Comparative analysis of miRNA expression profiles in flowering and non-flowering tissue of Crocus sativus L.

Crocus sativus is a valuable plant due to the presence of apocarotenoids in its stigma. Considerable work has been done in the past to understand the apocarotenoid biosynthetic pathway in saffron. However, the reports on understanding the regulation of flowering at the post-transcriptional level are meagre. The study aimed to discover the candidate miRNAs, target genes, transcription factors (TFs), and apocarotenoid biosynthetic pathway genes associated with the regulation and transition of flowering in C. sativus. In the present investigation, miRNA profiling was performed in flowering and non-flowering corms of saffron, along with expression analysis of apocarotenoid genes and transcription factors involved in the synthesis of secondary metabolites. Significant modulation in the expression of miR156, miR159, miR166, miR172, miR395, miR396, miR399, and miR408 gene families was observed. We obtained 36 known miRNAs (26 in flowering and 10 in non-flowering) and 64 novel miRNAs (40 in flowering and 24 in non-flowering) unique to specific tissues in our analysis. TFs, including CsMADS and CsMYb, showed significant modulation in expression in flowering tissue, followed by CsHB. Additionally, the miRNAs were predicted to be involved in carbohydrate metabolism, phytohormone signalling, regulation of flower development, and response to stress, cold, and defence. The comprehensive study has enhanced our understanding of the regulatory machinery comprising factors like phytohormones, abiotic stress, apocarotenoid genes, transcription factors, and miRNAs responsible for the synthesis of apocarotenoids and developmental processes during and after flowering.

Secretomic Insights into the Pathophysiology of Venturia inaequalis: The Causative Agent of Scab, a Devastating Apple Tree Disease.

Apple scab, caused by Venturia inaequalis, is one of the world's most commercially significant apple diseases. The fungi have a catastrophic impact on apples, causing considerable losses in fruit quality and productivity in many apple-growing locations despite numerous control agents. Fungi secrete various effectors and other virulence-associated proteins that suppress or alter the host's immune system, and several such proteins were discovered in this work. Using state-of-the-art bioinformatics techniques, we examined the V. inaequalis reference genome (EU-B04), resulting in the identification of 647 secreted proteins, of which 328 were classified as small secreted proteins (SSPs), with 76.52% of SSPs identified as anticipated effector proteins. The more prevalent CAZyme proteins were the enzymes engaged in plant cell wall disintegration (targeting pectin and xylanase), adhesion and penetration (Cutinases/acetyl xylan esterase), and reactive oxygen species formation (multicopper oxidases). Furthermore, members of the S9 prolyl oligopeptidase family were identified as the most abundant host defense peptidases. Several known effector proteins were discovered to be expressed during the V. inaequalis infection process on apple leaves. The present study provides valuable data that can be used to develop new strategies for controlling apple scab.

Review: Understanding Rare Genetic Diseases in Low Resource Regions Like Jammu and Kashmir - India.

Rare diseases (RDs) are the clinical conditions affecting a few percentage of individuals in a general population compared to other diseases. Limited clinical information and a lack of reliable epidemiological data make their timely diagnosis and therapeutic management difficult. Emerging Next-Generation DNA Sequencing technologies have enhanced our horizons on patho-physiological understanding of many of the RDs and ushered us into an era of diagnostic and therapeutic research related to this ignored health challenge. Unfortunately, relevant research is meager in developing countries which lack a reliable estimate of the exact burden of most of the RDs. India is to be considered as the "Pandora's Box of genetic disorders." Owing to its huge population heterogeneity and high inbreeding or endogamy rates, a higher burden of rare recessive genetic diseases is expected and supported by the literature findings that endogamy is highly detrimental to health as it enhances the degree of homozygosity of recessive alleles in the general population. The population of a low resource region Jammu and Kashmir (J&K) - India, is highly inbred. Some of its population groups variably practice consanguinity. In context with the region's typical geographical topography, highly inbred population structure and unique but heterogeneous gene pool, a huge burden of known and uncharacterized genetic disorders is expected. Unfortunately, many suspected cases of genetic disorders remain undiagnosed or misdiagnosed due to lack of appropriate clinical as well as diagnostic resources in the region, causing patients to face a huge psycho-socio-economic crisis and many a time suffer life-long with their ailment. In this review, the major challenges associated with RDs are highlighted in general and an account on the methods that can be adopted for conducting fruitful molecular genetic studies in genetically vulnerable and low resource regions is also provided, with an example of a region like J&K - India.

Development of a system for efficient callus production, somatic embryogenesis and gene editing using CRISPR/Cas9 in Saffron (Crocus sativus L.).

BACKGROUND: Crocus sativus is a recalcitrant plant for genetic transformation and genetic improvement, largely due to difficulties in Agrobacterium mediated transformation and vegetative reproduction. Effective genome editing requires proficient callus production and an efficient method to deliver Cas9 and sgRNAs into the plant. Here, we demonstrate Agrobacterium-mediated transformation of saffron. Further, we developed a CRISPR-Cas9 based system in this plant, for efficient gene knockout or edits in future. RESULTS: Efficient callus production and regeneration confers important benefits in developing competent transformation system in plants. More than 70% multiplication rate of callus initiation was achieved from corm slices of saffron subjected to a two-step sterilization procedure and grown on complete MS medium supplemented with 2,4-D (0.5 mg/L), BAP (1 mg/L), IAA (1 mg/L), photoperiod of 16/8 h and 45% relative humidity at 20 ± 2 °C. In vitro cormlet generation was accomplished in 8 weeks by using mature somatic embryos on MS medium supplemented with TDZ (0.5 mg/L) + IAA (1 mg/L) + Activated charcoal (0.1 g/L) at 15 ± 2 °C. The attempt of using Agrobacterium-mediated transformation resulted in successful integration of the binary vector into the somatic embryos of saffron with a transformation efficiency of 4%. PCR and Southern blot analysis confirmed the integration of Cas9 into saffron. CONCLUSION: The protocol for callus production, somatic embryogenesis and regeneration was standardised. Successful demonstration of integrated Cas9 in this study constitutes first step in developing strategies for genetic manipulation of saffron, which has so far been considered recalcitrant. Furthering the development of this technology holds significant potential for advancing genetic research in saffron by integrating multigene targeting and/or use of recyclable cassettes.

Transcriptome analysis of Snow Mountain Garlic for unraveling the organosulfur metabolic pathway.

Snow Mountain Garlic grows in the high altitudes of the Himalayas under low temperature conditions. It contains various bioactive compounds whose metabolic pathways have not been worked out at genomic level. The present work is the first report on the transcriptome sequencing of this plant. >43 million paired-end reads (301 × 2) were generated using Illumina Miseq sequencing technology. Assembling of the sequencing data resulted in 326,785 transcripts. Differentially expressed genes between the clove and leaf tissues were identified and characterized. Besides, greater emphasis was laid on the genes, which were highly expressed in clove since the latter is assumed to contain high content of the bioactive compounds. Further analysis led to the identification of the genes plausibly involved in the organosulfur metabolism. We also identified several simple sequence repeats and single nucleotide polymorphism. These constitute valuable genetic resource for research and further genetic improvement of the plant.

Plant carotenoid cleavage oxygenases: structure-function relationships and role in development and metabolism.

A plant communicates within itself and with the outside world by deploying an array of agents that include several attractants by virtue of their color and smell. In this category, the contribution of 'carotenoids and apocarotenoids' is very significant. Apocarotenoids, the carotenoid-derived compounds, show wide representation among organisms. Their biosynthesis occurs by oxidative cleavage of carotenoids, a high-value reaction, mediated by carotenoid cleavage oxygenases or carotenoid cleavage dioxygenases (CCDs)-a family of non-heme iron enzymes. Structurally, this protein family displays wide diversity but is limited in its distribution among plants. Functionally, this protein family has been recognized to offer a role in phytohormones, volatiles and signal production. Further, their wide presence and clade-specific functional disparity demands a comprehensive account. This review focuses on the critical assessment of CCDs of higher plants, describing recent progress in their functional aspects and regulatory mechanisms, domain architecture, classification and localization. The work also highlights the relevant discussion for further exploration of this multi-prospective protein family for the betterment of its functional understanding and improvement of crops.

Transcript profiling of carotenoid/apocarotenoid biosynthesis genes during corm development of saffron (Crocus sativus L.).

The dried stigmas of saffron constitute the world's costliest spice. Saffron has many therapeutic applications due to the presence of apocarotenoids. The latter are synthesized at different stages of development, and the biosynthetic pathway involves several genes encoding different enzymes. In order to understand the differential expression of various genes of the pathway, eight distinct developmental stages (S1-early to S8-late) were identified. The corms were assorted into three groups (I, II, and III) based on corm weight. Expression profiles of 12 carotenoid/apocarotenoid genes were studied. The expression of all genes was minimum/least in groups I and II corms during bud development. Lowest expression of carotenogenic genes (CsPSY, CsPDS, CsZDS, CsCRTISO, CsLYC-ß1, CsLYC-ε, CsBCH2, and CsNCED) was observed during early stages (S1-S3) of corm growth (dormant period). In group III corms, increased expression of apocarotenoid genes (CsZCO, CsCCD2, CsUGT, and CsALDH) was observed during S4 to S8 stages (reproductive period, floral differentiation). Besides, expression profiles of genes in apical and axillary buds were also examined. Of all the genes studied, apocarotenoid biosynthesis genes (CsBCH2, CsZCO, CsCCD2, CsALDH, and CsUGT) were found to be upregulated in apical bud than in the axillary bud. The results indicated that interaction of phytohormones and sugars, mother corm reserves and the influence of internal and external factors may be contributing to the growth of saffron corm/bud. The study has laid a foundation for further research on the molecular mechanisms underlying bud dormancy/growth in saffron.

Epigenetic dynamics: role of epimarks and underlying machinery in plants exposed to abiotic stress.

Abiotic stress induces several changes in plants at physiological and molecular level. Plants have evolved regulatory mechanisms guided towards establishment of stress tolerance in which epigenetic modifications play a pivotal role. We provide examples of gene expression changes that are brought about by conversion of active chromatin to silent heterochromatin and vice versa. Methylation of CG sites and specific modification of histone tail determine whether a particular locus is transcriptionally active or silent. We present a lucid review of epigenetic machinery and epigenetic alterations involving DNA methylation, histone tail modifications, chromatin remodeling, and RNA directed epigenetic changes.

Genetic and epigenetic instability of amplification-prone sequences of a novel B chromosome induced by tissue culture in Plantago lagopus L.

Gene amplification is prevalent in many eukaryotes and has been found linked to various phenomena such as ontogenesis, carcinogenesis, in vitro culturing, neoplasia and drug resistance. Earlier, we reported a novel B chromosome in Plantago lagopus L., which was found to have arisen as a result of massive amplification of 5S rDNA. In addition, the chromosome is also composed of 45S rDNA and transposable elements. While the importance of gene amplification cannot be underestimated, its mechanism of origin is still unclear. Therefore, the aim of the present study was to determine whether amplification can be reactivated in the novel B chromosome. For this purpose, in vitro culture was used as stress. Three modes of tissue culture, i.e., direct, indirect and somatic embryogenesis were used for raising in vitro cultures. The variations due to genetic and epigenetic mechanisms were assessed in regenerants using molecular techniques, namely, PCR-RFLP, SSAP and MSAP. The retrotransposon-based molecular markers were applied to detect the polymorphism within transposable elements of in vitro regenerated and mother plants. We detected the variations that may be due to genetic changes either because of element recombination or activation of transposable elements which can lead to increase in the copy number. MSAP analysis revealed the differences in the DNA methylation pattern of the regenerants derived from novel chromosome bearing mother plants. Some regenerated plants were associated with increase and decrease in DNA methylation of both internal and external cytosine of the CCGG sequence.