Genomic and Transcriptomic Analyses of Nutrient Transporters in Plants

ABSTRACT/nGenomic and Transcriptomic Analyses of Nutrient Transporters in Plants/nby/nDhondup Lhamo/nDoctor of Philosophy in Plant Biology/nUniversity of California, Berkeley/nProfessor Sheng Luan, Chair/nPlants require Nitrogen (N), phosphate (P), potassium (K) to support numerous physiological processes. These nutrients are acquired and transported by large families of nutrient transporters found in various tissues and cell types. Decades of research on nutrient channels and transporters in the model plant, Arabidopsis thaliana, have expanded our understanding of how these nutrients are transported, stored, remobilized, utilized and recycled within plants. However, our knowledge of the nutrient transport systems in crops are lacking. Whole-genome sequencing and transcriptomic analyses of diverse plant species are increasing exponentially each year, which can be utilized for comparative genomic analyses to understand the evolution of specific family of nutrient channels and transporters, their functional conservation and divergence at the sequence and tissue- and cell-type-specific levels. Camelina sativa is an emerging oilseed crop that contributes to food, feeds and fuels. It is considered to be tolerant to various harsh environments including low-nutrient stresses. However, it is not known how this polyploid might have evolved these adaptive traits at a gene level. To initiate functional genomic studies in this crop in response to low-nutrient stresses, we identified all the major nutrient channels/transporters of P and K present in the Camelina genome, and performed comprehensive and comparative genomic and transcriptomic analyses. We found that a whole-genome triplication event was the major driving force for the gene expansion, with three homoeologs for each Arabidopsis ortholog. In addition, tandem gene duplications have further expanded a specific nutrient transporter family in the Camelina genome. We also examined the phylogenetic relationship of nutrient transporters between Camelina and Arabidopsis, and analyzed their gene structures and protein domains to determine potential functional conservation and divergence at the sequence level. In silico RNA-seq analysis revealed potential candidate nutrient transporter genes that might function in specific tissue or organ for nutrient uptake, translocation and/or distribution. These studies represent the first effort in characterizing nutrient transporters in Camelina, and provide opportunities for future functional studies. The expression of nutrient transporter genes in specific cell, tissue and in response to various nutrient stresses are under the control of transcriptional factors (TFs). In Arabidopsis, AtSTOP1, a zinc finger TF is known to be involved in different abiotic stress response by modulating the transcript accumulation of different transporters. However, its involvement in low-K response have not been evaluated. Our study in Arabidopsis have revealed it is indeed involved, but the mechanism of its regulation is not understood. We isolated T-DNA insertion mutants of AtSTOP1 homolog in rice, named “Sensitive to K starvation 1” (sks1) based on its phenotype. RNA-seq analysis was performed in root and shoot tissues of wild type (WT) and sks1 mutants under low-K and the control conditions to understand its transcriptional program in low-K response. We identified a large number of genes to be mis-regulated in the mutants that are potentially involved in signal transduction, ROS production, immune response, cell wall synthesis, ion transport and homeostasis. Several low-K-inducible genes in WT such as TFs and signaling genes were controlled by SKS1 in roots, whereas many metabolic enzymes in shoots. We additionally found that SKS1 might be involved in signal integrations between nutrients, and between roots and shoots in response to low-K stress. While all these studies focus on gene expression of nutrient transporters and regulators at a tissue level, our understanding of the diverse transcriptional programs present in different cell types of a specific tissue is sparse. Recent advances in single-cell transcriptomics of Arabidopsis roots provided us with the opportunity to dissect the distribution of large sets of nutrient channels and transporters at a single cell resolution that work together to transfer nutrients from the soil to different cell-types of root cells and eventually reach vasculature for a massive flow. For this, we profiled the transcriptional patterns of putative nutrient transporters in different cell types of roots using the single-cell transcriptomics datasets from Arabidopsis root. Such analyses identified a number of NPK transporters expressed in the root epidermis to mediate NPK uptake and distribution to the adjacent cells. Some transport genes showed cortex- and endodermis-specific expression to direct the nutrient flow towards the vasculature. For long distance transport, a variety of transporters were shown to express and potentially function in the xylem and phloem. In the context of subcellular distribution of mineral nutrients, the NPK transporters at subcellular compartments were often found to show ubiquitous expression pattern, which suggests function in house-keeping processes. Overall, these single cell transcriptomic analyses provide a map of nutrient transport route from the epidermis across the cortex to the vasculature, which can be further tested experimentally in the future.

Advisor: Luan, Sheng