Meta-analysis of transcriptomics studies identifies novel attributes and set of genes involved in iron homeostasis in rice.

Iron (Fe) is an important micronutrient for humans as well as for plant growth and development. Rice employs multiple mechanisms to counteract the negative effects of Fe deficiency and Fe toxicity. Previously, many transcriptomics studies have identified hundreds of genes affected by Fe deficiency and/or Fe toxicity. These studies are highly valuable to identify novel genes involved in Fe homeostasis. However, in the absence of their systematic integration, they remain underutilized. A systematic meta-analysis of transcriptomics data from such ten previous studies was performed here to identify various common attributes. From this meta-analysis, it is revealed that under Fe deficiency conditions, root transcriptome is more sensitive and exhibits greater similarity across multiple studies than the shoot transcriptome. Furthermore, under Fe toxicity conditions, upregulated genes are more reliable and consistent than downregulated genes in susceptible cultivars. The integration of data from Fe deficiency and Fe toxicity conditions helped to identify key marker genes for Fe stress. As a proof-of-concept of the analysis, among the genes consistently regulated in opposite directions under Fe deficiency and toxicity conditions, two genes were selected: a proton-dependent oligopeptide transporter (POT) family protein and Vacuolar Iron Transporter (VIT)-Like (VTL) gene, and validated their expression and sub-cellular localization. Since VIT genes are known to play an important role in Fe homeostasis in plants, the entire OsVTL gene family in rice was characterized. This meta-analysis has identified many novel candidate genes that exhibit consistent expression patterns across multiple tissues, conditions, and studies. This makes them potential targets for future research aimed at developing Fe-biofortified rice varieties, as well as varieties tolerant to sub-optimal Fe levels in soil.

Negative regulators of grain yield and mineral contents in rice: potential targets for CRISPR-Cas9-mediated genome editing.

Rice is a major global staple food crop, and improving its grain yield and nutritional quality has been a major thrust research area since last decades. Yield and nutritional quality are complex traits which are controlled by multiple signaling pathways. Sincere efforts during past decades of research have identified several key genetic and molecular regulators that governed these complex traits. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene knockout approaches has accelerated the development of improved varieties; however, finding out target gene with negative regulatory function in particular trait without giving any pleiotropic effect remains a challenge. Here, we have reviewed past and recent literature and identified important negative regulators of grain yield and mineral contents which could be potential targets for CRISPR-Cas9-mediated gene knockout. Additionally, we have also compiled a list of microRNAs (miRNAs), which target positive regulators of grain yield, plant stress tolerance, and grain mineral contents. Knocking out these miRNAs could help to increase expression of such positive regulators and thus improve the plant trait. The knowledge presented in this review would help to further accelerate the CRISPR-Cas9-mediated trait improvement in rice.

An integrated analysis of cell-type specific gene expression reveals genes regulated by REVOLUTA and KANADI1 in the Arabidopsis shoot apical meristem.

In the Arabidopsis thaliana shoot apical meristem (SAM) the expression domains of Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI (KAN) genes are separated by a narrow boundary region from which new organs are initiated. Disruption of this boundary through either loss of function or ectopic expression of HD-ZIPIII and KAN causes ectopic or suppression of organ formation respectively, raising the question of how these transcription factors regulate organogenesis at a molecular level. In this study we develop a multi-channel FACS/RNA-seq approach to characterize global patterns of gene expression across the HD-ZIPIII-KAN1 SAM boundary. We then combine FACS, RNA-seq and perturbations of HD-ZIPIII and KAN expression to identify genes that are both responsive to REV and KAN1 and normally expressed in patterns that correlate with REV and KAN1. Our data reveal that a significant number of genes responsive to REV are regulated in opposite ways depending on time after induction, with genes associated with auxin response and synthesis upregulated initially, but later repressed. We also characterize the cell type specific expression patterns of auxin responsive genes and identify a set of genes involved in organogenesis repressed by both REV and KAN1.

Rice lipases: a conundrum in rice bran stabilization: a review on their impact and biotechnological interventions.

Rice is a primary food and is one of the most important constituents of diets all around the world. Rice bran is a valuable component of rice, containing many oil-soluble vitamins, minerals, and oil. It is known for its ability to improve the economic value of rice. Further, it contains substantial quantities of minerals like potassium, calcium, magnesium, iron and antioxidants like tocopherols, tocotrienols, and γ-oryzanol, indicating that rice bran can be utilized effectively against several life-threatening disorders. It is difficult to fully utilize the necessary nutrients due to the presence of lipases in rice bran. These lipases break down lipids, specifically Triacylglycerol, into free fatty acids and glycerol. This review discusses physicochemical properties, mechanism of action, distribution, and activity of lipases in various components of rice seeds. The phylogenetic and gene expression analysis helped to understand the differential expression pattern of lipase genes at different growth phases of rice plant. Further, this review discusses various genetic and biotechnological approaches to decrease lipase activity in rice and other plants, which could potentially prevent the degradation of bran oil. The goal is to establish whether lipases are a major contributor to this issue and to develop rice varieties with improved bran stability. This information sets the stage for upcoming molecular research in this area. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01343-3.

Dissecting the nutrient partitioning mechanism in rice grain using spatially resolved gene expression profiling.

Rice, a staple food worldwide, contains varying amounts of nutrients in different grain tissues. The underlying molecular mechanism of such distinct nutrient partitioning remains poorly investigated. Here, an optimized rapid laser capture microdissection (LCM) approach was used to individually collect pericarp, aleurone, embryo and endosperm from grains 10 days after fertilization. Subsequent RNA-Seq analysis in these tissues identified 7760 differentially expressed genes. Analysis of promoter sequences of tissue-specific genes identified many known and novel cis-elements important for grain filling and seed development. Using the identified differentially expressed genes, comprehensive spatial gene expression pathways were built for accumulation of starch, proteins, lipids, and iron. The extensive transcriptomic analysis provided novel insights about nutrient partitioning mechanisms; for example, it revealed a gradient in seed storage protein accumulation across the four tissue types analysed. The analysis also revealed that the partitioning of various minerals, such as iron, is most likely regulated through transcriptional control of their transporters. We present the extensive analysis from this study as an interactive online tool that provides a much-needed resource for future functional genomics studies aimed to improve grain quality and seed development.

Vacuolar Iron Transporter (Like) proteins: Regulators of cellular iron accumulation in plants.

Iron is not only important for plant physiology, but also a very important micronutrient in human diets. The vacuole is the main site for accumulation of excess amounts of various nutrients and toxic substances in plant cells. During the past decade, many Vacuolar Iron Transporter (VIT) and VIT-Like (VTL) genes have been identified and shown to play important roles in iron homeostasis in different plants. Furthermore, recent reports identified novel roles of these transporter genes in symbiotic nitrogen fixation (SNF) in legume crops as well as in the blue coloration of petals in flowers. The literature indicates their universal role in Fe transport across different tissues (grains, nodules, flowers) to different biological processes (cellular iron homeostasis, SNF, petal coloration) in different plants. Here, we have systematically reviewed different aspects, such as structure, molecular evolution, expression, and function of VIT/VTL proteins. This will help future studies aimed at functional analysis of VIT/VTL genes in other plant species, vacuolar transportation mechanisms, and iron biofortification at large.

Heavy metal stress in rice: Uptake, transport, signaling, and tolerance mechanisms.

Heavy metal contamination of agricultural fields has become a global concern as it causes a direct impact on human health. Rice is the major food crop for almost half of the world population and is grown under diverse environmental conditions, including heavy metal-contaminated soil. In recent years, the impact of heavy metal contamination on rice yield and grain quality has been shown through multiple approaches. In this review article, different aspects of heavy metal stress, that is uptake, transport, signaling and tolerance mechanisms, are comprehensively discussed with special emphasis on rice. For uptake, some of the transporters have specificity to one or two metal ions, whereas many other transporters are able to transport many different ions. After uptake, the intercellular signaling is mediated through different signaling pathways involving the regulation of various hormones, alteration of calcium levels, and the activation of mitogen-activated protein kinases. Heavy metal stress signals from various intermediate molecules activate various transcription factors, which triggers the expression of various antioxidant enzymes. Activated antioxidant enzymes then scavenge various reactive oxygen species, which eventually leads to stress tolerance in plants. Non-enzymatic antioxidants, such as ascorbate, metalloids, and even metal-binding peptides (metallothionein and phytochelatin) can also help to reduce metal toxicity in plants. Genetic engineering has been successfully used in rice and many other crops to increase metal tolerance and reduce heavy metals accumulation. A comprehensive understanding of uptake, transport, signaling, and tolerance mechanisms will help to grow rice plants in agricultural fields with less heavy metal accumulation in grains.

Identification and molecular characterization of rice bran-specific lipases.

KEY MESSAGE: Among the 113 lipases present in rice genome, bran and endosperm-specific lipases were identified and lipase activity for one of the selected lipase gene is demonstrated in yeast. Rice bran is nutritionally superior than endosperm as it has major reservoirs of various minerals, vitamins, essential mineral oils and other bioactive compounds, however it is often under-utilized as a food product due to bran instability after milling. Various hydrolytic enzymes, such as lipases, present in bran causes degradation of the lipids present and are responsible for the bran instability. Here, in this study, we have systematically analyzed the 113 lipase genes present in rice genome, and identified 21 seed-specific lipases. By analyzing the expression of these genes in different seed tissues during seed development, we have identified three bran-specific and three endosperm-specific lipases, and one lipase which expresses in both bran and endosperm tissues. Further analysis of these genes during seed maturation and seed germination revealed that their expression increases during seed maturation and decreases during seed germination. Finally, we have shown the lipase activity for one of the selected genes, LOC_Os05g30900, in heterologous system yeast. The bran-specific lipases identified in this study would be very valuable for engineering designer rice varieties having increased bran stability in post-milling.

Functional interrelation of MYC2 and HY5 plays an important role in Arabidopsis seedling development.

Arabidopsis MYC2 bHLH transcription factor plays a negative regulatory role in blue light (BL)-mediated seedling development. HY5 bZIP protein works as a positive regulator of multiple wavelengths of light and promotes photomorphogenesis. Both MYC2 and HY5, belonging to two different classes of transcription factors, are the integrators of multiple signaling pathways. However, the functional interrelations of these two transcription factors in seedling development remain unknown. Additionally, whereas HY5-mediated regulation of gene expression has been investigated in detail, the transcriptional regulation of HY5 itself is yet to be understood. Here, we show that HY5 and MYC2 work in an antagonistic manner in Arabidopsis seedling development. Our results reveal that HY5 expression is negatively regulated by MYC2 predominantly in BL, and at various stages of development. On the other hand, HY5 negatively regulates the expression of MYC2 at various wavelengths of light. In vitro and in vivo DNA-protein interaction studies suggest that MYC2 binds to the E-box cis-acting element of HY5 promoter. Collectively, this study demonstrates a coordinated regulation of MYC2 and HY5 in blue-light-mediated Arabidopsis seedling development.

Spatio-temporal distribution of micronutrients in rice grains and its regulation.

Rice has been a staple food for more than half of the global population. Different parts of rice grains contain different amounts of macro- and micro-nutrients. Polished white rice, which is the main form of rice consumption, mainly contains starch, however, the bran and germ, which are removed during polishing, contain large amounts of micronutrients and bioactive compounds. To engineer nutritionally superior rice varieties, it is imperative to understand the spatial and temporal distribution of different nutrients in different parts of the rice grain. Keeping this in mind, in this review, we have performed a comprehensive literature review to put together all the recent findings regarding the spatio-temporal distribution of all the important micronutrients in different cell-layers/tissues of developing seeds and mature seed grains. Furthermore, we have overviewed the underlying cell-layer specific possible regulatory mechanism responsible for the loading/partitioning for each of the micronutrients into specific tissue types. Most of the nutrient filling occurs between 7 and 18 days after fertilization (DAF) through the dorsal vascular bundle and the aleurone layer. During the last few years, spatio-temporal distribution of various minerals and the role of their transporters has been studied in great detail. However, with regard to vitamins and other bioactive compounds, such studies are still very limited. Distribution of minerals in the grain is mainly regulated by the distribution of their ligands and transporters, whereas the accumulation of various vitamins is mainly metabolic enzyme activity. Collective knowledge discussed here in this niche area would help to design new studies to improve the micronutrient content located in the inner part of the seed.

Global gene regulatory network underlying miR165a in Arabidopsis shoot apical meristem.

Arabidopsis microRNA165a (miR165a) targets Class III Homeodomain Leucine-Zipper (HD-ZIPIII) transcription factors to regulate various aspects of plant development and stress response. Over-expression of miR165a mimics the loss-of-function phenotype of HD-ZIPIII genes and leading to ectopic organ formation, shoot apical meristem (SAM) termination, loss of leaf polarity, and defective vasculature development. However, the molecular mechanisms underlying these phenotypes remain unresolved. Here, we over-expressed miR165a in a dexamethasone inducible manner and identified differentially expressed genes in the SAM through RNA-Seq. Simultaneously, using multi-channel FACS combined with RNA-Seq approach, we characterized global transcriptome patterns in miR165a expressing cell-types compared to HD-ZIPIII expressing cell-types and other cell-types in SAM. By integrating our results we identified sets of genes which are up-regulated by miR165a as well have enriched expression in miR165a cell-types, and vice-versa. Known plant development related genes such as HD-ZIPIII and their targets LITTLE ZIPPERs, Like AUXIN RESISTANT 2, BEL1-like homeodomain 6, ROTUNDIFOLIA like 16 were found to be down-regulated. Among the up-regulated genes, GIBBERELLIN 2-OXIDASEs, various elemental transporters (YSL3, ZIFL1, SULTR), and other transporter genes were prominent. Thus, the genes identified in this study help to unravel the molecular mechanism of miR165a and HD-ZIPIII regulated plant development and stress response.

Reference gene identification for gene expression analysis in rice under different metal stress.

Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common approach to quantify changes in gene expression. Appropriate internal reference genes are essential for normalization of data of RT-qPCR. In the present study, we identified suitable reference genes for analysis of gene expression in rice seedlings subjected to different heavy metal stresses such as deficiencies of iron and zinc and toxicities of cobalt, cadmium and nickel. First, from publically available RNA-Seq data we identified 10 candidate genes having stable expression. We also included commonly used house-keeping gene OsUBQ5 (Ubiquitin 5) in our analysis. Expression stability of all the 11 genes was determined by two independent tools, NormFinder and geNorm. Our results show that selected candidate reference genes have higher stability in their expression compared to that of OsUBQ5. Genes with locus ID LOC_Os03g16690, encoding an oxysterol-binding protein (OsOBP) and LOC_Os01g56580, encoding Casein Kinase_1a.3 (OsCK1a.3) were identified to be the most stably expressed reference genes under most of the conditions tested. Finally, the study reveals that it is better to use a specific reference gene for a specific heavy metal stress condition rather than using a common reference gene for multiple heavy metal stress conditions. The reference genes identified here would be very useful for gene expression studies under heavy metal stresses in rice.

Gene Expression Pattern of Vacuolar-Iron Transporter-Like (VTL) Genes in Hexaploid Wheat during Metal Stress.

Iron is one of the important micronutrients that is required for crop productivity and yield-related traits. To address the Fe homeostasis in crop plants, multiple transporters belonging to the category of major facilitator superfamily are being explored. In this direction, earlier vacuolar iron transporters (VITs) have been reported and characterized functionally to address biofortification in cereal crops. In the present study, the identification and characterization of new members of vacuolar iron transporter-like proteins (VTL) was performed in wheat. Phylogenetic distribution demonstrated distinct clustering of the identified VTL genes from the previously known VIT genes. Our analysis identifies multiple VTL genes from hexaploid wheat with the highest number genes localized on chromosome 2. Quantitative expression analysis suggests that most of the VTL genes are induced mostly during the Fe surplus condition, thereby reinforcing their role in metal homeostasis. Interestingly, most of the wheat VTL genes were also significantly up-regulated in a tissue-specific manner under Zn, Mn and Cu deficiency. Although, no significant changes in expression of wheat VTL genes were observed in roots under heavy metals, but TaVTL2, TaVTL3 and TaVTL5 were upregulated in the presence of cobalt stress. Overall, this work deals with the detailed characterization of wheat VTL genes that could provide an important genetic framework for addressing metal homeostasis in bread wheat.

Insertional Mutagenesis Approaches and Their Use in Rice for Functional Genomics.

Insertional mutagenesis is an indispensable tool for engendering a mutant population using exogenous DNA as the mutagen. The advancement in the next-generation sequencing platform has allowed for faster screening and analysis of generated mutated populations. Rice is a major staple crop for more than half of the world's population; however, the functions of most of the genes in its genome are yet to be analyzed. Various mutant populations represent extremely valuable resources in order to achieve this goal. Here, we have reviewed different insertional mutagenesis approaches that have been used in rice, and have discussed their principles, strengths, and limitations. Comparisons between transfer DNA (T-DNA), transposons, and entrapment tagging approaches have highlighted their utilization in functional genomics studies in rice. We have also summarised different forward and reverse genetics approaches used for screening of insertional mutant populations. Furthermore, we have compiled information from several efforts made using insertional mutagenesis approaches in rice. The information presented here would serve as a database for rice insertional mutagenesis populations. We have also included various examples which illustrate how these populations have been useful for rice functional genomics studies. The information provided here will be very helpful for future functional genomics studies in rice aimed at its genetic improvement.

Molecular characterization and expression dynamics of MTP genes under various spatio-temporal stages and metal stress conditions in rice.

Metal Tolerance Proteins (MTPs) are the class of membrane proteins involved in the transport of metals, mainly Zn, Mn, Fe, Cd, Co and Ni, and confer metal tolerance in plants. In the present study, a comprehensive molecular analysis of rice MTP genes was performed to understand the evolution, distribution and expression dynamics of MTP genes. Exploration of the whole genome re-sequencing information available for three thousand rice genotypes highlighted the evolution and allelic diversity of MTP genes. Based on the presence of non-synonymous single nucleotide polymorphism (SNP), MTP1, MTP6, MTP8 and MTP9 were found to be the most conserved genes. Furthermore, results showed localization of MTP1, MTP8.1 and MTP9, and MTP11, respectively with QTLs/m-QTLs for Zn and Cd accumulation, making these genes promising candidates to understand the QTL regulation. Expression profiling of the entire set of 10 MTP genes revealed root and shoot specific expressions of MTP9 and MTP8.1, respectively, under all tested vegetative stages. Expression of seed-specific MTPs increased as seed maturation progressed, which revealed their potential role in transporting metals during seed filling. Upon exposure to harmful heavy metals, expression of most MTP genes decreased in root and increased in shoot, suggests that different mechanisms are being employed by MTPs in different tissues. Contrastingly, only a few MTPs were found to be responsive to Fe and/or Zn starvation conditions. The extensive analysis of MTPs presented here will be helpful in identifying candidate MTP genes for crop biofortification and bioremediation purposes.

Cell type boundaries organize plant development.

In plants the dorsoventral boundary of leaves defines an axis of symmetry through the centre of the organ separating the top (dorsal) and bottom (ventral) tissues. Although the positioning of this boundary is critical for leaf morphogenesis, how the boundary is established and how it influences development remains unclear. Using live-imaging and perturbation experiments we show that leaf orientation, morphology and position are pre-patterned by HD-ZIPIII and KAN gene expression in the shoot, leading to a model in which dorsoventral genes coordinate to regulate plant development by localizing auxin response between their expression domains. However we also find that auxin levels feedback on dorsoventral patterning by spatially organizing HD-ZIPIII and KAN expression in the shoot periphery. By demonstrating that the regulation of these genes by auxin also governs their response to wounds, our results also provide a parsimonious explanation for the influence of wounds on leaf dorsoventrality.