Abstract
Perennial ryegrass (Lolium perenne) is a forage and amenity grass species widely cultivated in temperate regions worldwide. As such, perennial ryegrass populations are exposed to a range of environmental conditions and stresses on a seasonal basis and from year to year. One source of potential stress is limitation on water availability. The ability of these perennial grasses to be able to withstand and recover after periods of water limitation or drought can be a key component of grassland performance. Thus, we were interested in looking at changes in patterns of gene expression associated with increasing water stress. Clones of a single genotype of perennial ryegrass were grown under non-flowering growth room conditions in vermiculite supplemented with nutrient solution. Leaf and root tissue was sampled at 4 times in quadruplicate relating to estimated water contents of 35%, 15%, 5% and 1%. RNA was extracted and RNAseq used to generate transcriptome profiles at each sampling point. Transcriptomes were assembled using the published reference genome sequence and differential gene expression analysed using 3 different programmes, DESeq2, edgeR and limma (with the voom transformation), individually and in combination, deriving Early, Middle and Late stage comparisons. Identified differentially expressed genes were then associated with enriched GO terms using BLAST2GO. For the leaf, up-regulated differentially expressed genes were strongly associated with GO terms only during the Early stage and the majority of GO terms were associated with only down-regulated genes at the Middle or Late stages. For the roots, few differentially expressed genes were identified at either Early or Middle stages. Only one replicate at 1% estimated water content produced high quality data for the root, however, this indicated a high level of differential expression. Again the majority of enriched GO terms were associated with down-regulated genes. The performance of the different analysis programmes and the annotations associated with identified differentially expressed genes is discussed.
Highlights
Perennial ryegrass (Lolium perenne) is a widely cultivated perennial forage and amenity grass in temperate areas worldwide and, as such, is either the major component or is a key constituent of many perennial pasture types
The analysis of variability among replicates in the form of principle component analysis (PCA) and heat maps using the count matrices showed that shoot replicates within each estimated water content (EWC) sampling point clustered together and the first two components of the PCA accounts for up to 69% of the overall variability (Fig 1)
The PCA and heat map plots generated from the root data, by contrast, indicated a higher degree of variability between replicates and a greater degree of overlap between the 35% and Complete 84.41 84.41 84.73 85.04 81.90 84.52 79.81 71.15
Summary
Perennial ryegrass (Lolium perenne) is a widely cultivated perennial forage and amenity grass in temperate areas worldwide and, as such, is either the major component or is a key constituent of many perennial pasture types. Numerous studies have reported relative changes in transcriptome profiles of model and crop plants in response to water-stress, often with a focus on the identification of differentially expressed genes (DEGs) distinguishing drought tolerant from susceptible genotypes. An aspect of the majority of these studies is that, for obvious practical reasons, they have isolated, analysed and compared transcriptomes from the aerial parts of the plant While this is experimentally more tractable, this has led to a relative lack of knowledge of the gene expression responses of roots to the progression of water stress. These include hydroponic systems, with osmotic stress being imposed using high molecular weight polyethylene glycol (PEG) [18,19,20,21] and the use of other suitable soil, sand [4, 13, 22, 23] and non-soil based hydrated media such as vermiculite [24,25,26,27,28], from which roots can be isolated relatively rapidly while minimising mechanical damage, at least for simpler root systems
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