Quantitative real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real‐time quantitative RT‐PCR in perennial ryegrass (Lolium perenne L.) during plant development. Partial sequences of nine L. perenne housekeeping genes were obtained by RT‐PCR using degenerate primers designed from the corresponding genes in closely related species. Primers for quantitative RT‐PCR were designed based on partial sequences. The housekeeping genes were evaluated for their expression stability in different tissues at various stages of development. The analysis found that eEF‐1α and eIF‐4a were the most stable and β‐TUB was the least stable of the genes tested when all tissues were analyzed together. Analysis by geNorm indicated that the four most stably expressed housekeeping genes (eEF‐1α, eIF‐4a, 25S rRNA, and GAPDH) should be utilized when normalizing gene expression during plant developmental studies. For root crown tissues at different stages of development, eIF‐4a and 25S rRNA were the most stably expressed of the housekeeping genes tested. In leaf tissues, eEF‐1α and UBQ5 were the most stably expressed of the housekeeping genes tested. We found that using two housekeeping genes as reference genes is sufficient during RT‐PCR gene expression studies when analyzing either root crown or leaf tissues during different stages of development.

Background Forage and turf grasses are routinely cut and grazed upon throughout their lifecycle. When grasses are cut or damaged, they rapidly release a volatile chemical cocktail called green leaf volatiles (GLV). Previously we have shown that mechanical wounding or exposure to GLV released from cut grass, activated a Lt 46 kDa mitogen-activated protein kinase (MAPK) within 3 min and a 44 kDa MAPK within 15–20 min in the model grass species Lolium temulentum ( Lt ). Currently very little is known concerning the perception, signaling or molecular responses associated with wound stress in grasses. Since GLV are released during wounding, we wanted to investigate what genes and signaling pathways would be induced in undamaged plants exposed to GLV. Results RNA-Seq generated transcriptome of Lolium plants exposed to GLV identified 4308 up- and 2794 down-regulated distinct differentially-expressed sequences (DES). Gene Ontology analysis revealed a strong emphasis on signaling, response to stimulus and stress related categories. Transcription factors and kinases comprise over 13% of the total DES found in the up-regulated dataset. The analysis showed a strong initial burst within the first hour of GLV exposure with over 60% of the up-regulated DES being induced. Specifically sequences annotated for enzymes involved in the biosynthesis of jasmonic acid and other plant hormones, mitogen-activated protein kinases and WRKY transcription factors were identified. Interestingly, eleven DES for ferric reductase oxidase, an enzyme involved in iron uptake and transport, were exclusively found in the down-regulated dataset. Twelve DES of interest were selected for qRT-PCR analysis; all displayed a rapid induction one hour after GLV exposure and were also strongly induced by mechanical wounding. Conclusion The information gained from the analysis of this transcriptome and previous studies suggests that GLV released from cut grasses transiently primes an undamaged plant’s wound stress pathways for potential oncoming damage, and may have a dual role for inter- as well as intra-plant signaling. Electronic supplementary material The online version of this article (10.1186/s12870-019-1799-6) contains supplementary material, which is available to authorized users.

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For forage and turf grasses, wounding is a predominant stress that often results in extensive loss of vegetative tissues followed by rapid regrowth. Currently, little is known concerning the perception, signaling, or molecular responses associated with wound stress in forage- and turf-related grasses. A transcriptome analysis of Lolium temulentum plants subjected to severe wounding revealed 9413 upregulated and 7704 downregulated, distinct, differentially expressed genes (DEGs). Categories related to signaling, transcription, and response to stimuli were enriched in the upregulated DEGs. Specifically, sequences annotated as enzymes involved in hormone biosynthesis/action and cell wall modifications, mitogen-activated protein kinases, WRKY transcription factors, proteinase inhibitors, and pathogen defense-related DEGs were identified. Surprisingly, DEGs related to heat shock and chaperones were more prevalent in the downregulated DEGs when compared with the upregulated DEGs. This wound transcriptome analysis is the first step in identifying the molecular components and pathways used by grasses in response to wounding. The information gained from the analysis will provide a valuable molecular resource that will be used to develop approaches that can improve the recovery, regrowth, and long-term fitness of forage and turf grasses before/after cutting or grazing.