Plant Metabolomics: Current Initiatives and Future Prospects.

Plant metabolomics is a rapidly advancing field of plant sciences and systems biology. It involves comprehensive analyses of small molecules (metabolites) in plant tissues and cells. These metabolites include a wide range of compounds, such as sugars, amino acids, organic acids, secondary metabolites (e.g., alkaloids and flavonoids), lipids, and more. Metabolomics allows an understanding of the functional roles of specific metabolites in plants' physiology, development, and responses to biotic and abiotic stresses. It can lead to the identification of metabolites linked with specific traits or functions. Plant metabolic networks and pathways can be better understood with the help of metabolomics. Researchers can determine how plants react to environmental cues or genetic modifications by examining how metabolite profiles change under various crop stages. Metabolomics plays a major role in crop improvement and biotechnology. Integrating metabolomics data with other omics data (genomics, transcriptomics, and proteomics) provides a more comprehensive perspective of plant biology. This systems biology approach enables researchers to understand the complex interactions within organisms.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Marker Assisted Introgression of Resistance Genes and Phenotypic Evaluation Enabled Identification of Durable and Broad-Spectrum Blast Resistance in Elite Rice Cultivar, CO 51.

Across the globe, rice cultivation is seriously affected by blast disease, caused by Magnaporthe oryzae. This disease has caused heavy yield loss to farmers over the past few years. In this background, the most affordable and eco-friendly strategy is to introgress blast-resistant genes from donors into elite rice cultivars. However, it is not only challenging to evolve such resistance lines using conventional breeding approaches, but also a time-consuming process. Therefore, the marker-assisted introduction of resistance genes has been proposed as a rapid strategy to develop durable and broad-spectrum resistance in rice cultivars. The current study highlights the successful introgression of a blast resistance gene, i.e., Pi9, into CO 51, an elite rice cultivar which already has another resistance gene named Pi54. The presence of two blast resistance genes in the advanced backcross breeding materials (BC2F2:3) was confirmed in this study through a foreground selection method using functional markers such as NBS4 and Pi54MAS. The selected positive introgressed lines were further genotyped for background selection with 55 SSR markers that are specific to CO 51. Consequently, both Pi9 as well as Pi54 pyramided lines, with 82.7% to 88.1% of the recurrent parent genome recovery, were identified and the selected lines were evaluated under hotspot. The analysis outcomes found that both the lines possessed a high level of resistance against blast disease during the seedling stage itself. In addition to this, it was also noticed that the advanced breeding rice lines that carry Pi9 + Pi54 were effective in nature and exhibited a higher degree of resistance against blast disease compared to the lines that were introgressed with a single blast resistance gene. Thus, the current study demonstrates a rapid and a successful introgression and pyramiding of two blast resistance genes, with the help of markers, into a susceptible yet high-yielding elite rice cultivar within a short period of time. Those gene pyramided rice lines can be employed as donors to introgress the blast-resistant genes in other popular susceptible cultivars.

Development of ß-carotene, lysine, and tryptophan-rich maize (Zea mays) inbreds through marker-assisted gene pyramiding.

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased ß-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in ß-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1-BC3F1, and ICF1-ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3'TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45-88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the ß-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-ß1σ4-4-8-8, DBT-IC-ß1σ4-9-21-21, DBT-IC-ß1σ4-10-1-1, DBT-IC-ß2σ5-9-51-51, DBT-IC-ß2σ5-9-52-52 and DBT-IC-ß2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78-99.31% for DBT-IC-ß1σ4 population and 85.71-99.51% for DBT-IC-ß2σ5 population) like the recurrent parents and ß-carotene (14.21-14.35 µg/g for DBT-IC-ß1σ4 and 13.28-13.62 µg/g for DBT-IC-ß2σ5), lysine (0.31-0.33% for DBT-IC-ß1σ4 and 0.31-0.34% for DBT-IC-ß2σ5), and tryptophan (0.079-0.082% for DBT-IC-ß1σ4 and 0.078-0.083% for DBT-IC-ß2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs.

A Comparative Metabolomic Analysis Reveals the Nutritional and Therapeutic Potential of Grains of the Traditional Rice Variety Mappillai Samba.

Rice (Oryza sativa L.) is the staple food of the majority of the population, particularly in Asia and Africa. Enriching rice with nutritional and therapeutic contents can improve its benefits for patients with lifestyle disorders. This study aimed to profile the phytochemical contents of the therapeutically known traditional rice Mappillai Samba against white rice CBMAS 14065 using non-targeted gas chromatography-mass spectrometry (GC-MS/MS). An analysis of the data using a mass spectrometry-data independent analysis (MS-DIAL) and MetaboAnalyst identified 113 metabolites belonging to 21 different classes of metabolites. A partial least square-discriminant analysis (PLS-DA) revealed 43 variable importance in projection (VIP) metabolites. This study identified therapeutically important metabolites, including phenylpropanoids, phytosterols, flavonoids, and polyamines, in the grains of Mappillai Samba. Three significant metabolic pathways, viz., phenylpropanoid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, and steroid biosynthesis, were responsible for the grain metabolome variation between CBMAS 14065 and Mappillai Samba. Overall, the results of this study unravelled the biochemical complexity of Mappillai Samba, paving the way for the genetic mapping of the therapeutic compound accumulation in rice and the development of similar therapeutic rice varieties through molecular breeding.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for ß-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in ß-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285ß+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the ß-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74-90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and ß-carotene content. The agronomic performance of the four lines was similar (78.83-99.44%) to that of the recurrent parent, and ß-carotene content (7.541-8.711 µg/g) was on par with the donor parent. Our study is the first to improve the ß-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.

OsARD4 encoding an acireductone dioxygenase improves root architecture in rice by promoting development of secondary roots.

This study was aimed at unravelling the molecular basis of root growth behavior in a drought-tolerant upland rice genotype, Nootripathu. Root tips of Nootripathu were found to possess shorter root caps and a greater number of dividing cells, favoring faster elongation compared to shallow-rooted IR20. Width and length of cortical cells in the roots of rapidly growing Nootripathu were found to be two to three times higher than IR20. Evaluation of shallow-rooted IR20, deep-rooted Nootripathu and their Recombinant Inbred Lines (RILs) for root characteristics revealed the presence of genetic variation for root traits among RILs. 2D-PAGE analysis of proteins in roots of IR20, Nootripathu and bulks of extreme RILs differing in root traits resulted in the identification of proteins co-segregating with root growth behavior and co-localized with QTLs for root traits. A putative candidate gene, OsARD4, encoding an "acireductone dioxygenase" was validated for its role in modulating the root growth pattern through genetic transformation. Transgenic ASD16 rice plants engineered for the overexpression of OsARD4 exhibited root growth characteristics similar to those of Nootripathu, including faster radical emergence, more rapid elongation of primary roots, early initiation of crown/lateral roots, and higher root biomass than the non-transgenic plants.