Prioritization of candidate genes for major QTLs governing yield traits employing integrated multi-omics approach in rice (Oryza sativa L.).

Rapidly identifying candidate genes underlying major QTLs is crucial for improving rice (Oryza sativa L.). In this study, we developed a workflow to rapidly prioritize candidate genes underpinning 99 major QTLs governing yield component traits. This workflow integrates multiomics databases, including sequence variation, gene expression, gene ontology, co-expression analysis, and protein-protein interaction. We predicted 206 candidate genes for 99 reported QTLs governing ten economically important yield-contributing traits using this approach. Among these, transcription factors belonging to families of MADS-box, WRKY, helix-loop-helix, TCP, MYB, GRAS, auxin response factor, and nuclear transcription factor Y subunit were promising. Validation of key prioritized candidate genes in contrasting rice genotypes for sequence variation and differential expression identified Leucine-Rich Repeat family protein (LOC_Os03g28270) and cytochrome P450 (LOC_Os02g57290) as candidate genes for the major QTLs GL1 and pl2.1, which govern grain length and panicle length, respectively. In conclusion, this study demonstrates that our workflow can significantly narrow down a large number of annotated genes in a QTL to a very small number of the most probable candidates, achieving approximately a 21-fold reduction. These candidate genes have potential implications for enhancing rice yield.

Development of miRNA-SSR and target-SSR markers from yield-associate genes and their applicability in the assessment of genetic diversity and association mapping in rice (Oryza sativa L.).

The gene-derived functional markers are considered effective to use in marker-assisted breeding and genetic diversity analysis. As of now, no functional markers have been identified from miRNAs regulating yield traits. The miRNAs play a key role as regulators in controlling the candidate genes involved in grain yield improvement in rice. In this study, 13 miRNA-SSR and their target gene SSR markers were mined from 29 yield-responsive miRNA along with their 29 target genes in rice. The validation of these markers showed that four miRNA-SSRs and one target gene SSR markers had shown polymorphism among 120 diverse rice genotypes. The PIC values ranged from 0.25 (OsARF18-SSR) to 0.72 (miR408-SSR, miR172b-SSR, and miR396f-SSR) with an average value of 0.57. These polymorphic markers grouped 120 rice genotypes into 3 main clusters based on the levels of high genetic diversity. These markers also showed significant association with key yield traits. Among all, miR172b-SSR showed a strong association with plant height in two seasons. This investigation suggests that this new class of molecular markers has great potential in the characterization of rice germplasm by genetic diversity and population structure and in marker-assisted breeding for the development of high-yielding varieties. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-024-01462-z.

Panicle transcriptome of high-yield mutant indica rice reveals physiological mechanisms and novel candidate regulatory genes for yield under reproductive stage drought stress.

BACKGROUND: Reproductive stage drought stress (RDS) is a major global threat to rice production. Due to climate change, water scarcity is becoming an increasingly common phenomenon in major rice-growing areas worldwide. Understanding RDS mechanisms will allow candidate gene identification to generate novel rice genotypes tolerant to RDS. RESULTS: To generate novel rice genotypes that can sustain yield under RDS, we performed gamma-irradiation mediated mutation breeding in the drought stress susceptible mega rice variety, MTU1010. One of the mutant MM11 (MTU1010 derived mutant11) shows consistently increased performance in yield-related traits under field conditions consecutively for four generations. In addition, compared to MTU1010, the yield of MM11 is sustained in prolonged drought imposed during the reproductive stage under field and in pot culture conditions. A comparative emerged panicle transcriptome analysis of the MTU1010 and MM11 suggested metabolic adjustment, enhanced photosynthetic ability, and hormone interplay in regulating yield under drought responses during emerged panicle development. Regulatory network analysis revealed few putative significant transcription factor (TF)-target interactions involved in integrated signalling between panicle development, yield and drought stress. CONCLUSIONS: A gamma-irradiate rice mutant MM11 was identified by mutation breeding, and it showed higher potential to sustain yield under reproductive stage drought stress in field and pot culture conditions. Further, a comparative panicle transcriptome revealed significant biological processes and molecular regulators involved in emerged panicle development, yield and drought stress integration. The study extends our understanding of the physiological mechanisms and candidate genes involved in sustaining yield under drought stress.

The proteome landscape of the root cap reveals a role for the jacalin-associated lectin JAL10 in the salt-induced endoplasmic reticulum stress pathway.

Rapid climate change has led to enhanced soil salinity, one of the major determinants of land degradation, resulting in low agricultural productivity. This has a strong negative impact on food security and environmental sustainability. Plants display various physiological, developmental, and cellular responses to deal with salt stress. Recent studies have highlighted the root cap as the primary stress sensor and revealed its crucial role in halotropism. The root cap covers the primary root meristem and is the first cell type to sense and respond to soil salinity, relaying the signal to neighboring cell types. However, it remains unclear how root-cap cells perceive salt stress and contribute to the salt-stress response. Here, we performed a root-cap cell-specific proteomics study to identify changes in the proteome caused by salt stress. The study revealed a very specific salt-stress response pattern in root-cap cells compared with non-root-cap cells and identified several novel proteins unique to the root cap. Root-cap-specific protein-protein interaction (PPI) networks derived by superimposing proteomics data onto known global PPI networks revealed that the endoplasmic reticulum (ER) stress pathway is specifically activated in root-cap cells upon salt stress. Importantly, we identified root-cap-specific jacalin-associated lectins (JALs) expressed in response to salt stress. A JAL10-GFP fusion protein was shown to be localized to the ER. Analysis of jal10 mutants indicated a role for JAL10 in regulating the ER stress pathway in response to salt. Taken together, our findings highlight the participation of specific root-cap proteins in salt-stress response pathways. Furthermore, root-cap-specific JAL proteins and their role in the salt-mediated ER stress pathway open a new avenue for exploring tolerance mechanisms and devising better strategies to increase plant salinity tolerance and enhance agricultural productivity.

Root Cap to Soil Interface: A Driving Force Toward Plant Adaptation and Development.

Land plants have developed robust roots to grow in diverse soil ecosystems. The distal end of the root tip has a specialized organ called the 'root cap'. The root cap assists the roots in penetrating the ground, absorbing water and minerals, avoiding heavy metals and regulating the rhizosphere microbiota. Furthermore, root-cap-derived auxin governs the lateral root patterning and directs root growth under varying soil conditions. The root cap formation is hypothesized as one of the key innovations during root evolution. Morphologically diversified root caps in early land plant lineage and later in angiosperms aid in improving the adaptation of roots and, thereby, plants in diverse soil environments. This review article presents a retrospective view of the root cap's important morphological and physiological characteristics for the root-soil interaction and their response toward various abiotic and biotic stimuli. Recent single-cell RNAseq data shed light on root cap cell-type-enriched genes. We compiled root cap cell-type-enriched genes from Arabidopsis, rice, maize and tomato and analyzed their transcription factor (TF) binding site enrichment. Further, the putative gene regulatory networks derived from root-cap-enriched genes and their TF regulators highlight the species-specific biological functions of root cap genes across the four plant species.

Yield-associated putative gene regulatory networks in Oryza sativa L. subsp. indica and their association with high-yielding genotypes.

BACKGROUND: With the increase in population and economies of developing countries in Asia and Africa, the research towards securing future food demands is an imminent need. Among japonica and indica genotypes, indica rice varieties are largely cultivated across the globe. However, our present understanding of yield-contributing gene information stems mainly from japonica and studies on the yield potential of indica genotypes are limited. METHODS AND RESULTS: In the present study, yield contributing orthologous genes previously characterized from japonica varieties were identified in the indica genome and analysed with binGO tool for GO biological processes categorization. Transcription factor binding site enrichment analysis in the promoters of yield-related genes of indica was performed with MEME-AME tool that revealed putative common TF regulators are enriched in flower development, two-component signalling and water deprivation biological processes. Gene regulatory networks revealed important TF-target interactions that might govern yield-related traits. Some of the identified candidate genes were validated by qRT-PCR analysis for their expression and association with yield-related traits among 16 widely cultivated popular indica genotypes. Further, SNP-metabolite-trait association analysis was performed using high-yielding indica variety Rasi. This resulted in the identification of putative SNP variations in TF regulators and targeted yield genes significantly linked with metabolite accumulation. CONCLUSIONS: The study suggests some of the high yielding indica genotypes such as Ravi003, Rasi and Kavya could be used as potential donors in breeding programs based on yield gene expression analysis and SNP-metabolites associations.

The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress.

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but protein levels increased in response to the treatment, particularly in the root tip. Expression of HvHorcH in Arabidopsis thaliana root tips increased salt tolerance. Hence, we conclude that this protein is involved in the adaptation of plants to salinity.

Root engineering in maize by increasing cytokinin degradation causes enhanced root growth and leaf mineral enrichment.

KEY MESSAGE: Root-specific expression of a cytokinin-degrading CKX gene in maize roots causes formation of a larger root system leading to higher element content in shoot organs. The size and architecture of the root system is functionally relevant for the access to water and soil nutrients. A great number of mostly unknown genes are involved in regulating root architecture complicating targeted breeding of plants with a larger root system. Here, we have explored whether root-specific degradation of the hormone cytokinin, which is a negative regulator of root growth, can be used to genetically engineer maize (Zea mays L.) plants with a larger root system. Root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene of Arabidopsis caused the formation of up to 46% more root dry weight while shoot growth of these transgenic lines was similar as in non-transgenic control plants. The concentration of several elements, in particular of those with low soil mobility (K, P, Mo, Zn), was increased in leaves of transgenic lines. In kernels, the changes in concentration of most elements were less pronounced, but the concentrations of Cu, Mn and Zn were significantly increased in at least one of the three independent lines. Our data illustrate the potential of an increased root system as part of efforts towards achieving biofortification. Taken together, this work has shown that root-specific expression of a CKX gene can be used to engineer the root system of maize and alter shoot element composition.

Classification of Grain Amaranths Using Chromosome-Level Genome Assembly of Ramdana, A. hypochondriacus.

In the age of genomics-based crop improvement, a high-quality genome of a local landrace adapted to the local environmental conditions is critically important. Grain amaranths produce highly nutritional grains with a multitude of desirable properties including C4 photosynthesis highly sought-after in other crops. For improving the agronomic traits of grain amaranth and for the transfer of desirable traits to dicot crops, a reference genome of a local landrace is necessary. Toward this end, our lab had initiated sequencing the genome of Amaranthus (A.) hypochondriacus (A.hyp_K_white) and had reported a draft genome in 2014. We selected this landrace because it is well adapted for cultivation in India during the last century and is currently a candidate for TILLING-based crop improvement. More recently, a high-quality chromosome-level assembly of A. hypochondriacus (PI558499, Plainsman) was reported. Here, we report a chromosome-level assembly of A.hyp_K_white (AhKP) using low-coverage PacBio reads, contigs from the reported draft genome of A.hyp_K_white, raw HiC data and reference genome of Plainsman (A.hyp.V.2.1). The placement of A.hyp_K_white on the phylogenetic tree of grain amaranths of known accessions clearly suggests that A.hyp_K_white is genetically distal from Plainsman and is most closely related to the accession PI619259 from Nepal (Ramdana). Furthermore, the classification of another accession, Suvarna, adapted to the local environment and selected for yield and other desirable traits, is clearly Amaranthus cruentus. A classification based on hundreds of thousands of SNPs validated taxonomy-based classification for a majority of the accessions providing the opportunity for reclassification of a few.

Uncovering of natural allelic variants of key yield contributing genes by targeted resequencing in rice (Oryza sativa L.).

In rice (Oryza sativa L.), during the course of domestication, numerous beneficial alleles remain untapped in the progenitor wild species and landraces. This study aims at uncovering these promising alleles of six key genes influencing the yield, such as DEP1, Ghd7, Gn1a, GS3, qSW5 and sd1 by targeted resequencing of the 200 rice genotypes. In all, 543 nucleotide variations including single nucleotide polymorphisms and insertion and deletion polymorphisms were identified from the targeted genes. Of them, 225 were novel alleles, which identified in the present study only and 91 were beneficial alleles that showed significant association with the yield traits. Besides, we uncovered 128 population-specific alleles with indica being the highest of 79 alleles. The neutrality tests revealed that pleiotropic gene, Ghd7 and major grain size contributing gene, GS3 showed positive and balanced selection, respectively during the domestication. Further, the haplotype analysis revealed that some of the rice genotypes found to have rare haplotypes, especially the high yielding variety, BPT1768 has showed maximum of three genes such as Gn1a-8, qSW5-12 and GS3-29. The rice varieties with novel and beneficial alleles along with the rare haplotypes identified in the present study could be of immense value for yield improvement in the rice breeding programs.

Root enhancement in cytokinin-deficient oilseed rape causes leaf mineral enrichment, increases the chlorophyll concentration under nutrient limitation and enhances the phytoremediation capacity.

BACKGROUND: Cytokinin is a negative regulator of root growth, and a reduction of the cytokinin content or signalling causes the formation a larger root system in model plants, improves their growth under drought and nutrient limitation and causes increased accumulation of elements in the shoot. Roots are an important but understudied target of plant breeding. Here we have therefore explored whether root enhancement by lowering the cytokinin content can also be achieved in oilseed rape (Brassica napus L.) plants. RESULTS: Transgenic plants overexpressing the CKX2 gene of Arabidopsis thaliana encoding a cytokinin-degrading cytokinin oxidase/dehydrogenase showed higher CKX activity and a strongly reduced cytokinin content. Cytokinin deficiency led to the formation of a larger root system under different growth conditions, which was mainly due to an increased number of lateral and adventitious roots. In contrast, shoot growth was comparable to wild type, which caused an enhanced root-to-shoot ratio. Transgenic plants accumulated in their leaves higher concentrations of macro- and microelements including P, Ca, Mg, S, Zn, Cu, Mo and Mn. They formed more chlorophyll under Mg- and S-deficiency and accumulated a larger amount of Cd and Zn from contaminated medium and soil. CONCLUSIONS: These findings demonstrate the usefulness of ectopic CKX gene expression to achieve root enhancement in oilseed rape and underpin the functional relevance of a larger root system. Furthermore, the lack of major developmental consequences on shoot growth in cytokinin-deficient oilseed rape indicates species-specific differences of CKX gene and/or cytokinin action.

Zn-fortified cereal grains in field-grown barley by enhanced root cytokinin breakdown.

Zinc (Zn) is an essential element in human nutrition. The concentration of Zn in cereals, which is a staple food in developing countries, is often too low thus contributing to Zn malnutrition in nearly two billion people worldwide. We have reported recently that transgenic barley plants expressing a cytokinin-degrading CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene in their roots form a larger root system and accumulate a higher concentration of Zn in their grains when grown under greenhouse conditions. Here, we have tested this trait under field conditions. Four independent pEPP:CKX lines accumulated an up to 30% higher Zn concentration in their grains as compared to the untransformed control suggesting that this is a stable trait. The increased Zn concentration exceeded the limit set by the HarvestPlus program for wheat. We, therefore, propose that root enhancement achieved by increased degradation of cytokinin in roots can be a sustainable strategy to combat malnutrition caused by Zn deficiency.

Root Engineering in Barley: Increasing Cytokinin Degradation Produces a Larger Root System, Mineral Enrichment in the Shoot and Improved Drought Tolerance.

Root size and architecture are important crop plant traits, as they determine access to water and soil nutrients. The plant hormone cytokinin is a negative regulator of root growth and branching. Here, we generated transgenic barley (Hordeum vulgare) plants with an enlarged root system by enhancing cytokinin degradation in roots to explore the potential of cytokinin modulations in improving root functions. This was achieved through root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE gene. Enhanced biomass allocation to roots did not penalize shoot growth or seed yield, indicating that these plants were not source limited. In leaves of transgenic lines, the concentrations of several macroelements and microelements were increased, particularly those with low soil mobility (phosphorus, manganese, and zinc). Importantly, seeds contained up to 44% more zinc, which is beneficial for human nutrition. Transgenic lines also demonstrated dampened stress responses to long-term drought conditions, indicating lower drought sensitivity. Taken together, this work demonstrates that root engineering of cereals is a promising strategy to improve nutrient efficiency, biofortification, and drought tolerance.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

Shoot- and root-borne cytokinin influences arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is functionally important for the nutrition and growth of most terrestrial plants. Nearly all phytohormones are employed by plants to regulate the symbiosis with AM fungi, but the regulatory role of cytokinin (CK) is not well understood. Here, we used transgenic tobacco (Nicotiana tabacum) with a root-specific or constitutive expression of CK-degrading CKX genes and the corresponding wild-type to investigate whether a lowered content of CK in roots or in both roots and shoots influences the interaction with the AM fungus Rhizophagus irregularis. Our data indicates that shoot CK has a positive impact on AM fungal development in roots and on the root transcript level of an AM-responsive phosphate transporter gene (NtPT4). A reduced CK content in roots caused shoot and root growth depression following AM colonization, while neither the uptake of phosphorus or nitrogen nor the root transcript levels of NtPT4 were significantly affected. This suggests that root CK may restrict the C availability from the roots to the fungus thus averting parasitism by AM fungi. Taken together, our study indicates that shoot- and root-borne CK have distinct roles in AM symbiosis. We propose a model illustrating how plants may employ CK to regulate nutrient exchange with the ubiquitous AM fungi.

Cytokinin Determines Thiol-Mediated Arsenic Tolerance and Accumulation.

The presence of arsenic in soil and water is a constant threat to plant growth in many regions of the world. Phytohormones act in the integration of growth control and stress response, but their role in plant responses to arsenic remains to be elucidated. Here, we show that arsenate [As(V)], the most prevalent arsenic chemical species in nature, causes severe depletion of endogenous cytokinins (CKs) in the model plant Arabidopsis (Arabidopsis thaliana). We found that CK signaling mutants and transgenic plants with reduced endogenous CK levels showed an As(V)-tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V), transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild-type plants. In contrast, CK depletion provoked the coordinated activation of As(V) tolerance mechanisms, leading to the accumulation of thiol compounds such as phytochelatins and glutathione, which are essential for arsenic sequestration. Transgenic CK-deficient Arabidopsis and tobacco lines show a marked increase in arsenic accumulation. Our findings indicate that CK is an important regulatory factor in plant adaptation to arsenic stress.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Cytokinin as a positional cue regulating lateral root spacing in Arabidopsis.

The root systems of plants have developed adaptive architectures to exploit soil resources. The formation of lateral roots (LRs) contributes to root system architecture. Roots of plants with a lower cytokinin status form LR primordia (LRP) in unusually close proximity, indicating a role for the hormone in regulating the positioning of LRs along the main root axis. Data obtained from cytokinin-synthesis mutants of Arabidopsis thaliana combined with gene expression analysis indicate that cytokinin synthesis by IPT5 and LOG4 occurring early during LRP initiation generates a local cytokinin signal abbreviating LRP formation in neighbouring pericycle cells. In addition, IPT3, IPT5, and IPT7 contribute to cytokinin synthesis in the vicinity of existing LRP, thus suppressing initiation of new LRs. Interestingly, mutation of CYP735A genes required for trans-zeatin biosynthesis caused strong defects in LR positioning, indicating an important role for this cytokinin metabolite in regulating LR spacing. Further it is shown that cytokinin and a known regulator of LR spacing, the receptor-like kinase ARABIDOPSIS CRINKLY4 (ACR4), operate in a non-hierarchical manner but might exert reciprocal control at the transcript level. Taken together, the results suggest that cytokinin acts as a paracrine hormonal signal in regulating root system architecture.

Cytokinin as a mediator for regulating root system architecture in response to environmental cues.

Root system architecture (RSA) is developmentally controlled by genetic pathways and their interaction with various environmental cues, in particular soil conditions. One important player in shaping RSA is the hormone cytokinin, which acts as a negative regulator of root elongation and branching. The redundant roles of cytokinin metabolism and signaling genes of Arabidopsis thaliana in regulating early stages of lateral root formation has recently been shown and it has been proposed that this redundancy reflects a role in mediating different environmental cues. Here we report that the transcript levels of cytokinin genes in the root responds to changes in nutrient availability in distinct ways. IPT3, IPT5, CYP735A2, LOG5, and CKX4 are particularly responsive cytokinin metabolism genes, genes encoding different type-A response regulators and the transcriptions factor genes ARR10 and CRF6 are among the most responsive signaling genes. This finding supports the hypothesis that environmental cues operate through fine-tuned transcriptional regulation of cytokinin genes to modulate root development.