California State University, Long Beach (CSULB)
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Mature source leaves maintain a protein-rich photosynthetic apparatus that produces sugars for export. As leaves age, photosynthesis decreases and protein catabolism occurs, leading to export of nitrogen via amino acid transport. This orderly molecular transition is necessary for high grain yields as the bulk of seed nitrogen is derived from senescing leaves. RNA-Seq analysis of aging Arabidopsis thaliana leaves provides an unbiased list of genes with significant changes in expression that can foster specific inquiry. Specific questions to address include identifying transcription factors, transporters, degradation enzymes, and protective enzymes and regulatory factors that balance the degradation process during leaf senescence. Our lab is interested in epigenetic changes that accompany leaf senescence and histone acetylase mutants were screened for leaf senescence phenotypes. Two different hac1 alleles were noted have darker green leaves with higher levels of total protein than wildtype in older leaves. The HAC1 gene encodes a histone acetylase of the CREB family. The higher levels of chlorophyll and protein suggested that the hac1 alleles displayed delayed senescence. Surprisingly, preliminary gene expression analyses indicated that two senescence up-regulated genes were expressed at higher levels in the mutant, suggesting that senescence was accelerated at the molecular level. Consistent with an acceleration of senescence, leaf tips became yellow earlier in the hac1 mutant compared to WT. In this RNA-Seq experiment, fully-expanded leaves from 50 day old plants were isolated from WT and the two hac1 alleles. Genes with significant changes in expression will be identified and tested for changes in histone acetylation in the hac1 alleles. These RNA-seq data can be used to mine for genes that change expression in the hac1 alleles and that may be regulated by histone acetylation.
We are conducting RNA-Seq for two types of experiments. In one, we are studying the gene expression changes over a time course as Arabidopsis thaliana leaves undergo senescence. In the other, we are studying the effect of mutant hac1 alleles in leaf senescence.
Leaf Senescence Time Course: Fully-expanded rosette leaves were harvested at four time points: 29, 35, 42 and 57 days of plant growth. At 29 days, the inflorescence has just emerged and at 57 days, the leaves are turning yellow at the tips. Three samples were harvested at each time point from one growth experiment. RNA was extracted with Trizol and the Illumina TruSeq kit (v.2) was used for library prep. Single-end reads (50 bases) were obtained from an Illumina HiSeq machine. Three samples (all different ages) were run per lane and demultiplexed. File names for these datasets are: 29d-1, 29d-2, 29d-3; 35d-1, 35d-2, 35d-3; 42d-1, 42d-2, 42d-3; 57d-1, 57d-2, 57d-3 and will be made available at a time to be determined.
Role of hac1 in leaf senescence: HAC1 (At1g79000) encodes a histone acetylase from the CREB family. Two independent hac1 alleles, hac1-136 (SALK_136314) and hac1-080 (SALK_080380) were noted to have ~30% higher total chlorophyll and protein levels in older leaves when compared to WT suggesting a staygreen or poor allocation phenotype. At the same time, leaves displayed yellow tips earlier than in the WT. Initial gene expression analyses done with real-time qPCR suggested that the mutants were displaying accelerated senescence at the molecular level. RNA-Seq was carried out for 2 samples of WT, hac1-136 and hac1-080. RNA was isolated by Trizol, and the library was made using a stranded TruSeq kit and sequenced on a NextSeq Illumina machine. 76 bp of paired end reads were obtained. Data will be made available at a time to be determined.
General Genetics is an introductory genetics class that includes a weekly lab with about 200 Biology and Biochemistry majors. In lecture, students learn about NGS technologies and ½ of one lab period will be spent viewing a completed run on the Green Line to introduce the immense size of the data sets and the steps involved in RNA-seq data processing.
The Cell and Molecular Biology Lab Class is an upper-division course with 16 students. Students will spend six weeks of the semester running RNA-Seq data from Arabidopsis thaliana (aging leaves or hac1 alleles) through the RNA-seq pipeline using the Discovery Environment. Students will change parameters and determine how this affects CuffDiff output. Students will then pick a gene of interest after biological process enrichment analysis, design and optimize real-time qPCR primers, and run real-time reactions on a biological replicate of the RNA–Seq experiment to confirm changes in gene expression.
