is positively associated with drought and salinity tolerance.

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.

Functional characterization of secondary wall deposition regulating transcription factors MusaVND2 and MusaVND3 in transgenic banana plants.

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.