Transcriptome wide identification, characterization and expression analysis of PHD gene family in Crocus sativus.

Crocus sativus L., of the Iridaceae family, yields world's most prized spice, saffron. Saffron is well known for its distinctive aroma, odour and colour, which are imputed to the presence of some specific glycosylated apocarotenoids. Even though the main biosynthetic pathway and most of the enzymes leading to apocarotenoid production have been identified, the regulatory mechanisms that govern the developmental stage and tissue specific production of apocarotenoids in Crocus remain comparatively unravelled. Towards this, we report identification, and characterization of plant homeodomain (PHD) finger transcription factor family in Crocus sativus. We also report cloning and characterisation of CstPHD27 from C. sativus. CstPHD27 recorded highest expression in stigma throughout flower development. CstPHD27 exhibited expression pattern which corresponded to the apocarotenoid accumulation in Crocus stigmas. CstPHD27 is nuclear localized and transcriptionally active in yeast Y187 strain. Over-expression of CstPHD27 in Crocus stigmas enhanced apocarotenoid content by upregulating the biosynthetic pathway genes. This report on PHD finger transcription factor family from C. sativus may offer a basis for elucidating role of this gene family in this traditionally and industrially prized crop. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01410-3.

CstMYB1R1, a REVEILLE-8-like transcription factor, regulates diurnal clock-specific anthocyanin biosynthesis and response to abiotic stress in Crocus sativus L.

KEY MESSAGE: CstMYB1R1 acts as a positive regulator of Crocus anthocyanin biosynthesis and abiotic stress tolerance which was experimentally demonstrated through molecular analysis and over-expression studies in Crocus and Nicotiana. Regulatory mechanics of flavonoid/anthocyanin biosynthesis in Crocus floral tissues along the diurnal clock has not been studied to date. MYB proteins represent the most dominant, functionally diverse and versatile type of plant transcription factors which regulate key metabolic and physiological processes in planta. Transcriptome analysis revealed that MYB family is the most dominant transcription factor family in C. sativus. Considering this, a MYB-related REVEILLE-8 type transcription factor, CstMYB1R1, was explored for its possible role in regulating Crocus flavonoid and anthocyanin biosynthetic pathway. CstMYB1R1 was highly expressed in Crocus floral tissues, particularly tepals and its expression was shown to peak at dawn and dusk time points. Anthocyanin accumulation also peaked at dawn and dusk and was minimum at night. Moreover, the diurnal expression pattern of CstMYB1R1 was shown to highly correlate with Crocus ANS/LDOX gene expression among the late anthocyanin pathway genes. CstMYB1R1 was shown to be nuclear localized and transcriptionally active. CstMYB1R1 over-expression in Crocus tepals enhanced anthocyanin levels and upregulated transcripts of Crocus flavonoid and anthocyanin biosynthetic pathway genes. Yeast one hybrid (Y1H) and GUS reporter assay confirmed that CstMYB1R1 interacts with the promoter of Crocus LDOX gene to directly regulate its transcription. In addition, the expression of CstMYB1R1 in Nicotiana plants significantly enhanced flavonoid and anthocyanin levels and improved their abiotic stress tolerance. The present study, thus, confirmed positive role of CstMYB1R1 in regulating Crocus anthocyanin biosynthetic pathway in a diurnal clock-specific fashion together with its involvement in the regulation of abiotic stress response.

CstPIF4 Integrates Temperature and Circadian Signals and Interacts with CstMYB16 to Repress Anthocyanins in Crocus.

Crocus sativus has emerged as an important crop because it is the only commercial source of saffron that contains unique apocarotenoids. Saffron is composed of dried stigmas of Crocus flower and constitutes the most priced spice of the world. Crocus floral organs are dominated by different classes of metabolites. While stigmas are characterized by the presence of apocarotenoids, tepals are rich in flavonoids and anthocyanins. Therefore, an intricate regulatory network might play a role in allowing different compounds to dominate in different organs. Work so far done on Crocus is focussed on apocarotenoid metabolism and its regulation. There are no reports describing the regulation of flavonoids and anthocyanins in Crocus tepals. In this context, we identified an R2R3 transcription factor, CstMYB16, which resembles subgroup 4 (SG4) repressors of Arabidopsis. CstMYB16 is nuclear localized and acts as a repressor. Overexpression of CstMYB16 in Crocus downregulated anthocyanin biosynthesis. The C2/EAR motif was responsible for the repressor activity of CstMYB16. CstMYB16 binds to the promoter of the anthocyanin biosynthetic pathway gene (LDOX) and reduces its expression. CstMYB16 also physically interacts with CstPIF4, which in turn is regulated by temperature and circadian clock. Thus, CstPIF4 integrates these signals and forms a repressor complex with CstMYB16, which is involved in the negative regulation of anthocyanin biosynthesis in Crocus. Independent of CstPIF4, CstMYB16 also represses CstPAP1 expression, which is a component of the MYB-bHLH-WD40 (MBW) complex and positively controls anthocyanin biosynthesis. This is the first report on identifying and describing regulators of anthocyanin biosynthesis in Crocus.

CstMYB14 links ROS signaling, apocarotenoid metabolism, and stress response in Crocus sativus L.

Reactive oxygen species (ROS) behave as signaling molecules and induce biosynthesis of many secondary metabolites, including apocarotenoids, which play critical roles in stress tolerance through radical scavenging. However, the mechanism that regulates ROS responsive apocarotenoid metabolism and subsequent stress response is unknown. In this study, an R2R3-MYB transcription factor (CstMYB14) was identified from Crocus sativus L., which acts as a regulator of apocarotenoid biosynthesis. CstMYB14 expression increases in response to H2 O2 in a concentration and time-dependent manner. CstMYB14 localizes to the nucleus and acts as a transcriptional activator. Over-expression of CstMYB14 in Crocus stigmas enhanced apocarotenoid biosynthesis. Yeast-one-hybrid demonstrated binding of CstMYB14 to promoters of two apocarotenoid pathway genes (phytoene synthase and carotenoid cleavage dioxygenase 2). Nicotiana benthamiana plants overexpressing CstMYB14 showed better growth and higher stress tolerance than wild type plants. Higher antioxidant activity in CstMYB14-Ox plants indicated that stress tolerance might be due to ROS scavenging. These results establish a molecular link between ROS signaling, apocarotenoid metabolism and stress tolerance. Further, CstMYB14 is shown to act as a key regulator which modulates ROS responsive biosynthesis of apocarotenoids which in turn impart stress tolerance through ROS scavenging.

Crocus transcription factors CstMYB1 and CstMYB1R2 modulate apocarotenoid metabolism by regulating carotenogenic genes.

KEY MESSAGE: Two MYB genes have been identified which regulate apocarotenoid metabolism in Crocus sativus. Apocarotenoids like crocin, picrocrocin and safranal are restricted to genus Crocus and are synthesized by oxidative cleavage of zeaxanthin followed by glycosylation reactions. In Crocus sativus, these apocarotenoids are synthesized in stigma part of the flower in developmentally regulated manner. Most of the genes of apocarotenoid pathway are known, however, the mechanism that regulates its tissue and stage specific biosynthesis remains elusive. MYB family was identified as the largest transcription factor family from Crocus transciptome which indicated its possible role in apocarotenoid regulation besides regulating other metabolic pathways. Towards this, we started with identification of 150 MYB genes from Crocus transcriptome databases. The phylogenetic analysis of Crocus MYB genes divided them into 27 clusters. Domain analysis resulted in identification of four groups of MYBs depending upon the number of R repeats present. Expression profiling indicated that 12 MYBs are upregulated in stigma out of which expression of four genes CstMYB1, CstMYB14, CstMYB16 and CstMYB1R2 correlated with crocin accumulation. Transient overexpression of two nuclear localized MYB genes (CstMYB1 and CstMYB1R2) in Crocus confirmed their role in regulating carotenoid metabolism. Yeast-one-hybrid confirmed that CstMYB1 binds to carotenoid cleavage dioxygenase 2 (CCD2) promoter while CstMYB1R2 binds to phytoene synthase (PSY) and CCD2 promoters. Overall, our study established that CstMYB1 and CstMYB1R2 regulate apocarotenoid biosynthesis by directly binding to promoters of pathway genes.

Functional characterization of flavonoid 3'-hydroxylase, CsF3'H, from Crocus sativus L: Insights into substrate specificity and role in abiotic stress.

Stress-responsive dihydroxy flavonoids exhibit capability to inhibit the accretion of reactive oxygen species (ROS). The formation of these dihydroxy flavonols is catalyzed by flavonoid hydroxylases which are among the rate limiting enzymes of flavonoid biosynthesis pathway. Although flavonoid hydroxylases have been identified in several plant species but their role in abiotic stress is not explicitly documented. In the present study we report identification of all the flavonoid biosynthesis pathway genes of Crocus sativus and their expression profiling. We also report functional characterization of flavonoid 3' hydroxylase (CsF3'H) and attempt to explore its physiological role in vitro and in planta. The results indicated that CsF3'H is 1608 bp long encoding 535 amino acids. Docking and enzyme kinetic studies revealed that CsF3'H catalyzes hydroxylation of naringenin and dihydrokaempferol to eriodictoyl and dihydroquercetin respectively, but exhibits higher affinity for naringenin. Further, CsF3'H showed comparatively higher expression in floral tissues particularly stigma and its expression was significantly enhanced in response to UV-B, dehydration and salinity stress indicative of its role in stress. The expression of CsF3'H was associated with concomitant accumulation of eriodictoyl and dihydroquercetin. Transient overexpression of CsF3'H in Nicotiana benthamiana leads to the accumulation of substantial amounts of eriodictoyl and dihydroquercetin. Further, it was observed that transient expression of CsF3'H conferred tolerance to UV-B and dehydration stress as was evident from higher chlorophyll and soluble sugar and lower MDA contents. Taken together, these results suggest that CsF3'H confers tolerance to UV-B and dehydration in planta through synthesis of dihydroflavonols.

Integrative network analyses of wilt transcriptome in chickpea reveal genotype dependent regulatory hubs in immunity and susceptibility.

Host specific resistance and non-host resistance are two plant immune responses to counter pathogen invasion. Gene network organizing principles leading to quantitative differences in resistant and susceptible host during host specific resistance are poorly understood. Vascular wilt caused by root pathogen Fusarium species is complex and governed by host specific resistance in crop plants, including chickpea. Here, we temporally profiled two contrasting chickpea genotypes in disease and immune state to better understand gene expression switches in host specific resistance. Integrative gene-regulatory network elucidated tangible insight into interaction coordinators leading to pathway determination governing distinct (disease or immune) phenotypes. Global network analysis identified five major hubs with 389 co-regulated genes. Functional enrichment revealed immunome containing three subnetworks involving CTI, PTI and ETI and wilt diseasome encompassing four subnetworks highlighting pathogen perception, penetration, colonization and disease establishment. These subnetworks likely represent key components that coordinate various biological processes favouring defence or disease. Furthermore, we identified core 76 disease/immunity related genes through subcellular analysis. Our regularized network with robust statistical assessment captured known and unexpected gene interaction, candidate novel regulators as future biomarkers and first time showed system-wide quantitative architecture corresponding to genotypic characteristics in wilt landscape.

Porostereum sp., Associated with Saffron (Crocus sativus L.), is a Latent Pathogen Capable of Producing Phytotoxic Chlorinated Aromatic Compounds.

Saffron (Crocus sativus L.) is one of the most expensive spices in the world due to its medicinal and aromatic value. However, saffron production is severely affected by the corm rot disease throughout the saffron producing countries. In this study, we report a basidiomycetous latent pathogen of saffron, designated as CSE26, capable of producing phytotoxic compounds. CSE26 is a highly odorous basidiomycete with monomitic hyphal system. Molecular phylogeny of ITS and 28S ribosomal gene sequence of CSE26 assigned it as Porostereum spadiceum. It was found to produce corm rot in C. sativus under in vivo and field conditions, with a disease severity index of 0.7 and 0.5, respectively. CSE26 was found to produce chlorinated aromatic compounds (CAMs) having phytotoxic activity against Arabidopsis plants. Therefore, these compounds may be acting as pathogenic determinants of CSE26. However, there is a need to study the level of production of these CAMs by this fungus in the natural environment and their effects on plant health.

Mortierella alpina CS10E4, an oleaginous fungal endophyte of Crocus sativus L. enhances apocarotenoid biosynthesis and stress tolerance in the host plant.

Crocus sativus is the only plant species which produces apocarotenoids like crocin, picrocrocin and safranal in significant amounts. These compounds impart organoleptic properties to saffron (dried stigmas of Crocus flower) making it world's costliest spice. Crocus apocarotenoids have tremendous medicinal properties as well. Effect of endophytes on Crocus apocarotenoid production and the molecular mechanism involved has not been reported so far. Here we studied the effect of an oleaginous fungal endophyte, Mortierella alpina CS10E4 on Crocus growth, apocarotenoid metabolism and tolerance to corm rot disease. The results demonstrated that there was a significant improvement in many morphological and physiological traits in endophyte treated Crocus plants including total biomass and size of corms, stigma biomass, number of apical sprouting buds, and number of adventitious roots. The endophyte also shifted metabolic flux towards enhanced production of apocarotenoids by modulating the expression of key pathway genes. Further, M. alpina CS10E4 enhanced tolerance to corm rot disease by releasing arachidonic acid which acts as conserved defense signal and induces jasmonic acid production in endophyte treated Crocus corms. This is first report on effect of a fungal endophyte on Crocus apocarotenoid metabolism and stress tolerance.

Transcriptome wide identification, phylogenetic analysis, and expression profiling of zinc-finger transcription factors from Crocus sativus L.

Crocus sativus belongs to Iridaceae family and is the only plant species which produces apocarotenoids like crocin, picrocrocin, and safranal in significant quantities. Besides their organoleptic properties, Crocus apocarotenoids have been found to possess remarkable pharmacological potential. Although apocarotenoid biosynthetic pathway has been worked out to a great degree, but the mechanism that regulates the tissue and developmental stage-specific production of Crocus apocarotenoids is not known. To identify the genes regulating apocarotenoid biosynthesis in Crocus, transcriptome wide identification of zinc-finger transcription factors was undertaken. 81 zinc-finger transcription factors were identified which grouped into eight subfamilies. C2H2, C3H, and AN20/AN1 were the major subfamilies with 29, 20, and 14 members, respectively. Expression profiling revealed CsSAP09 as a potential candidate for regulation of apocarotenoid biosynthesis. CsSAP09 was found to be highly expressed in stigma at anthesis stage corroborating with the accumulation pattern of apocarotenoids. CsSAP09 was nuclear localized and activated reporter gene transcription in yeast. It was highly induced in response to oxidative, salt and dehydration stresses, ABA and methyl jasmonate. Furthermore, upstream region of CsSAP09 was found to contain stress and light responsive elements. To our knowledge, this is the first report on the study of a gene family in C. sativus and may provide basic insights into the putative role of zinc finger genes. It may also serve as a valuable resource for functional characterization of these genes aimed towards unraveling their role in regulation of apocarotenoid biosynthesis.

Functional Characterization of CsBGlu12, a ß-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols.

Glycosylation and deglycosylation are impressive mechanisms that allow plants to regulate the biological activity of an array of secondary metabolites. Although glycosylation improves solubility and renders the metabolites suitable for transport and sequestration, deglycosylation activates them to carry out biological functions. Herein, we report the functional characterization of CsBGlu12, a ß-glucosidase from Crocus sativus. CsBGlu12 has a characteristic glucoside hydrolase 1 family (α/ß)8 triose-phosphate isomerase (TIM) barrel structure with a highly conserved active site. In vitro enzyme activity revealed that CsBGlu12 catalyzes the hydrolysis of flavonol ß-glucosides and cello-oligosaccharides. Site-directed mutagenesis of any of the two conserved catalytic glutamic acid residues (Glu200 and Glu414) of the active site completely abolishes the ß-glucosidase activity. Transcript analysis revealed that Csbglu12 is highly induced in response to UV-B, dehydration, NaCl, methyl jasmonate, and abscisic acid treatments indicating its possible role in plant stress response. Transient overexpression of CsBGlu12 leads to the accumulation of antioxidant flavonols in Nicotiana benthamiana and confers tolerance to abiotic stresses. Antioxidant assays indicated that accumulation of flavonols alleviated the accretion of reactive oxygen species during abiotic stress conditions. ß-Glucosidases are known to play a role in abiotic stresses, particularly dehydration through abscisic acid; however, their role through accumulation of reactive oxygen species (ROS) scavenging flavonols has not been established. Furthermore, only one ß-glucosidase 12 homolog has been characterized so far. Therefore, this work presents an important report on characterization of CsBGlu12 and its role in abiotic stress through ROS scavenging.

Overexpression of Crocus carotenoid cleavage dioxygenase, CsCCD4b, in Arabidopsis imparts tolerance to dehydration, salt and oxidative stresses by modulating ROS machinery.

Apocarotenoids modulate vital physiological and developmental processes in plants. These molecules are formed by the cleavage of carotenoids, a reaction catalyzed by a family of enzymes called carotenoid cleavage dioxygenases (CCDs). Apocarotenoids like ß-ionone and ß-cyclocitral have been reported to act as stress signal molecules during high light stress in many plant species. In Crocus sativus, these two apocarotenoids are formed by enzymatic cleavage of ß-carotene at 9, 10 and 7, 8 bonds by CsCCD4 enzymes. In the present study three isoforms of CsCCD4 were subjected to molecular modeling and docking analysis to determine their substrate specificity and all the three isoforms displayed high substrate specificity for ß-carotene. Further, expression of these three CsCCD4 isoforms investigated in response to various stresses revealed that CsCCD4a and CsCCD4b exhibit enhanced expression in response to dehydration, salt and methylviologen, providing a clue towards their role in mediating plant defense response. This was confirmed by overexpressing CsCCD4b in Arabidopsis. The transgenic plants developed longer roots and possessed higher number of lateral roots. Further, overexpression of CsCCD4b imparted enhanced tolerance to salt, dehydration and oxidative stresses as was evidenced by higher survival rate, increased relative root length and biomass in transgenic plants as compared to wild type. Transgenic plants also displayed higher activity and expression of reactive oxygen species (ROS) metabolizing enzymes. This indicates that ß-ionone and ß-cyclocitral which are enzymatic products of CsCCD4b may act as stress signals and mediate reprogramming of stress responsive genes which ultimately leads to plant defense.

Comprehensive transcriptome analysis of Crocus sativus for discovery and expression of genes involved in apocarotenoid biosynthesis.

BACKGROUND: Crocus sativus stigmas form rich source of apocarotenoids like crocin, picrocrocin and saffranal which besides imparting color, flavour and aroma to saffron spice also have tremendous pharmacological properties. Inspite of their importance, the biosynthetic pathway of Crocus apocarotenoids is not fully elucidated. Moreover, the mechanism of their stigma specific accumulation remains unknown. Therefore, deep transcriptome sequencing of Crocus stigma and rest of the flower tissue was done to identify the genes and transcriptional regulators involved in the biosynthesis of these compounds. RESULTS: Transcriptome of stigma and rest of the flower tissue was sequenced using Illumina Genome Analyzer IIx platform which generated 64,604,402 flower and 51,350,714 stigma reads. Sequences were assembled de novo using trinity resulting in 64,438 transcripts which were classified into 32,204 unigenes comprising of 9853 clusters and 22,351 singletons. A comprehensive functional annotation and gene ontology (GO) analysis was carried out. 58.5 % of the transcripts showed similarity to sequences present in public databases while rest could be specific to Crocus. 5789 transcripts showed similarity to transcription factors representing 76 families out of which Myb family was most abundant. Many genes involved in carotenoid/apocarotenoid pathway were identified for the first time in this study which includes zeta-carotene isomerase and desaturase, carotenoid isomerase and lycopene epsilon-cyclase. GO analysis showed that the predominant classes in biological process category include metabolic process followed by cellular process and primary metabolic process. KEGG mapping analysis indicated that pathways involved in ribosome, carbon and starch and sucrose metabolism were highly represented. Differential expression analysis indicated that key carotenoid/apocarotenoid pathway genes including phytoene synthase, phytoene desaturase and carotenoid cleavage dioxygenase 2 are enriched in stigma thereby providing molecular proof for stigma to be the site of apocarotenoid biosynthesis. CONCLUSIONS: This data would provide a rich source for understanding the carotenoid/apocarotenoid metabolism in Crocus. The database would also help in investigating many questions related to saffron biology including flower development.

Plant-endophyte symbiosis, an ecological perspective.

Endophytism is the phenomenon of mutualistic association of a plant with a microorganism wherein the microbe lives within the tissues of the plant without causing any symptoms of disease. In addition to being a treasured biological resource, endophytes play diverse indispensable functions in nature for plant growth, development, stress tolerance, and adaptation. Our understanding of endophytism and its ecological aspects are overtly limited, and we have only recently started to appreciate its essence. Endophytes may impact plant biology through the production of diverse chemical entities including, but not limited to, plant growth hormones and by modulating the gene expression of defense and other secondary metabolic pathways of the host. Studies have shown differential recruitment of endophytes in endophytic populations of plants growing in the same locations, indicating host specificity and that endophytes evolve in a coordinated fashion with the host plants. Endophytic technology can be employed for the efficient production of agricultural and economically important plants and plant products. The rational application of endophytes to manipulate the microbiota, intimately associated with plants, can help in enhancement of production of agricultural produce, increased production of key metabolites in medicinal and aromatic plants, as well as adaption to new bio-geographic regions through tolerance to various biotic and abiotic conditions. However, the potential of endophytic biology can be judiciously harnessed only when we obtain insight into the molecular mechanism of this unique mutualistic relationship. In this paper, we present a discussion on endophytes, endophytism, their significance, and diverse functions in nature as unraveled by the latest research to understand this universal natural phenomenon.

Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis.

BACKGROUND: Crocus sativus is a triploid sterile plant with long red stigmas which form commercial saffron. Saffron is the site for synthesis and accumulation of apocarotenoids like crocin, picrocrin and safranal which are responsible for its color, flavour and aroma making it world's most expensive spice. These compounds are formed by oxidative cleavage of zeaxanthin by carotenoid cleavage dioxygenases. Although the biosynthetic pathway of apocarotenoids is known to a considerable extent, the mechanism that regulates its tissue and developmental stage specific expression is not known. RESULTS: In the present work, we identified, cloned and characterized ultrapetala transcription factor called CsULT1 from Crocus. The gene contains an 80 amino acid long conserved SAND domain. The CsULT1 transcript was more abundant in stigma and showed increase in expression from pre anthesis stage till anthesis and decreased in post anthesis stage which corroborated with the accumulation pattern of crocin indicating its possible role in regulation of apocarotenoid biosynthesis. CsULT1 was found to be transcriptionally active and localized in nucleus. Its expression is induced in response to phytohormones like auxin, methyljasmonate and salicylic acid. Overexpression of CsULT1 in Crocus calli resulted in enhanced expression of key pathway genes like phytoene synthase (PSY), phytoene desaturase (PDS), beta carotene hydroxylase (BCH) and carotenoid cleavage dioxygenases (CCDs) indicating its role in regulation of apocarotenoid biosynthesis. CONCLUSION: This work presents first report on isolation and characterization of ultrapetala gene from Crocus. Our results suggest that CsULT1 is a novel regulator of Crocus apocarotenoid biosynthesis. We show for the first time involvement of plant SAND domain proteins in regulating secondary metabolic pathways.

Molecular characterization of UGT94F2 and UGT86C4, two glycosyltransferases from Picrorhiza kurrooa: comparative structural insight and evaluation of substrate recognition.

Uridine diphosphate glycosyltransferases (UGTs) are pivotal in the process of glycosylation for decorating natural products with sugars. It is one of the versatile mechanisms in determining chemical complexity and diversity for the production of suite of pharmacologically active plant natural products. Picrorhiza kurrooa is a highly reputed medicinal herb known for its hepato-protective properties which are attributed to a novel group of iridoid glycosides known as picrosides. Although the plant is well studied in terms of its pharmacological properties, very little is known about the biosynthesis of these important secondary metabolites. In this study, we identified two family-1 glucosyltransferases from P. kurrooa. The full length cDNAs of UGT94F4 and UGT86C4 contained open reading frames of 1455 and 1422 nucleotides, encoding polypeptides of 484 and 473 amino acids respectively. UGT94F2 and UGT86C4 showed differential expression pattern in leaves, rhizomes and inflorescence. To elucidate whether the differential expression pattern of the two Picrorhiza UGTs correlate with transcriptional regulation via their promoters and to identify elements that could be recognized by known iridoid-specific transcription factors, upstream regions of each gene were isolated and scanned for putative cis-regulatory elements. Interestingly, the presence of cis-regulatory elements within the promoter regions of each gene correlated positively with their expression profiles in response to different phytohormones. HPLC analysis of picrosides extracted from different tissues and elicitor-treated samples showed a significant increase in picroside levels, corroborating well with the expression profile of UGT94F2 possibly indicating its implication in picroside biosynthesis. Using homology modeling and molecular docking studies, we provide an insight into the donor and acceptor specificities of both UGTs identified in this study. UGT94F2 was predicted to be an iridoid-specific glucosyltransferase having maximum binding affinity towards 7-deoxyloganetin while as UGT86C4 was predicted to be a kaempferol-specific glucosyltransferase. These are the first UGTs being reported from P. kurrooa.

Comparative analyses of genotype dependent expressed sequence tags and stress-responsive transcriptome of chickpea wilt illustrate predicted and unexpected genes and novel regulators of plant immunity.

BACKGROUND: The ultimate phenome of any organism is modulated by regulated transcription of many genes. Characterization of genetic makeup is thus crucial for understanding the molecular basis of phenotypic diversity, evolution and response to intra- and extra-cellular stimuli. Chickpea is the world's third most important food legume grown in over 40 countries representing all the continents. Despite its importance in plant evolution, role in human nutrition and stress adaptation, very little ESTs and differential transcriptome data is available, let alone genotype-specific gene signatures. Present study focuses on Fusarium wilt responsive gene expression in chickpea. RESULTS: We report 6272 gene sequences of immune-response pathway that would provide genotype-dependent spatial information on the presence and relative abundance of each gene. The sequence assembly led to the identification of a CaUnigene set of 2013 transcripts comprising of 973 contigs and 1040 singletons, two-third of which represent new chickpea genes hitherto undiscovered. We identified 209 gene families and 262 genotype-specific SNPs. Further, several novel transcription regulators were identified indicating their possible role in immune response. The transcriptomic analysis revealed 649 non-cannonical genes besides many unexpected candidates with known biochemical functions, which have never been associated with pathostress-responsive transcriptome. CONCLUSION: Our study establishes a comprehensive catalogue of the immune-responsive root transcriptome with insight into their identity and function. The development, detailed analysis of CaEST datasets and global gene expression by microarray provide new insight into the commonality and diversity of organ-specific immune-responsive transcript signatures and their regulated expression shaping the species specificity at genotype level. This is the first report on differential transcriptome of an unsequenced genome during vascular wilt.