Growing Conditions and Varietal Ecologies Differently Regulates the Growth-regulating-factor (GRFs) Gene Family in Rice.

Background: Growth-regulating factors (GRFs) are crucial in rice for controlling plant growth and development. Among the rice cultivation practices, aerobic methods are water efficient but result in significant yield reduction relative to non-aerobic cultivation. Therefore, mechanistic insights into aerobic rice cultivation are important for improving the aerobic performance of rice. Objectives: This study aimed to examine the evolution of GRFs in different rice species, analyse the phenotypic differences between aerobic and non-aerobic conditions in three rice varieties, and assess the expression of GRFs in these varieties under both aerobic and non-aerobic conditions. Materials and Methods: This study comprehensively examined the GRFs gene family in 11 rice species (Oryza barthii, Oryza brachyantha, Oryza glaberrima, Oryza glumipatula, Oryza sativa subsp. indica, Oryza longistaminata, Oryza meridionalis, Oryza nivara, Oryza punctata, Oryza rufipogon, Oryza sativa subsp. japonica) focusing on phylogenetic analysis. Additionally, the expression patterns of 12 GRFs were investigated in three distinct genotypes of O. sativa subsp. indica rice, under both non-aerobic and aerobic conditions. Results: Three major phylogenetic clades were formed based on conserved motifs in the 123 GRFs proteins in eleven rice species. Further, novel motifs were identified especially in O. longistaminata indicative of the species level evolutionary differences in rice. Among the trait performance, the number of tillers was reduced by ~ 36% under aerobic conditions, but the reduction was found to be less in CR Dhan 201, an aerobic variety. Besides, three GRFs namely GRF3, GRF4, and GRF7 were found to be distinct in expression between aerobic and non-aerobic conditions. Conclusion: Three GRF genes namely GRF3, GRF4, and GRF7 could be associated with the aerobic adaptation in rice.

Strigolactone GR24-mediated mitigation of phosphorus deficiency through mycorrhization in aerobic rice.

Strigolactones (SLs) are a new class of plant hormones that play a significant role in regulating various aspects of plant growth promotion, stress tolerance and influence the rhizospheric microbiome. GR24 is a synthetic SL analog used in scientific research to understand the effects of SL on plants and to act as a plant growth promoter. This study aimed to conduct hormonal seed priming at different concentrations of GR24 (0.1, 0.5, 1.0, 5.0 and 10.0 µM with and without arbuscular mycorrhizal fungi (AMF) inoculation in selected aerobic rice varieties (CR Dhan 201, CR Dhan 204, CR Dhan 205, and CR Dhan 207), Kasalath-IC459373 (P-tolerant check), and IR-36 (P-susceptible check) under phosphorus (P)-deficient conditions to understand the enhancement of growth and priming effects in mycorrhization. Our findings showed that seed priming with 5.0 µM SL GR24 enhanced the performance of mycorrhization in CR Dhan 205 (88.91 %), followed by CR Dhan 204 and 207, and AMF sporulation in CR Dhan 201 (31.98 spores / 10 gm soil) and CR Dhan 207 (30.29 spores / 10 g soil), as well as rice growth. The study showed that the highly responsive variety CR Dhan 207 followed by CR Dhan 204, 205, 201, and Kasalath IC459373 showed higher P uptake than the control, and AMF treated with 5.0 µM SL GR24 varieties CR Dhan 205 followed by CR Dhan 207 and 204 showed the best performance in plant growth, chlorophyll content, and soil functional properties, such as acid and alkaline phosphatase activity, soil microbial biomass carbon (MBC), dehydrogenase activity (DHA), and fluorescein diacetate activity (FDA). Overall, AMF intervention with SL GR24 significantly increased plant growth, soil enzyme activity, and uptake of P compared to the control. Under P-deficient conditions, seed priming with 5.0 µM strigolactone GR24 and AMF inoculum significantly increased selected aerobic rice growth, P uptake, and soil enzyme activities. Application of SLs formulations with AMF inoculum in selected aerobic rice varieties, CR Dhan 207, CR Dhan 204, and CR Dhan 205, will play an important role in mycorrhization, growth, and enhancement of P utilization under P- nutrient deficient conditions.

Long-term impact of pulses crop rotation on soil fungal diversity in aerobic and wetland rice cultivation.

Pulse crop rotation in rice cultivation is a widely accepted agronomic practice. Depending upon the water regime, rice cultivation has been classified into wetland and aerobic practices. However, no studies have been conducted so far to understand the impact of pulse crop rotation and rice mono-cropping on fungal diversity, particularly in aerobic soil. A targeted metagenomic study was conducted to compare the effects of crop rotations (rice-rice and rice-pulse) on fungal diversity in wetland and aerobic rice soils. Out of 445 OTUs, 41.80% was unknown and 58.20% were assigned to six phyla, namely Ascomycota (56.57%), Basidiomycota (1.32%), Zygomycota (0.22%), Chytridiomycota (0.04%), Glomeromycota (0.03%), and Blastocladiomycota (0.02%). Functional trait analysis found wetland rice-pulse rotation increased symbiotrophs (36.7%) and saprotrophs (62.1%) population, whereas higher pathotrophs were found in aerobic rice-rice (62.8%) and rice-pulse (61.4%) cropping system. Certain soil nutrients played a major role in shaping the fungal community; Ca had significant (p < 0.05) positive impact on saprotroph, symbiotroph and endophytes, whereas Cu had significant (p < 0.05) negative impact on pathotrophs. This study showed that rice-pulse crop rotation could enhance the saprophytic and symbiotic fungal diversity in wetland and reduce the population of pathogens in aerobic rice cultivation.

Arbuscular Mycorrhizal Fungi Response on Soil Phosphorus Utilization and Enzymes Activities in Aerobic Rice under Phosphorus-Deficient Conditions.

The prominence of arbuscular mycorrhizal fungi (AMF) in sustainable rice production has long been recognized. However, there is little information about AMF response in aerobic rice cultivation under phosphorus (P)-deficient conditions. The aim of this experiment was to compare and determine the preeminent AMF effects on rice mycorrhizal colonization, responsiveness, P utilization, and different growth-promoting traits under P-deficient conditions. Different AMF genera viz. (Funneliformis sp., Rhizophagus sp., Glomus sp., Acaulospora sp., and Claroideoglomus sp.) in four different aerobic rice varieties developed by ICAR-NRRI, India (CR Dhan 201, CR Dhan 204, CR Dhan 205, and CR Dhan 207) were investigated using the check P-susceptible variety (IR 36) and the P-tolerant variety (Kasalath IC459373). Data analyzed through linear modeling approaches and bivariate associations found that AMF colonization was highly correlated with soil enzymes, particularly fluorescein diacetate (FDA) and plant P uptake. The microbial biomass carbon (MBC) and FDA content were significantly changed among rice varieties treated with AMF compared to uninoculated control. Out of four different rice varieties, CR Dhan 207 inoculated with AMF showed higher plant P uptake compared to other varieties. In all the rice varieties, AMF colonization had higher correlation coefficients with soil enzymes (FDA), MBC, and plant P uptake than uninoculated control. The present study indicates that AMF intervention in aerobic rice cultivation under P-deficient conditions significantly increased plant P uptake, soil enzymes activities and plant growth promotion. Thus, the information gathered from this study will help us to develop a viable AMF package for sustainable aerobic rice cultivation.

Mitochondrial markers differentiate two distinct phylogenetic groups in indigenous rice landraces of northeast India: an evolutionary insight.

The inheritance of the mitochondria genome and its diversity is unique for genetic and evolutionary studies relative to nuclear genomes. Northeast India and Himalayan regions are considered as one of the centres of indica rice origin. Also, rice diversity in northeast India is very distinct and highly suited for evolutionary studies. Although reports are available on the genetic diversity of indigenous northeast rice landraces, its relationship with the wild relatives is not yet properly explored and understood. In an attempt, mitochondrial markers were used to study the evolutionary relationship between the 68 landraces of northeast India and wild relatives (O. rufipogon and O. nivara) along with IR64 (indica) and Nipponbare (japonica) were taken as reference cultivars. Phylogenetically, the findings include two distinct clusters in the indigenous northeast India landraces representing indica and japonica groups. Further, the wild relatives and ~60% of northeast India landraces were identified to be closely related to the Nipponbare cluster. Besides, landraces of northeast India grouping with the indica group (IR64) are characterized by the absence of wild relatives. This indicates that there are two distinct evolutionary paths in the origin of northeast Indian rice landraces based on mitochondrial markers diversity and it is proposed that the inheritance of mitochondria, mitonuclear genome interactions, and bottleneck events could have genetically separated these two phylogenetically unique groups of northeast rice landraces.

Genome Organization and Comparative Evolutionary Mitochondriomics of Brown Planthopper, Nilaparvata lugens Biotype 4 Using Next Generation Sequencing (NGS).

Nilaparvata lugens is the main rice pest in India. Until now, the Indian N. lugens mitochondrial genome has not been sequenced, which is a very important basis for population genetics and phylogenetic evolution studies. An attempt was made to sequence two examples of the whole mitochondrial genome of N. lugens biotype 4 from the Indian population for the first time. The mitogenomes of N. lugens are 16,072 and 16,081 bp long with 77.50% and 77.45% A + T contents, respectively, for both of the samples. The mitochondrial genome of N. lugens contains 37 genes, including 13 protein-coding genes (PCGs) (cox1-3, atp6, atp8, nad1-6, nad4l, and cob), 22 transfer RNA genes, and two ribosomal RNA (rrnS and rrnL) subunits genes, which are typical of metazoan mitogenomes. However, both samples of N. lugens mitogenome in the present study retained one extra copy of the trnC gene. Additionally, we also found 93 bp lengths for the atp8 gene in both of the samples, which were 60-70 bp less than that of the other sequenced mitogenomes of hemipteran insects. The phylogenetic analysis of the 19 delphacids mitogenome dataset yielded two identical topologies when rooted with Ugyops sp. in one clade, and the remaining species formed another clade with P. maidis and M. muiri being sisters to the remaining species. Further, the genus Nilaparvata formed a separate subclade with the other genera (Sogatella, Laodelphax, Changeondelphax, and Unkanodes) of Delphacidae. Additionally, the relationship among the biotypes of N. lugens was recovered as the present study samples (biotype-4) were separated from the three biotypes reported earlier. The present study provides the reference mitogenome for N. lugens biotype 4 that may be utilized for biotype differentiation and molecular-aspect-based future studies of N. lugens.

In-silico evolutionary analysis of plant-OBERON proteins during compatible MYMV infection in respect of improving host resistance.

Yellow mosaic disease (YMD) of pulses caused by mungbean yellow mosaic virus is a major threat to crop production. An infection that is compatible with regulating and interacting host proteins and the virus causes YMD. Oberon families of proteins OBE1-4 and VIN1-4 are imperative for plants, functions in meristem and vascular development, and were also regulated during compatible disease infection. Furthermore, in-silico expression results suggested the involvement of OBE1 and OBE2 proteins during virus infection of Vigna, Arabidopsis and soybean. Moreover, a common ancestor for the meristem and virus movement related Oberons was inferred through phylogenetic analysis. Protein interaction studies showed three amino acids (Aspartate, glutamate and lysine) in the plant homeodomain (PHD), involved in interaction with the N-terminal region of the virus movement protein and were also conserved in both monocot and dicots. Additionally, major differences in the nuclear localization signal (NLS) showing clade specific conservation and significant variation between dicots and monocots were ascertained in meristem and virus movement related Oberons. Consequently, a combination of PHD, CCD and their interactions with the VPg viral domain increases the susceptibility to YMD. Further, modification in the NLS regions of the viral movement clade Oberons, to knock out allele generation in the OBE1 and OBE2 homologs through genome-editing approaches could be established as alternate strategies for the improvement of host resistance and control yellow mosaic disease in plants, especially in pulse crops.

Role of histone deacetylase inhibitors in androgenic callus induction of Oryza sativa sub indica, in sight into evolution and mode of action of histone deacetylase genes.

BACKGROUND: The potential of paddy breeding has reached its pinnacle, and hybrids have been the principal research outcome. Hence, our hypothesis was based on improvising the callus induction efficiency of recalcitrant Oryza sativa sub. indica hybrids by intervening into their cellular functions like cell division and histone regulation for the production of doubled haploids, a better output compared to hybrids. METHODOLOGY AND RESULTS: Insight into the mechanism of cell division is the foremost concern in altering the same and hence studies on evolution, expression and action of histone deacetylase and its 12 genes (9 HDA and 3 HD-tunin genes) were chosen in the hypothesis. Expression of HDA genes at three stages (anther dehiscence, 1st callusing and second callusing stages) with inhibitor (trichostatin-A) interventions indicated 1st callusing stage as the most important in influencing callus induction and also the genes HDA19, 6, 15 and 5 were the most important. TSA alone had a significant impact on the regulation of the genes HDT 702, HDA19, HDA9, and HDA6. Higher expression of HDA19 and HDA6 was involved in maximizing callus induction; HDA15 had an antagonistic expression compared to HDA19/6 and might be involved in chlorophyll regulation during regeneration. Results of evolutionary analysis on histone deacetylases indicated a long and single lineage of origin denoting its importance in the basic cellular functions. The tubulin deacetylation gene HDA5, which was exclusively found in dicotyledons, had a recent evolutionary history only from terrestrial plants, and also had significant conservation in its motifs and NLS region. CONCLUSION: By combating the recalcitrant nature of Indica cultivars, molecular editing on a combination of HDA genes will enhance the callus induction and regeneration efficiency of the next generation of doubled haploids, therby improving the total yield.

The potentiality of rice microsatellite markers in assessment of cross-species transferability and genetic diversity of rice and its wild relatives.

The main aim of this study is to assess the potentiality of SSR markers for the identification of the cross-species transferability frequency in a large set of the diverse genome types of wild relative rice along with cultivated rice. Here, we used 18 different rice genotypes representing nine different genome types with 70 SSR markers to investigate the potentiality of cross-species transferability rate. The overall cross-species transferability of SSR markers across the 18 rice genotypes ranged from 38.9% (RM280 and RM447) to 100% (RM490, RM318, RM279, RM18877 and RM20033, RM19303) with an average of 76.58%. Also, cross-species transferability across chromosome ranged from 54.4% (chromosome 4) to 86.5% (chromosome 2) with an average of 74.35%. The polymorphism information content of the markers varied from 0.198 (RM263) to 0.868 (RM510) with a mean of 0.549 ± 0.153, showing high discriminatory power. The highest rate of cross-transferability was observed in O. rufipogon (97%), The highest rate of cross-species transferability was in O. rufipogon (97.00%), followed by O. glaberrima (94.20%), O. nivara (92.80%), Swarna (92.80%), O. longistaminata (91.40%), O. eichingeri (90%), O. barthii (88.50%), O. alta (82.80%), O. australiensis (77.10%), O. grandiglumis (74.20%), O. officinalis (74.20%), Zizania latifolia (70.00%), O. latifolia (68.50%), O. brachyantha (62.80%), Leersia perrieri (57.10%) and O. ridleyi (41.40%) with least in O. coarctata (28.50%). A total of 341 alleles from 70 loci were detected with the number of alleles per locus ranged from 2 to 12. Based on dendrogram analysis, the AA genome groups was separated as distinct group from the rest of the genome types. Similarly, principal coordinate analysis and structure analysis clearly separated the AA genome type from the rest of the genome types. Through the analysis of molecular variance, more variance (51%) was observed among the individual, whereas less (14%) was observed among the population. Thus, our findings may offer a valuable resource for studying the genetic diversity and relationship to facilitate the understanding of the complex mechanism of the origin and evolutionary processes of different Oryza species and wild relative rice.

Host-mediated RNAi of a Notch-like receptor gene in Meloidogyne incognita induces nematode resistance.

GLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue of Caenorhabditis elegans glp-1 was identified in Meloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in the C. elegans gene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression of glp-1 throughout the life cycle of M. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy of Mi-glp-1, transgenic Arabidopsis plants carrying double-stranded RNA constructs of glp-1 were generated, and infection of these plants with M. incognita resulted in a 47-50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40-60% reductions in the transcript levels of the targeted glp-1 gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis in M. incognita and its potential use as a target in the management of nematode infestations in plants.

Effect of multiple allelic combinations of genes on regulating grain size in rice.

The grain size is one of the complex trait of rice yield controlled by a plethora of interaction of several genes in different pathways. The present study was undertaken to investigate the influence of seven known grain size regulating genes: DEP1, GS7, GS3, GW8, GL7, GS5 and GW2. A wide phenotypic variation for grain length, grain width and grain length-width ratio were observed in 89 germplasm. The correlation analysis showed a strong association among these three grain traits viz. GL, GW, GLWR and TGW which play important roles in determining the final rice grain size. Except for GW2, all six genes showed strong association with grain size traits. A total of 21 alleles were identified with an average of 2.1 allele/locus in 89 germplasm of which seven alleles were found to be favourable alleles for improving the grain size with the frequency range of 24 (26.97%) to 82 (92.13%); the largest was found in GS5 followed by GW8, GL7, DEP1, GS3 and GS7 genes. Through ANOVA, four markers (GS3-PstI, S9, GID76 and GID711) of three genes (GS3, DEP1 and GL7) were found significantly associated with all the three traits (GL, GLWR and TGW). Concurrent results of significant associations of grain size traits with other markers were observed in both analysis of variance and genetic association through the general linear model. Besides, the population structure analysis, cluster analysis and PCoA divided the entire germplasm into three sub-groups with the clear-cut demarcation of long and medium grain types. The present results would help in formulating strategies by selecting suitable candidate markers/genes for obtaining preferred grain shape/size and improving grain yield through marker-assisted breeding.

Genome-wide analysis identifies chickpea (Cicer arietinum) heat stress transcription factors (Hsfs) responsive to heat stress at the pod development stage.

The heat stress transcription factors (Hsfs) play a prominent role in thermotolerance and eliciting the heat stress response in plants. Identification and expression analysis of Hsfs gene family members in chickpea would provide valuable information on heat stress responsive Hsfs. A genome-wide analysis of Hsfs gene family resulted in the identification of 22 Hsf genes in chickpea in both desi and kabuli genome. Phylogenetic analysis distinctly separated 12 A, 9 B, and 1 C class Hsfs, respectively. An analysis of cis-regulatory elements in the upstream region of the genes identified many stress responsive elements such as heat stress elements (HSE), abscisic acid responsive element (ABRE) etc. In silico expression analysis showed nine and three Hsfs were also expressed in drought and salinity stresses, respectively. Q-PCR expression analysis of Hsfs under heat stress at pod development and at 15 days old seedling stage showed that CarHsfA2, A6, and B2 were significantly upregulated in both the stages of crop growth and other four Hsfs (CarHsfA2, A6a, A6c, B2a) showed early transcriptional upregulation for heat stress at seedling stage of chickpea. These subclasses of Hsfs identified in this study can be further evaluated as candidate genes in the characterization of heat stress response in chickpea.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

NAC transcription factor genes: genome-wide identification, phylogenetic, motif and cis-regulatory element analysis in pigeonpea (Cajanus cajan (L.) Millsp.).

The NAC (NAM, ATAF and CUC) proteins are plant-specific transcription factors implicated in development and stress responses. In the present study 88 pigeonpea NAC genes were identified from the recently published draft genome of pigeonpea by using homology based and de novo prediction programmes. These sequences were further subjected to phylogenetic, motif and promoter analyses. In motif analysis, highly conserved motifs were identified in the NAC domain and also in the C-terminal region of the NAC proteins. A phylogenetic reconstruction using pigeonpea, Arabidopsis and soybean NAC genes revealed 33 putative stress-responsive pigeonpea NAC genes. Several stress-responsive cis-elements were identified through in silico analysis of the promoters of these putative stress-responsive genes. This analysis is the first report of NAC gene family in pigeonpea and will be useful for the identification and selection of candidate genes associated with stress tolerance.