Molecular genetics and phenotypic assessment of foxtail millet (Setaria italica (L.) P. Beauv.) landraces revealed remarkable variability of morpho-physiological, yield, and yield-related traits.

Foxtail millet (Setaria italica (L.) P. Beauv.) is highly valued for nutritional traits, stress tolerance and sustainability in resource-poor dryland agriculture. However, the low productivity of this crop in semi-arid regions of Southern India, is further threatened by climate stress. Landraces are valuable genetic resources, regionally adapted in form of novel alleles that are responsible for cope up the adverse conditions used by local farmers. In recent years, there is an erosion of genetic diversity. We have hypothesized that plant genetic resources collected from the semi-arid climatic zone would serve as a source of novel alleles for the development of climate resilience foxtail millet lines with enhanced yield. Keeping in view, there is an urgent need for conservation of genetic resources. To explore the genetic diversity, to identify superior genotypes and novel alleles, we collected a heterogeneous mixture of foxtail millet landraces from farmer fields. In an extensive multi-year study, we developed twenty genetically fixed foxtail millet landraces by single seed descent method. These landraces characterized along with four released cultivars with agro-morphological, physiological, yield and yield-related traits assessed genetic diversity and population structure. The landraces showed significant diversity in all the studied traits. We identified landraces S3G5, Red, Black and S1C1 that showed outstanding grain yield with earlier flowering, and maturity as compared to released cultivars. Diversity analysis using 67 simple sequence repeat microsatellite and other markers detected 127 alleles including 11 rare alleles, averaging 1.89 alleles per locus, expected heterozygosity of 0.26 and an average polymorphism information content of 0.23, collectively indicating a moderate genetic diversity in the landrace populations. Euclidean Ward's clustering, based on the molecular markers, principal coordinate analysis and structure analysis concordantly distinguished the genotypes into two to three sub-populations. A significant phenotypic and genotypic diversity observed in the landraces indicates a diverse gene pool that can be utilized for sustainable foxtail millet crop improvement.

Potassium Chloroaurate-Mediated In Vitro Synthesis of Gold Nanoparticles Improved Root Growth by Crosstalk with Sucrose and Nutrient-Dependent Auxin Homeostasis in Arabidopsis thaliana.

In a hydroponic system, potassium chloroaurate (KAuCl4) triggers the in vitro sucrose (Suc)-dependent formation of gold nanoparticles (AuNPs). AuNPs stimulate the growth of the root system, but their molecular mechanism has not been deciphered. The root system of Arabidopsis (Arabidopsis thaliana) exhibits developmental plasticity in response to the availability of various nutrients, Suc, and auxin. Here, we showed the roles of Suc, phosphorus (P), and nitrogen (N) in facilitating a AuNPs-mediated increase in root growth. Furthermore, the recuperating effects of KAuCl4 on the natural (IAA) auxin-mediated perturbation of the root system were demonstrated. Arabidopsis seedlings harboring the cell division marker CycB1;1::CDB-GUS provided evidence of the restoration efficacy of KAuCl4 on the IAA-mediated inhibitory effect on meristematic cell proliferation of the primary and lateral roots. Arabidopsis harboring synthetic auxin DR5rev::GFP exhibited a reinstating effect of KAuCl4 on IAA-mediated aberration in auxin subcellular localization in the root. KAuCl4 also exerted significant and differential recuperating effects on the IAA-mediated altered expression of the genes involved in auxin signaling and biosynthetic pathways in roots. Our results highlight the crosstalk between KAuCl4-mediated improved root growth and Suc and nutrient-dependent auxin homeostasis in Arabidopsis.

ATL8, a RING E3 ligase, modulates root growth and phosphate homeostasis in Arabidopsis.

Ubiquitination-mediated post-translational modification of proteins is a pivotal regulatory mechanism involved in the growth and development of the plant. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of RING-type ubiquitin ligases (E3) and ATL8 is a membrane-localized protein. Here, a reverse genetics approach was used to elucidate the role of ATL8 in phosphate (Pi) homeostasis. Deficiencies of Pi and sucrose (Suc) enhanced the relative expression level of ATL8 in different tissues of the wild-type (Wt). The relative expression level of ATL8 was attenuated and augmented in the mutant (atl8) and overexpression lines (Oe1 and Oe2), respectively. There were significant reductions in different root traits, root hairs, root to shoot ratio, and total Pi content in atl8 compared with the Wt under different Pi regimes. On the contrary, Oe1 and Oe2 lines exhibited enhancement in some of these traits. Noticeably, anthocyanin content was significantly reduced in Oe1 and Oe2 compared with the Wt and atl8 under P- condition. Abscisic acid (ABA) treatment led to an increase in the primary root length of atl8 compared with the Wt, suggesting a cross-talk between ABA and ATL8 on root growth. Furthermore, the relative expression levels of the genes involved in the maintenance of Pi homeostasis (WRKY75, RNS1, E3L, and ACP5) were differentially modulated in atl8, Oe1, and Oe2 compared with the Wt under different Pi regimes. The results revealed the pivotal role of ATL8 in mediating morphophysiological and molecular adaptive responses to Pi deficiency.

Revealing the effect of seed phosphorus concentration on seedling vigour and growth of rice using mutagenesis approach.

The harvested plant products, specifically, the grains of cereals are major drivers of soil phosphorus (P) depletion. However, the breeding or biotechnology efforts to develop low P seeds have not been attempted because of possible adverse effects on seedling vigour and crop establishment. Several studies have contradictory observations on influence of seed P on seedling vigour. Lack of appropriate genetic material has been the major bottleneck in reaching the consensus. In this study, we used 30 EMS induced mutants of rice cultivar Nagina22 to understand the role of seed P on seedling vigour and associated physiological processes. Seedling vigour, morpho-physiological characteristics, acid phosphatases, alpha-amylase, and expression of P transporter genes were analyzed in seedlings obtained from seeds of high and low grain P mutants. The study suggests that seed P has a significant role on seedling vigour, chlorophyll content and photosynthesis process of young seedlings, and P transport from roots. Notably, we identified few mutants such as NH4791, NH4785, NH4714, NH4663, NH4614, and NH4618 which showed least influence of low seed P on seedling vigour and other metabolic processes. Therefore, these mutants can be used in breeding programs aiming for development of low P grains. Also, these and other identified mutants can be used to decipher the genetic and molecular mechanisms regulating the differential response of seed P on germination, seedling vigour and several other physiological processes influencing the crop growth and establishment.

Differential efficacy of water lily cultivars in phytoremediation of eutrophic water contaminated with phosphorus and nitrogen.

Excessive inputs of phosphorus (P) and nitrogen (N) trigger eutrophication of the water bodies, which promotes the undesirable growth of algal bloom and deterioration of the water quality, and aquatic biodiversity. Macrophytes provide an environmentally benign and economically viable paradigm for the ecological restoration of eutrophic aquatic ecosystems. Water lily (Nymphaea) is largely used as ornamental plant for landscaping, and it has been documented that water lily possesses the potentiality in mitigating polluted aquatic environments. In the present study, water lily cultivars Nymphaea Texas Dawn (NTD), Nymphaea Colorado (NC), Nymphaea Madame Wilfron Gonnère (NMWG), and Nymphaea Sunshine Princess (NSP) were investigated for their potency in alleviating the eutrophication. The concentrations of total P and total N were significantly higher in the leaves of NC and NSP compared with NTD and NMWG. Therefore, NC and NSP were selected for subsequent studies to decipher their recuperation efficacy on eutrophic waters at different growth stages. NC and NSP significantly reduced the concentrations of eutrophication indicators i.e., total P, NH4+-N, and chemical oxygen demand in different gradients of the simulated eutrophic water in a growth-dependent manner. On the contrary, NC and NSP triggered a significant increase in the concentration of dissolved oxygen particularly at the seedling stage. Notably, the concentrations of total P (shoot and root) and total N (root) were relatively higher in NSP than NC. The study thus revealed a growth-dependent differential efficacy of NSP and NC in mitigating the different eutrophic waters.

PAP90, a novel rice protein plays a critical role in regulation of D1 protein stability of PSII.

Introduction: Photosystem II (PSII) protein complex plays an essential role in the entire photosynthesis process. Various known and unknown protein factors are involved in the dynamics of the PSII complex that need to be characterized in crop plants for enhancing photosynthesis efficiency and productivity. Objectives: The experiments were conducted to decipher the regulatory proteins involved in PSII dynamics of rice crop. Methods: A novel rice regulatory protein PAP90 (PSII auxiliary protein ~90 kDa) was characterized by generating a loss-of-function mutant pap90. The mutation was characterized at molecular level followed by various experiments to analyze the morphological, physiological and biochemical processes of mutant under control and abiotic stresses. Results: The pap90 mutant showed reduced photosynthesis due to D1 protein instability that subsequently causes inadequate accumulation of thylakoid membrane complexes, especially PSII and decreases PSII functional efficiency. Expression of OsFtsH family genes and proteins were induced in the mutant, which are known to play a key role in D1 protein degradation and turnover. The reduced D1 protein accumulation in the mutant increased the production of reactive oxygen species (ROS). The accumulation of ROS along with the increased activity of antioxidant enzymes and induced expression of stress-associated genes and proteins in pap90 mutant contributed to its water-limited stress tolerance ability. Conclusion: We propose that PAP90 is a key auxiliary protein that interacts with D1 protein and maintains its stability, thereby promoting subsequent assembly of the PSII and associated membrane complexes.

CRISPR mediated genome engineering to develop climate smart rice: Challenges and opportunities.

Rice is a staple food crop, which ensures the calorie requirement of half of the world's population. With the continued increase in population, rice will play a key role in achieving the food security. However, in the constantly shrinking scenario of rice fields, the necessity of these extra grains of rice must be met by reducing the yield loss due to various abiotic and biotic stresses. The adverse effects of climate impact both quality and quantity of rice production. One of the most desirable applications of CRISPR/Cas technology would be to develop climate smart rice crop to sustain and enhance its productivity in the changing environment. In this review, we analyze the desirable phenotypes and responsible genetic factors, which can be utilized to develop tolerance against major abiotic stresses imposed by climate change through genome engineering. The possibility of utilizing the information from wild resources to engineer the corresponding alleles of cultivated rice has been presented. We have also shed light on available resources for generating genome edited rice lines. The CRISPR/Cas mediated genome editing strategies for engineering of novel genes were proposed to create a plant phenotype, which can face the adversities of climate change. Further, challenges of off-targets and undesirable phenotype were discussed.

The potential application of genome editing by using CRISPR/Cas9, and its engineered and ortholog variants for studying the transcription factors involved in the maintenance of phosphate homeostasis in model plants.

Phosphorus (P), an essential macronutrient, is pivotal for growth and development of plants. Availability of phosphate (Pi), the only assimilable P, is often suboptimal in rhizospheres. Pi deficiency triggers an array of spatiotemporal adaptive responses including the differential regulation of several transcription factors (TFs). Studies on MYB TF PHR1 in Arabidopsis thaliana (Arabidopsis) and its orthologs OsPHRs in Oryza sativa (rice) have provided empirical evidence of their significant roles in the maintenance of Pi homeostasis. Since the functional characterization of PHR1 in 2001, several other TFs have now been identified in these model plants. This raised a pertinent question whether there are any likely interactions across these TFs. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has provided an attractive paradigm for editing genome in plants. Here, we review the applications and challenges of this technique for genome editing of the TFs for deciphering the function and plausible interactions across them. This technology could thus provide a much-needed fillip towards engineering TFs for generating Pi use efficient plants for sustainable agriculture. Furthermore, we contemplate whether this technology could be a viable alternative to the controversial genetically modified (GM) rice or it may also eventually embroil into a limbo.

Characterization of the loss-of-function mutant NH101 for yield under phosphate deficiency from EMS-induced mutants of rice variety Nagina22.

In earlier studies at IIRR, Hyderabad, screening of ∼2000 EMS mutants of the rice variety Nagina22 (N22) resulted in the identification of 11 loss-of-function mutants with zero grain yield in Pi-deprived soil under field condition. Among these mutants, NH101 was selected for comparative analyses with N22 for various morphophysiological and/or molecular traits during growth in a hydroponic system (7 d) and in a pot soil (50% flowering) under different Pi regime. The total length of the seminal and adventitious roots, agronomic traits (panicle length and unfilled spikelet/panicle), activities of the antioxidant enzymes (SOD, POD, and APX), and the relative expression levels of the genes involved in the maintenance of Pi homeostasis (MPH) i.e., OsPHR2, SPX1/2 OsPT4, 6, and 8 showed significant increase in the Pi-deprived mutant compared with N22. Whereas, some of the traits showed significant reduction in NH101 than N22 such as number of tillers and filled spikelets/panicle, yield, contents of Pi and externally secreted APase, activity of CAT, and the relative expression levels of MPH genes i.e., OsmiR399a, OsPHO1;2, OsIPS1, OsPAP10a, OsPT2, 9, and 10. The study highlighted wide spectrum differential effects of the mutation in NH101 on various traits that play important roles governing the maintenance of Pi homeostasis. This mutant thus provides a rich repository of genetic material amenable for the identification of the genes that are pivotal for Pi use efficiency.

Activation-tagging in indica rice identifies a novel transcription factor subunit, NF-YC13 associated with salt tolerance.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with three distinct NF-YA, NF-YB and NF-YC subunits. It plays important roles in plant growth, development and stress responses. We have reported earlier on development of gain-of-function mutants in an indica rice cultivar, BPT-5204. Now, we screened 927 seeds from 70 Ac/Ds plants for salinity tolerance and identified one activation-tagged salt tolerant DS plant (DS-16, T3 generation) that showed enhanced expression of a novel 'histone-like transcription factor' belonging to rice NF-Y subfamily C and was named as OsNF-YC13. Localization studies using GFP-fusion showed that the protein is localized to nucleus and cytoplasm. Real time expression analysis confirmed upregulation of transcript levels of OsNF-YC13 during salt treatment in a tissue specific manner. Biochemical and physiological characterization of the DS-16 revealed enhanced K+/Na+ ratio, proline content, chlorophyll content, enzymes with antioxidant activity etc. DS-16 also showed transcriptional up-regulation of genes that are involved in salinity tolerance. In-silico analysis of OsNF-YC13 promoter region evidenced the presence of various key stress-responsive cis-regulatory elements. OsNF-YC13 subunit alone does not appear to have the capacity for direct transcription activation, but appears to interact with the B- subunits in the process of transactivation.