Browsing by Author "Ranjan, Aashish"

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A cytokinin-auxin antagonistic module participates in nitrogen-triggered tiller outgrowth in rice
(Oxford University Press, 2026) Chatterjee, Sourav; Dwivedi, Aditi; Sarkar, Ananda K; Ranjan, Aashish
Tillering is a key trait that shapes rice (Oryza sativa L.) shoot architecture and directly influences yield. While tiller bud formation is largely genetically determined, bud outgrowth into functional tillers is highly responsive to environmental cues. However, integration of environmental signals with genetic regulators to determine tiller bud fate remains poorly understood. Here, we investigated the effects of nitrogen on early stages of tiller bud outgrowth. Comprehensive phenotyping and temporal transcriptomic analyses demonstrated that both nitrate and ammonium promote bud outgrowth and elicit overlapping transcriptional responses, with nitrate acting more slowly. Gene regulatory network analysis identified phytohormone signaling as a key interface for nitrogen- triggered tiller outgrowth. Pharmacological and molecular experiments demonstrated the involvement of cytokinin–auxin antagonism in nitrogen-mediated tillering. Cytokinin promoted bud activation by repressing the critical bud dormancy regulators rice TEOSINTE BRANCHED 1 (OsTB1) and a homolog of PIN-FORMED 1 (OsPIN1a) through the Cytokinin Response Factors OsERF53/54. In contrast, auxin maintained dormancy by inducing OsTB1 and OsPIN1a expression through Auxin Response Factors OsARF11/16. Consistently, OsTB1 overexpression lines showed reduced responsiveness to nitrogen and hormone treatments, placing OsTB1 downstream of these convergent inputs. Sequence and gene expression differences in OsERF53/54, along with phenotypic variations across contrasting rice accessions, further substantiated the crucial roles of OsERF53/54 in nitrogen-mediated tillering. Together, we identify a key regulatory role of the cytokinin-auxin antagonistic module for integrating nitrogen signals to determine tiller bud fate. Adequate nitrogen promotes cytokinin signaling while attenuating auxin signaling and transport in tiller buds, thereby releasing dormancy and initiating bud outgrowth.
Brassinosteroids-regulated nitrogen metabolism fine-tunes growth physiology and low nitrogen response in tomato
(Elsevier B.V., 2023) Yadav, Ritesh Kumar; Analin, Benedict; Panda, Mahesh Kumar; Ranjan, Aashish; Singh, Amar Pal
Nitrogen (N) is a crucial nutrient for plants and its limited availability in the soils significantly affects plant growth and development. To adapt under low N condition, plants undergo various changes such as root system reprogramming to explore deeper soil horizons and metabolic activity adjustment. These N dependent responses and the genetic factors governing them are poorly known in crop plants. In this study, we investigated the effect of BRs on N metabolism in tomato. BRs application improved N assimilation and metabolic responses. By using the transgenic approach, we demonstrated the essential role of tomato Brassinazole resistant (BES1/BZR1) homolog 4 (BEH4) protein in regulating N metabolic response, growth physiology, and fruit quality. Overexpression of BEH4 promoted deeper root system architecture and improved physiological performance by adjusting N metabolic activity and photosynthetic efficiency in low N-grown plants. The BEH4 transgenic lines exhibited increased expression of genes involved in N uptake and assimilation which are associated with the improved N content and assimilation (root and shoot). Altogether, data suggested an essential role of BRs in plant adaptation to altered N regimes and appears potential target for genetic manipulation to improve nitrogen use efficiency (NUE) and nutritional quality in crops.
Decoding the gene coexpression network underlying the ability of Gevuina avellana to live in diverse light conditions
(John Wiley & Sons, 2018) Ostria-Gallardo, Enrique; Ranjan, Aashish; Ichihashi, Yasunori; Corcuera, Luis J.; Sinha, Neelima R.
Gevuina avellana (Proteaceae) is a typical tree from the South American temperate rainforest. Although this species mostly regenerates in shaded understories, it exhibits an exceptional ecological breadth, being able to live under a wide range of light conditions. Here we studied the genetic basis that underlies physiological acclimation of the photosynthetic responses of G. avellana under contrasting light conditions. We analyzed carbon assimilation and light energy used for photochemical processes in plants acclimated to contrasting light conditions. Also, we used a transcriptional profile of leaf primordia from G. avellana saplings growing under different light environments in their natural habitat, to identify the gene coexpression network underpinning photosynthetic performance and light-related processes. The photosynthetic parameters revealed optimal performance regardless of light conditions. Strikingly, the mechanism involved in dissipation of excess light energy showed no significant differences between high- and low-light-acclimated plants. The gene coexpression network defined a community structure consistent with the photochemical responses, including genes involved mainly in assembly and functioning of photosystems, photoprotection, and retrograde signaling. This ecophysiological genomics approach improves our understanding of the intraspecific variability that allows G. avellana to have optimal photochemical and photoprotective mechanisms in the diverse light habitats it encounters in nature.
Drought stress exacerbates fungal colonization and endodermal invasion and dampens defense responses to increase dry root rot in chickpea
(American Phytopathological Society, 2022) Irulappan, Vadivelmurugan; Kandpal, Manu; Saini, Kumud; Rai, Avanish; Ranjan, Aashish; Sinharoy, Senjuti; Senthil-Kumar, Muthappa
Drought plays a central role in increasing the incidence and severity of dry root rot (DRR) disease in chickpea. This is an economically devastating disease, compromising chickpea yields particularly severely in recent years due to erratic rainfall patterns. Macrophomina phaseolina (formerly Rhizoctonia bataticola) is the causal agent of DRR disease in the chickpea plant. The infection pattern in chickpea roots under well-watered conditions and drought stress are poorly understood at present. This study provides detailed disease symptomatology and the characteristics of DRR fungus at morphological and molecular levels. Using microscopy techniques, the infection pattern of DRR fungus in susceptible chickpea roots was investigated under well-watered and drought stress conditions. Our observations suggested that drought stress intensifies the progression of already ongoing infection by weakening the endodermal barrier and overall defense. Transcriptomic analysis suggested that the plant’s innate immune defense program is downregulated in infected roots when subjected to drought stress. Further, genes involved in hormonal regulation are differentially expressed under drought stress. These findings provide hints in terms of potential chickpea genes to target in crop improvement programs to develop climate change-resilient cultivars.
Enhancing crop yield by optimizing plant developmental features
(The Company of Biologists, 2016) Mathan, Jyotirmaya; Bhattacharya, Juhi; Ranjan, Aashish
A number of plant features and traits, such as overall plant architecture, leaf structure and morphological features, vascular architecture and flowering time are important determinants of photosynthetic efficiency and hence the overall performance of crop plants. The optimization of such developmental traits thus has great potential to increase biomass and crop yield. Here, we provide a comprehensive review of these developmental traits in crop plants, summarizing their genetic regulation and highlighting the potential of manipulating these traits for crop improvement. We also briefly review the effects of domestication on the developmental features of crop plants. Finally, we discuss the potential of functional genomics-based approaches to optimize plant developmental traits to increase yield.
eQTL regulating transcript levels associated with diverse biological processes in tomato
(American Society of Plant Biologists, 2016) Ranjan, Aashish; Budke, Jessica; Rowland, Steven D.; Chitwood, Daniel H; Kumar, Ravi; Carriedo, Leonela G.; Ichihashi, Yasunori; Zumstein, Kristina; Maloof, Julin N.; Sinha, Neelima R.
Variation in gene expression, in addition to sequence polymorphisms, is known to influence developmental, physiological and metabolic traits in plants. Genetic mapping populations have facilitated identification of expression Quantitative Trait Loci (eQTL), the genetic determinants of variation in gene expression patterns. We used an introgression population developed from the wild desert-adapted Solanum pennellii and domesticated tomato Solanum lycopersicum to identify the genetic basis of transcript level variation. We established the effect of each introgression on the transcriptome, and identified ~7,200 eQTL regulating the steady state transcript levels of 5,300 genes. Barnes-Hut t-distributed stochastic neighbor embedding clustering identified 42 modules revealing novel associations between transcript level patterns and biological processes. The results showed a complex genetic architecture of global transcript abundance pattern in tomato. Several genetic hotspots regulating a large number of transcript level patterns relating to diverse biological processes such as plant defense and photosynthesis were identified. Important eQTL regulating transcript level patterns were related to leaf number and complexity, and hypocotyl length. Genes associated with leaf development showed an inverse correlation with photosynthetic gene expression but eQTL regulating genes associated with leaf development and photosynthesis were dispersed across the genome. This comprehensive expression QTL analysis details the influence of these loci on plant phenotypes, and will be a valuable community resource for investigations on the genetic effects of eQTL on phenotypic traits in tomato.
Genetic diversity of farmer-preferred cassava landraces in Tanzania based on morphological descriptors and single nucleotide polymorphisms
(Cambridge University Press, 2017) Mtunguja, M. K.; Ranjan, Aashish; Laswai, Laswai; Muzanila, Y.; Ndunguru, J.; Sinha, N. R.
Cassava germplasm collection is important for the preservation of genetic variability, allowing the development of improved cultivars with desirable traits such as drought and disease tolerance, better starch quality and yield. Therefore, the assessment of diversity in cassava germplasm maintained by farmers is important for maintaining biodiversity and crop improvement. Herein, we report genetic diversity relationships of 52 farmer-preferred cassava landraces from the eastern zone of Tanzania based on morphological descriptors and single nucleotide polymorphisms (SNPs). Cluster analysis was performed for both morphological traits (genetic distance 1.18–0.15) and SNPs (genetic distance 0.078–0.002). The analysis revealed that there were a total of 17,393 variant positions, and that several of the SNPs were distributed across all the chromosomes. The abundance of SNP varied remarkably among the 18 cassava chromosomes, with chromosome 2 having the highest number of SNPs (1335) and chromosome 18 having the lowest number of SNPs (734). The power of SNPs in distinguishing morphologically similar landraces was shown. Both analyses did not group landraces according to geographical locations, suggesting that farmers were moving cassava germplasm to different areas. Their diversity was mainly due to adaptation and preferential selection by farmers. This further implied that within a geographical location, the cultivars were more diverse and there was no misnaming of cassava cultivars by farmers. The collection revealed a wide range of genetic diversity, and represented a valuable resource for trait improvement, allowing the capture of farmer-preferred traits in future cassava breeding programmes.
Genome sequencing of Mesorhizobium Spp. NI-7, an efficient nitrogen-fixing microsymbiont of chickpea with potential to unravel the molecular mechanisms of symbiotic nitrogen fixation in legumes
(Springer Nature Publishing AG, 2024) Srivastava, Deevita; Ghosh, Asim K; Ranjan, Aashish; Sinharoy, Senjuti
Root nodule symbiosis is a major pillar of sustainable agriculture. The newly formed symbiotic organ in the legume root harbours rhizobacteria, which can fix atmospheric nitrogen into a bioavailable and reduced form, ammonia. Previously, we reported the isolation of an efficient Mesorhizobium spp. NI-7, from the interior of chickpea nodules. Here, we report the draft genome sequence of the Mesorhizobium spp. NI-7 and the comparative genomics among different Mesorhizobium strains that have adopted symbiosis during chickpea domestication. The draft genome of Mesorhizobium spp. NI-7 consists of a single 4.28 Mbp chromosome and a 359 Kbp plasmid. The 16 S rDNA sequence based phylogenetic analysis highlighted that Mesorhizobium spp. NI-7 belongs to a diverse Mesorhizobium clade that evolved during the domestication of chickpea. Comparative genomics among several Mesorhizobium strains identified 2193 common orthologous groups and several unique orthologous groups among the different Mesorhizobium pairs. The draft genome contains the essential nitrogen fixation genes along with the genes required for the nutrient exchange from the plant to the symbiont. Additionally, part of the symbiotic NOD-factor operon and Type III secretion system were also detected in the Mesorhizobium spp. NI-7 draft genome. The comparative genomics among the Mesorhizobium strains identified a subset of rhizobial genes that would have evolved during chickpea-Mesorhizobium adaptation to the Indian sub-continent. These genes are unique targets that can be validated in the future to understand the chickpea and Mesorhizobium adaptation. In summary, the draft genome sequencing of Mesorhizobium spp. NI-7 will equip the plant-microbe community with a chickpea-compatible Mesorhizobium strain isolated from India, suitable for both fundamental and advanced research on nodulation in chickpea, as well as for promoting sustainable agriculture in a comprehensive manner.
Global transcriptome and co-expression analysis reveals robust host defence pathway reprogramming and identifies key regulators of early phases of Cicer-Ascochyta interactions
(American Phytopathological Society, 2022) Singh, Ritu; Dwivedi, Aditi; Singh, Yeshveer; Kumar, Kamal; Ranjan, Aashish; Verma, Praveen K.
Ascochyta blight (AB) caused by a filamentous fungus Ascochyta rabiei is a major threat to global chickpea production. The mechanisms underlying chickpea response to A. rabiei remain elusive. Here, we investigated the comparative transcriptional dynamics of AB-resistant and susceptible chickpea genotypes upon A. rabiei infection to understand the early host defence response. Our findings revealed that AB-resistant plants underwent rapid and extensive transcriptional reprogramming compared to susceptible host. At early stage (24-hpi), mainly cell wall remodeling and secondary metabolite pathways were highly activated, while DEGs related with signaling components viz. protein kinases, transcription factors, and hormonal pathways show remarkable upsurge at 72-hpi, especially in resistant genotype. Notably, our data suggests imperative role of JA, ET, and ABA signaling in providing immunity against A. rabiei. Furthermore, gene co-expression networks and modules corroborated the importance of cell wall remodeling, signal transduction, and phytohormone pathways. The hub genes such as MYB14, PRE6, and MADS-SOC1 discovered in these modules might be the master regulators governing chickpea immunity. Overall, we not only provide novel insights for comprehensive understanding of immune signaling components mediating AB resistance/susceptibility at early Cicer-Ascochyta interactions, but also offer a valuable resource for developing AB-resistant chickpea.
High photosynthesis rate in two wild rice species is driven by leaf anatomy mediating high Rubisco activity and electron transport rate
(Oxford University Press, 2021) Mathan, Jyotirmaya; Singh, Anuradha; Jathar, Vikram; Ranjan, Aashish
The importance of increasing photosynthetic efficiency for sustainable crop yield increases to feed the growing world population is well recognized. The natural genetic variation for leaf photosynthesis in crop plants is largely unexploited for increasing genetic yield potential. The genus Oryza, including cultivated rice and wild relatives, offers tremendous genetic variability to explore photosynthetic differences, and underlying biochemical, photochemical, and developmental bases. We quantified leaf photosynthesis and related physiological parameters for six cultivated and three wild rice genotypes, and identified photosynthetically efficient wild rice accessions. Fitting A/Ci curves and biochemical analyses showed that the leaf photosynthesis in cultivated rice varieties, IR64 and Nipponbare, was limited due to leaf nitrogen content, Rubisco activity, and electron transport rate compared to photosynthetically efficient accessions of wild rice Oryza australiensis and Oryza latifolia. The selected wild rice accessions with high leaf photosynthesis per unit area had striking anatomical features, such as larger mesophyll cells with more chloroplasts, fewer mesophyll cells between two consecutive veins, and higher mesophyll cell and chloroplast surface area exposed to intercellular space. Our results show the existence of desirable variations in Rubisco activity, electron transport rate, and leaf anatomical features in the rice system itself that could be targeted for increasing the photosynthetic efficiency of cultivated rice varieties.
High temperature restricts cell division and leaf size by coordination of PIF4 and TCP4 transcription factors
(Oxford University Press, 2022) Saini, Kumud; Dwivedi, Aditi; Ranjan, Aashish
High ambient temperature suppresses Arabidopsis (Arabidopsis thaliana) rosette leaf area and elongates the stem and petiole. While the mechanism underlying the temperature-induced elongation response has been extensively studied, the genetic basis of temperature regulation of leaf size is largely unknown. Here, we show that warm temperature inhibits cell proliferation in Arabidopsis leaves, resulting in fewer cells compared to the control condition. Cellular phenotyping and genetic and biochemical analyses established the key roles of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and TEOSINTE BRANCHED1/CYCLOIDEA/PCF4 (TCP4) transcription factors in the suppression of Arabidopsis leaf area under high temperature by a reduction in cell number. We show that temperature-mediated suppression of cell proliferation requires PIF4, which interacts with TCP4 and regulates the expression of the cell cycle inhibitor KIP-RELATED PROTEIN1 (KRP1) to control leaf size under high temperature. Warm temperature induces binding of both PIF4 and TCP4 to the KRP1 promoter. PIF4 binding to KRP1 under high temperature is TCP4 dependent as TCP4 regulates PIF4 transcript levels under high temperature. We propose a model where a warm temperature-mediated accumulation of PIF4 in leaf cells promotes its binding to the KRP1 promoter in a TCP4-dependent way to regulate cell production and leaf size. Our finding of high-temperature-mediated transcriptional upregulation of KRP1 integrates a developmental signal with an environmental signal that converges on a basal cell regulatory process.
Identification of an RNA silencing suppressor encoded by an Indian citrus ringspot virus
(Springer Nature Publishing AG, 2025) Angira, Aniket; Baranwal, V. K.; Ranjan, Aashish; Choudhary, Nandlal
Plant viruses encode RNA silencing suppressor (RSS) proteins to counter the induced antiviral defense, an RNAi silencing mechanism of the host. Indian citrus ringspot virus (ICRSV) causes the ringspot disease, which leads to signifcant yield loss of kinnow orange. The ICRSV genome contains six open reading frames (ORFs), however, the ORF encoding the potential RSS is not yet known. In this study, we have attempted to identify the RSS protein of ICRSV. To this end, ORF 2,3,4,5 and 6 were cloned into pCAMBIA1302 (35s-GFP) vector, followed by transformation of Agrobacterium tumefaciens and agro-infltration into leaves of Nicotiana benthamiana 16c line. Only the leaves infltrated with 35s-GFP/ORF5 showed a GFP fuorescence signal similar to 35s-GFP/P19, a well-studied positive RSS. Usually, the induced host RNAi silencing is supposed to cleave the expressed GFP-RNA. However, it is suspected that ORF5-encoded protein was able to suppress the host silencing mechanism, leading to the retention of the GFP fuorescence signal. This fnding was further supported by beta-glucuronidase (GUS) histochemical assays by infltrating the construct expressing ORF5-GUS under 35s promoter in the leaves of N. benthamiana. Leaves infltrated with 35s-GUS/ORF5 formed diX-indigo precipitate similar to leaves infltrated with, indicating the RSS activity of ICRSV. Later, semi-quantitative PCR and quantitative reverse transcription PCR (qRT-PCR) assays showed a higher expression of GFP and GUS in ORF5 agro-infltrated leaves. Together, these results suggest that ORF5 encoded protein has the potential RSS function of ICRSV which successfully suppresses host RNAi silencing mechanism.
In-silico prediction of coat protein structure of Indian citrus ringspot virus and their interactions with the Argonaut2/DCL4 proteins
(Springer Nature Publishing AG, 2025) Angira, Aniket; Yadav, Siddharth; Mathur, Puniti; Baranwal, V. K.; Ranjan, Aashish; Choudhary, Nandlal
The RNA silencing mechanism is a crucial regulatory system in plants, particularly in antiviral defense. However, most of the plant viruses encode a specifc protein called RNA silencing suppressor protein that suppress the RNA silencing mechanism of host. This study employs the bioinformatics tools, including SWISS homology model and I-TASSER, to predict the coat protein (CP) tertiary structure of Indian citrus ringspot virus (ICRSV). Then, fve protein–protein docking servers (GRAMM, pyDockWEB, HawkDock, ZDOCK and ClusPro) were utilized to investigate interactions of CP of ICRSV with Argonaut2/Dicer-Like (DCL4) protein 4 of RNA silencing pathway of host. In blind docking experiments, the CP consistently engaged in docking interactions with DCL4, while with AGO2, it interacted near the PIWI and MID domains. The AGO2-CP cluster demonstrated 4 salt bridges, 30 hydrogen bonds, and 328 non-bonded contacts, with interface areas spanning 2529 in AGO2 and 2424 in CP, involving 50 and 51 interface residues, respectively. Similarly, the DCL4-CP cluster showed 5 hydrogen bonds and 122 non-bonded contacts, with interface areas spanning 965 in DCL4 and 987 in CP, involving 16 and 19 interface residues, respectively. The established phenomenon of CP interaction with AGO2/DCL4, may resulting in the inhibition of the RNA silencing mechanism and shedding light on the suppression mechanisms of host defense responses.
Induced post-invasive defenses in the nonhost plant Parthenium hysterophorus L. prevent root cortical colonization by Macrophomina phaseolina and impart resistance to dry root rot
(Elsevier B.V., 2025) Mirchandani, Rishabh; Kandpal, Manu; Ranjan, Aashish; Sinharoy, Senjuti; Senthil-Kumar, Muthappa
Dry root rot (DRR) of chickpea is caused by the broad-range necrotrophic fungus Macrophomina phaseolina. Chickpea germplasm does not provide durable resistance to DRR, which is particularly devastating under drought. Even moderately resistant chickpea varieties become susceptible under combined stress. We hypothesized that nonhost resistance (NHR) is durable even under combined stress. Using the blotter paper assay and stereomicroscopic observations, we identified the asterid weed Parthenium hysterophorus as a potential nonhost of M. phaseolina among 82 potential nonhosts. Epidermal necrotic lesions were prevented in P. hysterophorus. In planta fungal load was 0.195 and 0.007 ng/ng total DNA in chickpea and P. hysterophorus, respectively. M. phaseolina could not colonize the P. hysterophorus root while up to 6 cortical cell layers were colonized in chickpea. Further, NHR was durable under combined stress. Dual RNA sequencing revealed that M. phaseolina actively attempted to infect the nonhost and activated specific genes in the xenobiotics degradation pathway. P. hysterophorus also showed an active defense response with1958 and 2294 differentially expressed genes at 2 and 4 DAI, respectively, with 363 upregulated at both time points. Differential expression of cell wall synthesis, phytohormone signaling, and other defense response pathways likely contributes to NHR. Few genes in the phenylpropanoid biosynthesis pathways in P. hysterophorus were also upregulated, possibly because these metabolites are linked to the distinct changes in the fungus during nonhost infection. We therefore conclude that P. hysterophorus exhibits post-invasive NHR to M. phaseolina and that general defense, phytohormone signaling and secondary metabolic pathways contribute to NHR.
Integration of metabolite and transcriptome profiles of cultivated and wild rice to unveil gene regulatory networks and key genes determining rice source and sink strength
(Springer Nature Publishing AG, 2025) Singh, Anuradha; Mathan, Jyotirmaya; Dwivedi, Aditi; Rani, Ruchi; Ranjan, Aashish
Targeting source and sink strength for crop yield increase requires a comprehensive genetic and metabolic understanding of desirable source and sink features. We performed comprehensive metabolite and transcriptomic comparisons of the photosynthetic flag leaves and milky-stage developing grains of two cultivated rice varieties (Oryza sativa L. ssp. Indica cv. IR64 and Oryza sativa L. ssp. Japonica cv. Nipponbare) and two wild rice accessions (Oryza rufipogon and Oryza australiensis). The selected wild rice accessions had stronger source strength as evidenced by a higher photosynthesis rate and more abundance of primary metabolites in the photosynthetic leaves than the cultivated varieties. In contrast, cultivated varieties had efficient sink as grains were bigger and accumulated more sugars, amino acids, and fatty acids than the selected wild rice. Transcriptomic analyses identified 9,309 genes for efficient source in wild rice, enriched for biological pathways related to photosynthesis, carbohydrate metabolism, and sucrose transport. 7,062 genes, enriched for starch biosynthesis and lipid metabolism, were associated with the efficient sink strength in the cultivated varieties. Gene co-expression networks showed 267 hub genes for source strength in wild rice that included important genes for photosynthetic reactions and sucrose metabolism. 196 hub genes for sink strength in cultivated rice included genes involved in sucrose, amino acid, and fatty acid metabolism. Gene co-expression modules further identified the candidate transcription regulators, such as zinc finger proteins and NAC for source strength and MYB55/80 and MADS64 for sink strength. Moreover, our analyses suggested a complex interplay of phytohormones regulating rice source and sink strength.
Isolation, characterization, and expression analysis of NAC transcription factor from Andrographis paniculata (Burm. f.) nees and their role in andrographolide production
(MDPI AG, 2024) Kumar, Ramesh; Kumar, Chavlesh; Choudhury, Debjani Roy; Ranjan, Aashish; Raipuria, Ritesh Kumar; Dubey, Kaushik Kumar Dhar; Mishra, Ayushi; Kumar, Chetan; Manzoor, Malik Muzafar; Kumar, Ashok; Kumari, Abha; Singh, Kuldeep; Singh, Gyanendra Pratap; Singh, Rakesh
Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes (ApNAC83, ApNAC21 22 and ApNAC02) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant’s leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83. The transient expression of the isolated NAC genes in a heterologous system (Nicotiana benthamiana) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis.
Leaf growth in third dimension: a perspective of leaf thickness from genetic regulation to ecophysiology
(John Wiley & Sons, 2025) Aneja, Prakshi; Sanyal, Rajarshi; Ranjan, Aashish
Leaf thickness, the leaf growth in the third dimension as quantified by the distance between the adaxial and abaxial surface, is an indispensable aspect of leaf development. The fitness of a plant is strongly influenced by leaf thickness via modulation of major physiological processes, including photosynthesis and water use efficiency. The cellular basis of leaf thickness by alterations in either cell size or the number of cell layers is envisaged using Arabidopsis leaf thickness mutants, such as angustifolia (an) and rotundifolia (rot). Environmental factors coordinate with endogenous signaling mechanisms to exhibit leaf thickness plasticity. Plants growing in different ecological and environmental regimes show different leaf thickness attributes. However, genetic and molecular understandings of leaf thickness regulation remain largely limited. In this review, we highlight how cellular growth is transposed to fine-tune the leaf thickness via the integration of potential cues and molecular players. We further discuss the physiological significance of leaf thickness plasticity to the environmental cues that might serve as ecological adaptation enabling the plants to withstand future climatic conditions. Taken together, we seek to bridge the genetics and molecular biology of leaf thickness to its physiological significance so that leaf thickness can be systemically targeted in crop improvement programs.
Light-induced indeterminacy alters shade avoiding tomato leaf morphology
(American Society of Plant Biologists, 2015) Chitwood, Daniel H.; Kumar, Ravi; Ranjan, Aashish; Pelletier, Julie M.; Townsley, Brad T.; Ichihashi, Yasunori; Martinez, Ciera C.; Zumstein, Kristina; Harada, John J.; Maloof, Julin N.; Sinha, Neelima R.
Plants sense the foliar shade of competitors and alter their developmental programs through the shade-avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of more than 18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato (Solanum lycopersicum) and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier. Observing that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia, shade avoidance is not mediated through canonical pathways described in mature organs but rather through the expression of KNOTTED1-LIKE HOMEOBOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from the prolonged production of juvenile leaf types.
A new advanced backcross tomato population enables high resolution leaf QTL mapping and gene identification
(Genetics Society of America, 2016) Fulop, Daniel; Ranjan, Aashish; Ofner, Itai; Covington, Michael F.; Chitwood, Daniel H.; West, Donelly; Ichihashi, Yasunori; Headland, Lauren; Zamir, Daniel; Maloof, Julin N.; Sinha, Neelima R.
Quantitative Trait Loci (QTL) mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum) and its more distant interfertile relatives typically follow a near isogenic line (NIL) design, such as the S. pennellii Introgression Line (IL) population, with a single wild introgression per line in an otherwise domesticated genetic background. Here, we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar and S. pennellii This so-called Backcrossed Inbred Line (BIL) population is comprised of a mix of BC2 and BC3 lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existing S. pennellii IL population, with which it shares parents. Using the BILs, we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous hidden Markov model for genotyping the lines, and by using state-of-the-art sparse regression techniques for QTL mapping.
Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India
(Elsevier B.V., 2024) Angira, Aniket; Baranwal, V K; Ranjan, Aashish; Choudhary, Nandlal
Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507bp partial coat protein gene (pCPG) segment was amplified from infected kinnow tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL-1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.