Functional characterization of GhNAC2 promoter conferring hormone- and stress-induced expression: a potential tool to improve growth and stress tolerance in cotton.

The GhNAC2 transcription factor identified from G. herbaceum improves root growth and drought tolerance through transcriptional reprogramming of phytohormone signaling. The promoter of such a versatile gene could serve as an important genetic engineering tool for biotechnological application. In this study, we identified and characterized the promoter of GhNAC2 to understand its regulatory mechanism. GhNAC2 transcription factor increased in root tissues in response to GA, ethylene, auxin, ABA, mannitol, and NaCl. In silico analysis revealed an overrepresentation of cis-regulatory elements associated with hormone signaling, stress responses and root-, pollen-, and seed-specific promoter activity. To validate their role in GhNAC2 function/regulation, an 870-bp upstream regulatory sequence was fused with the GUS reporter gene (uidA) and expressed in Arabidopsis and cotton hairy roots for in planta characterization. Histochemical GUS staining indicated localized expression in root tips, root elongation zone, root primordia, and reproductive tissues under optimal growth conditions. Mannitol, NaCl, auxin, GA, and ABA, induced the promoter-driven GUS expression in all tissues while ethylene suppressed the promoter activity. The results show that the 870 nt fragment of the GhNAC2 promoter drives root-preferential expression and responds to phytohormonal and stress signals. In corroboration with promoter regulation, GA and ethylene pathways differentially regulated root growth in GhNAC2-expressing Arabidopsis. The findings suggest that differential promoter activity governs the expression of GhNAC2 in root growth and stress-related functions independently through specific promoter elements. This multifarious promoter can be utilized to develop yield and climate resilience in cotton by expanding the options to control gene regulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01411-2.

A GARP transcription factor SlGCC positively regulates lateral root development in tomato via auxin-ethylene interplay.

MAIN CONCLUSION: SlGCC, a GARP transcription factor, functions as a root-related transcriptional repressor. SlGCC synchronizes auxin and ethylene signaling involving SlPIN3 and SlIAA3 as intermediate targets sketching a molecular map for lateral root development in tomato. The root system is crucial for growth and development of plants as it performs basic functions such as providing mechanical support, nutrients and water uptake, pathogen resistance and responds to various stresses. SlGCC, a GARP family transcription factor (TF), exhibited predominant expression in age-dependent (initial to mature stages) tomato root. SlGCC is a transcriptional repressor and is regulated at a transcriptional and translational level by auxin and ethylene. Auxin and ethylene mediated SlGCC protein stability is governed via proteasome degradation pathway during lateral root (LR) growth development. SlGCC over-expressor (OE) and under-expressed (UE) tomato transgenic lines demonstrate its role in LR development. This study is an attempt to unravel the vital role of SlGCC in regulating tomato LR architecture.

The tomato EAR-motif repressor, SlERF36, accelerates growth transitions and reduces plant life cycle by regulating GA levels and responses.

Faster vegetative growth and early maturity/harvest reduce plant life cycle time and are important agricultural traits facilitating early crop rotation. GA is a key hormone governing developmental transitions that determine growth speed in plants. An EAR-motif repressor, SlERF36 that regulates various growth transitions, partly through regulation of the GA pathway and GA levels, was identified in tomato. Suppression of SlERF36 delayed germination, slowed down organ growth and delayed the onset of flowering time, fruit harvest and whole-plant senescence by 10-15 days. Its over-expression promoted faster growth by accelerating all these transitions besides increasing organ expansion and plant height substantially. The plant life cycle and fruit harvest were completed 20-30 days earlier than control without affecting yield, in glasshouse as well as net-house conditions, across seasons and generations. These changes in life cycle were associated with reciprocal changes in expression of GA pathway genes and basal GA levels between suppression and over-expression lines. SlERF36 interacted with the promoters of two GA2 oxidase genes, SlGA2ox3 and SlGA2ox4, and the DELLA gene, SlDELLA, reducing their transcription and causing a 3-5-fold increase in basal GA3/GA4 levels. Its suppression increased SlGA2ox3/4 transcript levels and reduced GA3/GA4 levels by 30%-50%. SlERF36 is conserved across families making it an important candidate in agricultural and horticultural crops for manipulation of plant growth and developmental transitions to reduce life cycles for faster harvest.

Novel roles of HSFs and HSPs, other than relating to heat stress, in temperature-mediated flowering.

The thermotolerant ability of heat shock factors (HSFs) and heat shock proteins (HSPs) in plants has been shown. Recently, focus has been on their function in plant growth and development under non-stress conditions. Their role in flowering has been suggested given that lower levels of HSF/HSPs resulted in altered flowering in Arabidopsis. Genetic and molecular studies of Arabidopsis HSF/HSP mutants advocated an association with temperature-mediated regulation of flowering, but the fundamental genetic mechanism behind this phenomenon remains obscure. Here we outline plausible integration between HSFs/HSPs and temperature-dependent pathways in plants regulating flowering. Moreover, we discuss how similar pathways can be present in thermoperiodic geophytic plants that require ambient high temperatures for flowering induction.

Phosphomevalonate kinase regulates the MVA/MEP pathway in mango during ripening.

Mango is a popular tropical fruit with a great diversity in taste and aroma, contributed primarily by terpenoids. Phosphomevalonate kinase (PMK) is a key enzyme for isoprenoid biosynthesis in the mevalonic acid (MVA) pathway responsible for terpenoids. In this study, two cultivars of mango, "Dashehari" and "Banganpalli", showing opposite spatio-temporal patterns of ripening polarity, were investigated for studying the role of MiPMK in aroma production. MiPMK transcription and enzyme activity increased during ripening in both varieties. Expression in the early-ripening inner zones preceded that in the later-ripening outer zones of "Dashehari" while it was higher in the early ripening outer zones in "Banganpalli". Polypeptide sequences of the two enzymes showed differences in a few amino acids that were also reflected in kinetic properties such as specific activity and pH optima. Silencing of MiPMK in "Dashehari" fruit by VIGS suppressed the kinase activity and led to changes in relative contributions of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways. This also altered the fruit metabolite profile with a reduction/disappearance of sesquiterpenes such as geranyl geraniol, trans-farnesol, ß-caryophyllene, ß-pinene, bisabolene and guaiane but the appearance of menthol and d-limonene in silenced fruit. The study shows that MiPMK levels may control downstream metabolite flux of the MVA pathway in mango.

Tomato (Solanum lycopersicum) WRKY23 enhances salt and osmotic stress tolerance by modulating the ethylene and auxin pathways in transgenic Arabidopsis.

Osmotic stress is one of the biggest problems in agriculture, which adversely affects crop productivity. Plants adopt several strategies to overcome osmotic stresses that include transcriptional reprogramming and activation of stress responses mediated by different transcription factors and phytohormones. We have identified a WRKY transcription factor from tomato, SlWRKY23, which is induced by mannitol and NaCl treatment. Over-expression of SlWRKY23 in transgenic Arabidopsis enhances osmotic stress tolerance to mannitol and NaCl and affects root growth and lateral root number. Transgenic Arabidopsis over-expressing SlWRKY23 showed reduced electrolyte leakage and higher relative water content than Col-0 plants upon mannitol and NaCl treatment. These lines also showed better membrane integrity with lower MDA content and higher proline content than Col-0. Responses to mannitol were governed by auxin as treatment with TIBA (auxin transport inhibitor) negatively affected the osmotic tolerance in transgenic lines by inhibiting lateral root growth. Similarly, responses to NaCl were controlled by ethylene as treatment with AgNO3 (ethylene perception inhibitor) inhibited the stress response to NaCl by suppressing primary and lateral root growth. The study shows that SlWRKY23, a osmotic stress inducible gene in tomato, imparts tolerance to mannitol and NaCl stress through interaction of the auxin and ethylene pathways.

SlDREB3, a negative regulator of ABA responses, controls seed germination, fruit size and the onset of ripening in tomato.

SlDREB3 was identified as a ripening up-regulated gene of the AP2/ERF-domain family of transcription factors. Its manipulation affects processes primarily governed by ABA. It negatively regulates ABA responses in tomato by altering ABA levels/signaling and is, in turn, negatively regulated by ABA. SlDREB3 over-expression lines show higher transcript levels of the ABA metabolism genes CYP707A3 and UGT75C1 and an 85% reduction in ABA levels leading to early seed germination. In contrast, suppression lines show decreased CYP707A3/UGT75C1 expression, 3-fold higher ABA levels and delayed germination. The expression of other ABA signaling and response genes is also affected. Suppression of SlDREB3 accelerates the onset of ripening by 4-5 days while its over-expression delays it and also reduces final fruit size. SlDREB3 manipulation effects large scale changes in the fruit transcriptome with suppression lines showing early increase in ABA levels and activation of most ripening pathway genes that govern ethylene, carotenoids and softening. Strikingly, key transcription factors like CNR, NOR, RIN, FUL1, governing ethylene-dependent and ethylene-independent aspects of ripening, are activated early upon SlDREB3 suppression suggesting their control by ABA. The studies identify SlDREB3 as a negative regulator of ABA responses across tissues and a key ripening regulator controlling ethylene-dependent and ethylene-independent aspects.

Identification of tomato root growth regulatory genes and transcription factors through comparative transcriptomic profiling of different tissues.

Tomato is an economically important vegetable crop and a model for development and stress response studies. Although studied extensively for understanding fruit ripening and pathogen responses, its role as a model for root development remains less explored. In this study, an Illumina-based comparative differential transcriptomic analysis of tomato root with different aerial tissues was carried out to identify genes that are predominantly expressed during root growth. Sequential comparisons revealed ~ 15,000 commonly expressed genes and ~ 3000 genes of several classes that were mainly expressed or regulated in roots. These included 1069 transcription factors (TFs) of which 100 were differentially regulated. Prominent amongst these were members of families encoding Zn finger, MYB, ARM, bHLH, AP2/ERF, WRKY and NAC proteins. A large number of kinases, phosphatases and F-box proteins were also expressed in the root transcriptome. The major hormones regulating root growth were represented by the auxin, ethylene, JA, ABA and GA pathways with root-specific expression of certain components. Genes encoding carbon metabolism and photosynthetic components showed reduced expression while several protease inhibitors were amongst the most highly expressed. Overall, the study sheds light on genes governing root growth in tomato and provides a resource for manipulation of root growth for plant improvement. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01015-0.

Expression of the tomato WRKY gene, SlWRKY23, alters root sensitivity to ethylene, auxin and JA and affects aerial architecture in transgenic Arabidopsis.

WRKY transcription factors (TFs) are a large plant-specific family of TFs that govern development and biotic/abiotic stress responses in plants. We have identified SlWRKY23 as a gene primarily expressed in roots. SlWRKY23 encodes a protein of 320 amino acids that functions as a transcriptional activator. It is transcriptionally up-regulated by ethylene, BAP and salicylic acid treatment but suppressed by IAA. Expression of SlWRKY23 in transgenic Arabidopsis affects sensitivity of roots to ethylene, JA and auxin with transgenic plants showing hypersensitivity to ethylene, JA and auxin-mediated primary root growth inhibition. This hypersensitivity is correlated with higher expression of ERF1 and ARF5 that mediate responses to these hormones. SlWRKY23 expression also affects aerial growth with transgenic plants showing greater number of leaves but smaller rosettes. Flowering time is reduced in transgenic lines and these plants also show a greater number of inflorescence branches, siliques and seeds. The siliques are longer and compactly packed with seeds but seeds are smaller in size. Root biomass shows a 25% decrease in transgenic SlWRKY23 Arabidopsis plants at harvest compared with controls. The studies show that SlWRKY23 regulates plant growth possibly through modulation of genes controlling hormone responses.

PGPR-induced OsASR6 improves plant growth and yield by altering root auxin sensitivity and the xylem structure in transgenic Arabidopsis thaliana.

Plant-growth-promoting rhizobacteria (PGPR) improve plant growth by altering the root architecture, although the mechanisms underlying this alteration have yet to be unravelled. Through microarray analysis of PGPR-treated rice roots, a large number of differentially regulated genes were identified. Ectopic expression of one of these genes, OsASR6 (ABA STRESS RIPENING6), had a remarkable effect on plant growth in Arabidopsis. Transgenic lines over-expressing OsASR6 had larger leaves, taller inflorescence bolts and greater numbers of siliques and seeds. The most prominent effect was observed in root growth, with the root biomass increasing four-fold compared with the shoot biomass increase of 1.7-fold. Transgenic OsASR6 over-expressing plants showed higher conductance, transpiration and photosynthesis rates, leading to an ˜30% higher seed yield compared with the control. Interestingly, OsASR6 expression led to alterations in the xylem structure, an increase in the xylem vessel size and altered lignification, which correlated with higher conductance. OsASR6 is activated by auxin and, in turn, increases auxin responses and root auxin sensitivity, as observed by the increased expression of auxin-responsive genes, such as SAUR32 and PINOID, and the key auxin transcription factor, ARF5. Collectively, these phenomena led to an increased root density. The effects of OsASR6 expression largely mimic the beneficial effects of PGPRs in rice, indicating that OsASR6 activation may be a key factor governing PGPR-mediated changes in rice. OsASR6 is a potential candidate for the manipulation of rice for improved productivity.

JcMYB1, a Jatropha R2R3MYB Transcription Factor Gene, Modulates Lipid Biosynthesis in Transgenic Plants.

The lipid biosynthesis pathway in plants has been studied in detail; however, the factors that regulate the pathway at the transcription level are largely unknown. LEAFY COTYLEDON1 (LEC1), WRINKLED1 (WRI1) and FUSCA3 (FUS3) are considered master regulators to control seed oil content in Arabidopsis. Beside these master regulators, several other transcription factors that may regulate the pathway in plants are poorly studied. In the present work, we have shown the involvement of an uncharacterized Jatropha curcas R2R3MYB gene (JcMYB1) in seed oil biosynthesis. Seed oil analysis and expression profiling of fatty acid (FA) and triacylglycerol (TAG) biosynthetic genes in transgenic Arabidopsis and tobacco plants revealed that JcMYB1 enhances seed oil accumulation and alters FA composition by regulating the expression of endogenous pathway genes in transgenics. Using virus-induced gene silencing (VIGS) in Jatropha, we demonstrated that the suppression of JcMYB1 reduced lipid content with altered FA composition. Agro-infiltration and yeast one-hybrid assay results showed that JcMYB1 protein directly binds to the diacylglycerol acyltransferase1 (DGAT1) promoter, a rate-limiting enzyme of TAG biosynthesis, and activates its expression. These results suggested that JcMYB1 may augment the lipid content by regulating lipid biosynthetic genes. Additionally, manipulation of JcMYB1 in oil crop plants may be used for the potential improvement of oil production and quality.

Tocopherol levels in different mango varieties correlate with MiHPPD expression and its over-expression elevates tocopherols in transgenic Arabidopsis and tomato.

Mango fruit tocopherol levels vary in different varieties during ripening. This study shows that tocopherol accumulation is highly correlated with its p-hydroxyphenyl pyruvate dioxygenase (MiHPPD) gene expression during ripening. MiHPPD transcript is ethylene induced and differentially expressed in four mango varieties used in this study. Higher/lower accumulation of tocopherol (mainly α-tocopherol) was achieved by heterologous expression of MiHPPD in Arabidopsis and tomato. The results suggest that tocopherol accumulation in mango fruit is correlated to MiHPPD gene expression. Over-expression of MiHPPD gene channelizes the flux towards tocophreol biosynthesis and could be used as a potential tool for metabolic engineering.

Heterologous expression of two GPATs from Jatropha curcas alters seed oil levels in transgenic Arabidopsis thaliana.

Oils and fats are stored in endosperm during seed development in the form of triacylglycerols. Three acyltransferases: glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidyl acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) are involved in the storage lipid biosynthesis and catalyze the stepwise acylation of glycerol backbone. In this study two members of GPAT gene family (JcGPAT1 and JcGPAT2) from Jatropha seeds were identified and characterized. Sequence analysis suggested that JcGPAT1 and JcGPAT2 are homologous to Arabidopsis acyltransferase-1 (ATS1) and AtGPAT9 respectively. The sub-cellular localization studies of these two GPATs showed that JcGPAT1 localizes into plastid whereas JcGPAT2 localizes in to endoplasmic reticulum. JcGPAT1 and JcGPAT2 expressed throughout the seed development with higher expression in fully matured seed compared to immature seed. The transcript levels of JcGPAT2 were higher in comparison to JcGPAT1 in different developmental stages of seed. Over-expression of JcGPAT1 and JcGPAT2 under constitutive and seed specific promoters in Arabidopsis thaliana increased total oil content. Transgenic seeds of JcGPAT2-OE lines accumulated 43-60% more oil than control seeds whereas seeds of Arabidopsis lines over-expressing plastidial GPAT lead to only 13-20% increase in oil content. Functional characterization of GPAT homologues of Jatropha in Arabidopsis suggested that these are involved in oil biosynthesis but might have specific roles in Jatropha.

Comparative transcriptome analysis of unripe and mid-ripe fruit of Mangifera indica (var. "Dashehari") unravels ripening associated genes.

Ripening in mango is under a complex control of ethylene. In an effort to understand the complex spatio-temporal control of ripening we have made use of a popular N. Indian variety "Dashehari" This variety ripens from the stone inside towards the peel outside and forms jelly in the pulp in ripe fruits. Through a combination of 454 and Illumina sequencing, a transcriptomic analysis of gene expression from unripe and midripe stages have been performed in triplicates. Overall 74,312 unique transcripts with ≥1 FPKM were obtained. The transcripts related to 127 pathways were identified in "Dashehari" mango transcriptome by the KEGG analysis. These pathways ranged from detoxification, ethylene biosynthesis, carbon metabolism and aromatic amino acid degradation. The transcriptome study reveals differences not only in expression of softening associated genes but also those that govern ethylene biosynthesis and other nutritional characteristics. This study could help to develop ripening related markers for selective breeding to reduce the problems of excess jelly formation during softening in the "Dashehari" variety.

Expression of GhNAC2 from G. herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis.

NAC proteins are plant-specific transcription factors that play essential roles in regulating development and responses to abiotic and biotic stresses. We show that over-expression of the cotton GhNAC2 under the CaMV35S promoter increases root growth in both Arabidopsis and cotton under unstressed conditions. Transgenic Arabidopsis plants also show improved root growth in presence of mannitol and NaCl while transgenic cotton expressing GhNAC2 show reduced leaf abscission and wilting upon water stress compared to control plants. Transgenic Arabidopsis plants also have larger leaves, higher seed number and size under well watered conditions, reduced transpiration and higher relative leaf water content. Micro-array analysis of transgenic plants over-expressing GhNAC2 reveals activation of the ABA/JA pathways and a suppression of the ethylene pathway at several levels to reduce expression of ERF6/ERF1/WRKY33/ MPK3/MKK9/ACS6 and their targets. This probably suppresses the ethylene-mediated inhibition of organ expansion, leading to larger leaves, better root growth and higher yields under unstressed conditions. Suppression of the ethylene pathway and activation of the ABA/JA pathways also primes the plant for improved stress tolerance by reduction in transpiration, greater stomatal control and suppression of growth retarding factors.

Over-expression of JcDGAT1 from Jatropha curcas increases seed oil levels and alters oil quality in transgenic Arabidopsis thaliana.

The increasing consumption of fossil fuels and petroleum products is leading to their rapid depletion and is a matter of concern around the globe. Substitutes of fossil fuels are required to sustain the pace of economic development. In this context, oil from the non food crops (biofuel) has shown potential to substitute fossil fuels. Jatropha curcas is an excellent shrub spread and naturalized across the globe. Its oil contains a high percentage of unsaturated fatty acids (about 78-84% of total fatty acid content) making the oil suitable for biodiesel production. Despite its high oil content, it has been poorly studied in terms of important enzymes/genes responsible for oil biosynthesis. Here, we describe the isolation of the full length cDNA clone of JcDGAT1, a key enzyme involved in oil biosynthesis, from J. curcas seeds and manipulation of oil content and composition in transgenic Arabidopsis plants by its expression. Transcript analysis of JcDGAT1 reveals a gradual increase from early seed development to its maturation. Homozygous transgenic Arabidopsis lines expressing JcDGAT1 both under CaMV35S promoter and a seed specific promoter show an enhanced level of total oil content (up by 30-41%) in seeds but do not show any phenotypic differences. In addition, our studies also show alterations in the oil composition through JcDGAT1 expression. While the levels of saturated FAs such as palmitate and stearate in the oil do not change, there is significant reproducible decrease in the levels of oleic acid and a concomitant increase in levels of linolenic acid both under the CaMV35S promoter as well as the seed specific promoter. Our studies thus confirm that DGAT is involved in flux control in oil biosynthesis and show that JcDGAT1 could be used specifically to manipulate and improve oil content and composition in plants.

Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango.

Mango is characterized by high tocopherol and carotenoid content during ripening. From a cDNA screen of differentially expressing genes during mango ripening, a full-length p-hydroxyphenylpyruvate dioxygenase (MiHPPD) gene homologue was isolated that encodes a key enzyme in the biosynthesis of tocopherols. The gene encoded a 432-amino-acid protein. Transcript analysis during different stages of ripening revealed that the gene is ripening related and rapidly induced by ethylene. The increase in MiHPPD transcript accumulation was followed by an increase in tocopherol levels during ripening. The ripening-related increase in MiHPPD expression was also seen in response to abscisic acid and to alesser extent to indole-3-acetic acid. The expression of MiHPPD was not restricted to fruits but was also seen in other tissues such as leaves particularly during senescence. The strong ethylene induction of MiHPPD was also seen in young leaves indicating that ethylene induction of MiHPPD is tissue independent. Promoter analysis of MiHPPD gene in tomato discs and leaves of stable transgenic lines of Arabidopsis showed that the cis elements for ripening-related, ethylene-responsive, and senescence-related expression resided within the 1590 nt region upstream of the ATG codon. Functionality of the gene was demonstrated by the ability of the expressed protein in bacteria to convert p-hydroxyphenylpyruvate to homogentisate. These results provide the first evidence for HPPD expression during ripening of a climacteric fruit.

Differential expression of the mango alcohol dehydrogenase gene family during ripening.

Alcohol dehydrogenases play an important role during fruit ripening and aroma production. Three full-length cDNAs (MiAdh1, 2 and 3) encoding alcohol dehydrogenases were obtained from mango fruit pulp using RT-PCR approaches. All three members displayed strong homology in the coding region when compared at the protein and nucleotide levels, however showed variations in untranslated regions. Expression patterns of these ADHs were different during fruit development and ripening. MiADH1 and MiADH2 transcripts accumulated at the onset of ripening in mango fruit whereas MiADH3 accumulated during early development of fruit. Expression analysis also indicated that mango ADHs were responsive to ethylene but regulated differently by ABA. MiADH1 was induced by ABA treatment whereas MiADH2 transcript was negatively regulated by ABA. MiADH3 did not respond to ABA in ripening fruit. Differences in substrate specificity for NADH and NADPH were also observed between the three enzymes. Total ADH enzyme activity correlated positively with increased transcript levels at the initiation of ripening.