IC4R006-RNA-Seq-2015-25644226

Project Title

Transcriptome analysis of nitrogen-starvation-responsive genes in rice

The Background of This Project

• The macronutrient nitrogen (N) is an essential component of numerous important compounds, including amino acids, proteins, nucleic acids, chlorophyll, and some plant hormones. During periods of N-starvation, various deficiency-responsive genes function to support plant survival by increasing the level of chlorophyll synthesis, altering root architecture, improving N-assimilation, enhancing lignin content, and changing the amounts of sugars and sugar phosphates.
• In this study, researchers performed deep transcriptomic investigations with rice plants and obtained detailed expression profiles for genes involved in responses to low-N stress.

Plant Culture & Treatment

• Oryza sativa L. ssp. japonica cv. Dongjin rice was used in all experiments. Seeds were surface-sterilized and germinated for two weeks in a Murashige and Skoog medium that lacked a nitrogen source. The seedlings were further grown in an N-sufficient nutrient solution at 28°C/25°C (day/night) under a 14-h photoperiod and 50 to 55% relative humidity.

Figure 4 Patterns of GUS expression in 5 DAG seedlings. (A-H) Preferential expression of genes in leaf sheaths from Lines 3A-60813 (a and b), 3A-51694 (c and d), 4A-02639 (e and f), and 4A-01614 (g and h). (a, c, e, and g) Seedlings were grown under N-sufficient conditions (left) or N-deficient conditions (right). (b, d, f, and h) Cross section of leaf sheath under N-starvation. (i and j) Preferential expression in vascular cylinders of roots from Line 1B-11001. (i) N-sufficient conditions. (j) N-deficient conditions. Bar = 200 μm.

• At the six-leaf stage, the seedlings were divided into two groups: 1) N-starvation, with the amount of NH 4 NO 3 in the solution reduced to 0.072 mM; and 2) N-sufficient, for which the nutrient solution contained the normal N concentration of 1.44 mM. At 12 h after the treatment began, the total roots and leaf sheaths were harvested from plants in both groups.

Illumina Sequencing

• Total RNA was prepared using RNAiso Reagent (Takara Bio Inc., Otsu, Japan). Quality was checked with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA (30 μg) was used for synthesizing complementary DNA (cDNA). After the libraries were constructed, the cDNA was sequenced with the Illumina HiSeq TM 2000 according to the manufacturer’s recommendations
• RNA-Seq reads were aligned to the rice reference genomes by the TopHat2 program. The BAM File was assembled by Cufflinks and then merged with reference transcriptome annotations into a unified annotation for further analysis. Expression levels for each gene were calculated by quantifying the Illumina reads according to the RPKM method.

Research Findings

• Eight cDNA libraries were constructed from two biological replicates of leaf sheaths and roots from plant grown under deficient or sufficient conditions. Sequencing those libraries resulted in the identification of 40,756,549 and 41,703,971 paired-end reads (202-nucleotide read length) from the sheaths and roots, respectively. In all, 86% of the reads from the sheaths and 69% from the roots were mapped to the reference genome, for which nearly 87% were correctly aligned and approximately 98% of them had unique locations in reference genome.
• Comparing transcript abundances revealed 1,650 transcripts that were differentially expressed (fold-change ≥2; p ≤ 0.05) due to a deficient N supply (Additional files 3 and 4). Among them, 1,158 were differentially expressed in the leaf sheaths and 492 in the roots. Of those identified in the N-deficient sheaths, 548 transcripts were up-regulated and 610 transcripts were down-regulated. In the N-deficient roots, 276 transcripts were up-regulated and 216 were down-regulated.
• The researhers classified the 1,650 differentially expressed genes into 54 functional groups by GO analysis (Figure 3). The dominant terms were 'cell part' (GO:0044464) in Cellular Component, 'binding' (GO:0005488) in Molecular Function, and 'cellular process' (GO:0009987) in Biological Process. In the third category, more than 30% of the genes for 'metabolic process' (GO:0008152), 'response to stimulus' (GO:0050896), and 'biological regulation' (GO:0065007) responded to N-starvation.

• The researhers used GUS assays of T-DNA gene trap lines to confirm the N-starvation-responsive TF genes.Five GUS-positive lines displayed N-responsive GUS activity. Although this activity was weak when plants were grown in a standard N-sufficient medium, it was rapidly induced by N-starvation. Under low-N con- ditions, four lines (3A-60813, 3A-51694, 4A-02639, and 4A-01614) showed preferential GUS-staining in the sheaths (vascular bundles) while one (1B-11001) showed staining in the roots (vascular cylinder).

Labs working on this Project

• Department of Plant Molecular Systems Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea.
• Department of Crop Genomics and Genetic Improvement, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
• National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI),Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.