CRISPR-Cas System, a Possible "Savior" of Rice Threatened by Climate Change: An Updated Review.

Climate change has significantly affected agriculture production, particularly the rice crop that is consumed by almost half of the world's population and contributes significantly to global food security. Rice is vulnerable to several abiotic and biotic stresses such as drought, heat, salinity, heavy metals, rice blast, and bacterial blight that cause huge yield losses in rice, thus threatening food security worldwide. In this regard, several plant breeding and biotechnological techniques have been used to raise such rice varieties that could tackle climate changes. Nowadays, gene editing (GE) technology has revolutionized crop improvement. Among GE technology, CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) system has emerged as one of the most convenient, robust, cost-effective, and less labor-intensive system due to which it has got more popularity among plant researchers, especially rice breeders and geneticists. Since 2013 (the year of first application of CRISPR/Cas-based GE system in rice), several trait-specific climate-resilient rice lines have been developed using CRISPR/Cas-based GE tools. Earlier, several reports have been published confirming the successful application of GE tools for rice improvement. However, this review particularly aims to provide an updated and well-synthesized brief discussion based on the recent studies (from 2020 to present) on the applications of GE tools, particularly CRISPR-based systems for developing CRISPR rice to tackle the current alarming situation of climate change, worldwide. Moreover, potential limitations and technical bottlenecks in the development of CRISPR rice, and prospects are also discussed.

Comparative transcriptomic and evolutionary analysis of FAD-like genes of Brassica species revealed their role in fatty acid biosynthesis and stress tolerance.

BACKGROUND: Fatty acid desaturases (FADs) are involved in regulating plant fatty acid composition by adding double bonds to growing hydrocarbon chain. Apart from regulating fatty acid composition FADs are of great importance, and are involved in stress responsiveness, plant development, and defense mechanisms. FADs have been extensively studied in crop plants, and are broadly classed into soluble and non-soluble fatty acids. However, FADs have not yet been characterized in Brassica carinata and its progenitors. RESULTS: Here we have performed comparative genome-wide identification of FADs and have identified 131 soluble and 28 non-soluble FADs in allotetraploid B. carinata and its diploid parents. Most soluble FAD proteins are predicted to be resided in endomembrane system, whereas FAB proteins were found to be localized in chloroplast. Phylogenetic analysis classed the soluble and non-soluble FAD proteins into seven and four clusters, respectively. Positive type of selection seemed to be dominant in both FADs suggesting the impact of evolution on these gene families. Upstream regions of both FADs were enriched in stress related cis-regulatory elements and among them ABRE type of elements were in abundance. Comparative transcriptomic data analysis output highlighted that FADs expression reduced gradually in mature seed and embryonic tissues. Moreover, under heat stress during seed and embryo development seven genes remained up-regulated regardless of external stress. Three FADs were only induced under elevated temperature whereas five genes were upregulated under Xanthomonas campestris stress suggesting their involvement in abiotic and biotic stress response. CONCLUSIONS: The current study provides insights into the evolution of FADs and their role in B. carinata under stress conditions. Moreover, the functional characterization of stress-related genes would exploit their utilization in future breeding programs of B. carinata and its progenitors.

Comparative phylogenomic insights of KCS and ELO gene families in Brassica species indicate their role in seed development and stress responsiveness.

Very long-chain fatty acids (VLCFAs) possess more than twenty carbon atoms and are the major components of seed storage oil, wax, and lipids. FAE (Fatty Acid Elongation) like genes take part in the biosynthesis of VLCFAs, growth regulation, and stress responses, and are further comprised of KCS (Ketoacyl-CoA synthase) and ELO (Elongation Defective Elongase) sub-gene families. The comparative genome-wide analysis and mode of evolution of KCS and ELO gene families have not been investigated in tetraploid Brassica carinata and its diploid progenitors. In this study, 53 KCS genes were identified in B. carinata compared to 32 and 33 KCS genes in B. nigra and B. oleracea respectively, which suggests that polyploidization might has impacted the fatty acid elongation process during Brassica evolution. Polyploidization has also increased the number of ELO genes in B. carinata (17) over its progenitors B. nigra (7) and B. oleracea (6). Based on comparative phylogenetics, KCS, and ELO proteins can be classified into eight and four major groups, respectively. The approximate date of divergence for duplicated KCS and ELO genes varied from 0.03 to 3.20 million years ago (MYA). Gene structure analysis indicated that the maximum number of genes were intron-less and remained conserved during evolution. The neutral type of selection seemed to be predominant in both KCS and ELO genes evolution. String-based protein-protein interaction analysis suggested that bZIP53, a transcription factor might be involved in the activation of transcription of ELO/KCS genes. The presence of biotic and abiotic stress-related cis-regulatory elements in the promoter region suggests that both KCS and ELO genes might also play their role in stress tolerance. The expression analysis of both gene family members reflect their preferential seed-specific expression, especially during the mature embryo development stage. Furthermore, some KCS and ELO genes were found to be specifically expressed under heat stress, phosphorus starvation, and Xanthomonas campestris infection. The current study provides a basis to understand the evolution of both KCS and ELO genes in fatty acid elongation and their role in stress tolerance.

Genome Wide Analysis of Family-1 UDP Glycosyltransferases in Populus trichocarpa Specifies Abiotic Stress Responsive Glycosylation Mechanisms.

Populus trichocarpa (Black cottonwood) is a dominant timber-yielding tree that has become a notable model plant for genome-level insights in forest trees. The efficient transport and solubility of various glycoside-associated compounds is linked to Family-1 UDP-glycosyltransferase (EC 2.4.1.x; UGTs) enzymes. These glycosyltransferase enzymes play a vital role in diverse plant functions, such as regulation of hormonal homeostasis, growth and development (seed, flower, fiber, root, etc.), xenobiotic detoxification, stress response (salt, drought, and oxidative), and biosynthesis of secondary metabolites. Here, we report a genome-wide analysis of the P. trichocarpa genome that identified 191 putative UGTs distributed across all chromosomes (with the exception of chromosome 20) based on 44 conserved plant secondary product glycosyltransferase (PSPG) motif amino acid sequences. Phylogenetic analysis of the 191 Populus UGTs together with 22 referenced UGTs from Arabidopsis and maize clustered the putative UGTs into 16 major groups (A-P). Whole-genome duplication events were the dominant pattern of duplication among UGTs in Populus. A well-conserved intron insertion was detected in most intron-containing UGTs across eight examined eudicots, including Populus. Most of the UGT genes were found preferentially expressed in leaf and root tissues in general. The regulation of putative UGT expression in response to drought, salt and heat stress was observed based on microarray and available RNA sequencing datasets. Up- and down-regulated UGT expression models were designed, based on transcripts per kilobase million values, confirmed their maximally varied expression under drought, salt and heat stresses. Co-expression networking of putative UGTs indicated their maximum co-expression with cytochrome P450 genes involved in triterpenoid biosynthesis. Our results provide an important resource for the identification of functional UGT genes to manipulate abiotic stress responsive glycosylation in Populus.

Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocols for Bi and Multilocular Varieties of Brassica rapa.

The regeneration of the high-yielding multilocular types has not been attempted, although successful regeneration and transformation in brassica have been done. Here, we report efficient regeneration and transformation protocols for two B. rapa genotypes; UAF11 and Toria. The B. rapa cv UAF11 is a multilocular, non-shattering, and high-yielding genotype, while Toria is the bilocular type. For UAF11 8 shoots and for Toria 7 shoots, explants were observed on MS supplemented with 3 mg/L BAP + 0.4 mg/L NAA + 0.01 mg/L GA3 + 5 mg/L AgNO3 + 0.75 mg/L Potassium Iodide (KI), MS salt supplemented with 1 mg/L IBA and 0.37 mg/L KI produced an equal number of roots (3) in UAF11 and Toria. For the establishment of transformation protocols, Agrobacterium-mediated floral dip transformation was attempted using different induction media, infection time, and flower stages. The induction medium III yielded a maximum of 7.2% transformants on half-opened flowers and 5.2% transformants on fully opened flowers in UAF11 and Toria, respectively, with 15 min of inoculation. This study would provide the basis for the improvement of tissue culture and transformation protocols in multilocular and bilocular Brassica genotypes.