Recipient Organization
UNIVERSITY OF WISCONSIN SYSTEM
2420 NICOLET DR CL835
GREEN BAY,WI 54311
Performing Department
(N/A)
Non Technical Summary
To improve nitrogen use efficiency (NUE), it is crucial to understand the dynamic gene regulation that allows plants to respond to a changing nitrogen environment. We recently reported differential expression of transcript isoforms in response to nitrogen, thus opening a new frontier in deciphering molecular basis of nitrogen response. Here, I propose to elucidate the regulatory and functional mechanisms of AMINO ACID VACUOLAR TRANSPORTER 1B (AVT1B) that undergoes prominent isoform switching in response to nitrogen. When nitrogen is present, AVT1B is expressed as the full-length mRNA that encodes a putative amino acid transporter; when nitrogen is limiting, a truncated mRNA is transcribed with the first four exons missing. To test the hypothesis that the transport activity of the full length AVT1B is modulated by the truncated AVT1B during nitrogen starvation to fine-tune plant nitrogen metabolism, I will probe the regulatory mechanisms underlying the isoform switch (objective 1), determine the biochemical function and interaction of the two isoforms (objective 2), and evaluate the role of both isoforms in planta (objective 3). The proposed study will uncover mechanisms tying environmental signals to transcript plasticity and likely identify new target genes for breeding and engineering to increase NUE. This proposed project will provide valuable training in gene regulation and functional genomics, which, combined with my background in metabolism and biochemistry, will allow me to develop a strong research program. The fellowship will support my development toward a well-rounded scientist and teacher with the skills necessary to be successful in a tenure-track position.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
To improve nitrogen use efficiency (NUE), it is crucial to understand the dynamic gene regulation that allows plants to respond to a changing nitrogen environment. This research works toelucidate the regulatory and functional mechanisms of AMINO ACID VACUOLAR TRANSPORTER 1B (AVT1B) that undergoes prominent isoform switching in response to nitrogen.Research Objective 1 (RO1): Probe the regulatory mechanism of alternative expression of isoforms of AVT1B.Overview: AVT1B is expressed at full length with sufficient nitrogen but as a short isoform when nitrogen is limiting. The goal of this aim is to understand how this isoform switch is regulated.Research Objective 2 (RO2): Determine the subcellular localization, biochemical function, and interaction of AVT1B isoforms.Overview: The yeast homolog of AVT1B is a vacuolar amino acid transporter, so I hypothesized that at least the long isoform, but possibly both isoforms, of AVT1B is also a vacuolar amino acid transporter. I also hypothesized that the transcript switching between two isoforms allow the protein product of the short isoform to physically interact with the long form thus affecting transport activity.Research Objective 3 (RO3): Elucidate the in planta function of both isoforms of AVT1B.Overview: The goal of this aim is to understand the role of both isoforms in planta using transgenic and physiological approaches. The original proposal included generation of transgenic lines, in planta N use assays, and RNA-Sequencing (RNA-Seq) to identify downstream targets of AVT1B.
Project Methods
In order to fulfill the research objectives of this project, the project director will employ the following methods:>RO1.1: Determine the TSS of transcripts of AVT1B. To experimentally validate that isoform switching of AVT1B is due to alternative TSSs, I determined the TSSs using RACE. Briefly, WT Arabidopsis seedlings treated with +N or -N were harvested for RNA extraction. TSSs were determined by 5'-RNA Ligase Mediated (RLM) RACE using the FirstChoice® RLM-Race Kit, followed by sequencing. I identified three major TSSs for the long isoform and two major TSSs for the short isoform.>RO1.2: Evaluate RNA polymerase II occupancy at AVT1B. To further differentiate between alternative TSSs and alternative splicing, I will determine whether Pol II occupancy is changed in response to N in a manner correlated with alternative transcription by ChIP-qPCR. ChIP will be performed using antibodies against Pol II Ser2P and Ser5P as previously described. I will evaluate Pol II occupancy, specifically Ser5P/Ser2P ratio, at each TSS by qPCR using gene-specific primer pairs. Expected and alternative outcomes: I expect to detect significant enrichment of Pol II Ser5P (high Ser5P/Ser2P) at TSS1 in +N, consistent with expression of AVT1Blong, and a high Ser5P/Ser2P ratio at TSS2 in -N, supporting that an alternative TSS is responsible for the isoform switch. Conversely, if high Ser5P/Ser2P is observed at TSS1 regardless of N conditions, it would indicate the isoform switch is caused by alternative splicing of the same pre-mRNA, requiring further studies into underlying mechanisms.>RO1.3: Profile the chromatin landscape at the AVT1B gene locus. Here, I will probe whether histone modifications could serve as a mechanism to affect alternative transcription of AVT1B. WT plants will be treated with +N or -N as described above. ChIP will be performed as in RO1.2 with antibodies against H3K4me3 and H3K9me2. Following ChIP, qPCR with primers for TSS1 and TSS2 will be used to determine their local histone modification patterns. Expected results: Since H3K4me3 and H3K9me2 are associated with activation and repression of transcription initiation, respectively, I expect that in the presence of N, H3K4me3 will peak around TSS1 and H3K9me2 will be detected around TSS2 to repress transcription of AVT1Bshort. In -N, H3K4me3 will peak around TSS2 and H3K9me2 will be observed around TSS1. This would support that N-dependent chromatin landscape could regulate alternative promoter use.>RO2.1: Evaluate the subcellular localization of both isoforms of AVT1B. I will construct four constructs - i) p35S::YFPAVT1Blong , ii) p35S::AVT1Blong-YFP; iii) p35S::RFP-AVT1Bshort , iv) p35S::AVT1Bshort-RFP,- and transform into the avt1b knockout background, to determine the localization of both isoforms. I will test the isoforms separately first, then test the two isoforms together to determine if AVT1Bshort affects the localization of AVT1Blong. To prevent masking of signals at either termini, the fluorescent protein will be fused to both N- and C-terminals. Different fluorescent markers will be used for AVT1Blong and AVT1Bshort to allow expressing both isoforms together to test their interaction. The use of avt1b mutant as background will minimize interference caused by endogenous AVT1B. Expected and alternative outcomes: This subaim will uncover the subcellular localization of the two isoforms with a few possible outcomes: i) both AVT1B isoforms are vacuolar, ii) AVT1Blong is localized to the vacuole, but presence of AVT1Bshort prevents it from reaching the vacuole, or iii) AVT1Blong is vacuolar, while AVT1Bshort is localized to another membrane. It is also possible that the transporters localize to other organelles or the cell membrane.>RO2.2: Determine transport activity of AVT1B isoforms. I will use a yeast system to determine the transport activity of AVT1B isoforms. Specifically, cDNA of AVT1Blong and AVT1Bshort will be cloned into pYES-DEST42 (Invitrogen). Resulting constructs (pYES-AVT1Blong and pYES-AVT1Bshort) will be transformed into yeast WT strain BY4741 and Δavt1 (obtained from Horizon Discovery). ScAvt1p is the closest yeast homolog to AtAVT1B, and thus knockout of this gene should allow for less background uptake of amino acids. Both yeast strains will be transformed with empty vector, pYES-AVT1Blong, pYES-AVT1Bshort, or both together. Vacuoles will be purified from transformed yeast via ultracentrifugation and incubated with 3H-labeled amino acid (or 3H-glucose as a negative control) for 1, 3, or 5 minutes. After washing, amount of radioactive amino acid will be quantified by scintillation counting. Detection of radioactivity in the vacuole that is significantly higher than the negative control after incubation with a specific 3H-labeled amino acid (e.g 3H-Gly) would support that the isoform transports that amino acid into the vacuole.>RO2.3: Determine whether AVT1Bshort and AVT1Blong physically interact. BiFC will be performed using BiFC vectors that contain the N- or C-terminal of YFP (referred to as NY and CY) from Walter et al. To avoid spatial effects that may cause false negatives, the fusions will be placed in both orientations. The pairwise combinations of constructs will then be transiently expressed in tobacco leaves by infiltration and fluorescence will be examined by confocal microscopy. A vector with the full coding sequence of YFP will be used as a positive control, and the empty vectors as a negative control. Expected results and alternative approaches: I expect to observe YFP fluorescence only when AVT1Blongand AVT1Bshort fused with complementing YN and YC are co-expressed, suggesting AVT1Blong and AVT1Bshort physically interact. This assay will also allow me to visualize where the interaction occurs in the plant cell. If there is weak expression or too much background expression of YFP, I will use split ubiquitin-based membrane yeast two-hybrid (MYTH) as an alternative, which is an adaption of traditional yeast two-hybrid used to determine interactions between membrane proteins.>RO3: Elucidate the in planta function of both isoforms of AVT1B. I am currently in the process of generating avt1b mutant lines. I am using a CRISPR-Cas9 approach to knockout the short isoform and both isoforms. As an alternative, I am also overexpressing the long and short isoform independently in wild type (WT) Arabidopsis. In planta assay of N-use traits: WT, avt1b mutants, and the isoform-specific transgenic lines will be first grown in normal growth conditions and then treated with either sufficient N (5 mM KNO3 ) or N depletion (5 mM KCl) for six days to determine the effect of AVT1B isoforms on plant N use. After N treatments, shoot and root fresh weight will be measured, and chlorophyll content will be analyzed spectrophotometrically. Vacuoles will be purified from leaf-derived protoplasts, and amino acids will be extracted and quantified by GC-MS as previously described from leaves and purified vacuoles to determine the effect on amino acid profiles. In planta assay of transcriptome: In order to capture the downstream effects of alternative expression of AVT1B, I will evaluate transcriptomes of the mutants described above compared to WT in +N and -N conditions. Plants will be collected at 0 and 2 hours after N treatment. RNA will be extracted from three pooled seedlings for three biological replicates for each sample using the Qiagen RNeasy Plant Mini Kit and used for RNA-Seq library construction and sequencing with Novogene. RNA-Seq reads will be trimmed using Trimmomatic and aligned to the Arabidopsis genome using TopHat2. The gene counts will be generated using featureCount and normalized using EDASeq. Expression will be compared between mutants and WT with DESeq2 to determine the gene networks that are affected by the function of the two isoforms.