Overexpression of a Medicago truncutula homeodomain finger protein, MtPHD6, enhances drought tolerance in Arabidopsis

Purpose: We aimed to dissect function of Medicago truncutula homeodomain finger protein, MtPHD6 in Arabidopsis Methods: A total amount of 3 µg RNA was used for generation of sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Clean reads were obtained by removing low quality reads, reads containing adapter and ploy-N from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. Index of the Arabidopsis genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of drought stress versus control condition was performed using the DESeq R package (1.18.0). Results:In total, four samples with three biological replicates per genotype/treatment combination were used for RNA sequencing analysis. At least 2 G clean bases were generated for each sample. Comparative analysis revealed different networks which were modulated by drought stress and MtPHD6 transgene in Arabidopsis seedlings Overall design: In this researchstudy, M. truncatula Jemalong A17 and Arabidopsis A. thaliana Columbia were wild-type (WT) Col-0 was used as control. After surface-sterilization in 50% (v/v) bleach with 0.01% (v/v) TritonX-100, Arabidopsis seeds were stratified in deionized water at 4 °C for 3 d in darkness. Then the seeds were sown on the full-strength Murashige and Skoog (MS) medium containing 1% (w/v) sucrose and 0.8% (w/v) agar (pH 5.7) in a growth room. The growth room was maintained at 21~23 °C with an irradiance of 100~120 µmol photons m-2 s-1, 60% relative humidity, and a 16 h light/8 h dark cycle. After growth for 7 d on the MS medium, the seedlings, except those used in germination tests, were transferred into soil-filled plastic containers and irrigated with nutrient solution twice each week.