To select the most stable reference genes in annual ryegrass (Lolium multiflorum), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium.

Cyanogenic glucosides (CNglcs) play an important role in plant defense response, however, the regulation mechanism of CNglcs synthesis by external environment and endogenous hormones is largely unclear. In this study, we found that jasmonates (JAs) promoted the synthesis of CNglcs by activating the expression of CNglcs biosynthesis genes in Lotus japonicus. Several differentially expressed bHLH family genes related to the synthesis of cyanogenic glucosides were identified by RNA-seq. The LjbHLH7 can directly activate the expression of CYP79D3 gene, the first step of CNglcs synthesis, by binding to the G-box sequence of its promoter. Transgenic plants overexpressing LjbHLH7 exhibited higher relative CNglcs content and enhanced insect resistance than the wild type. Furthermore, the transcriptional activity of LjbHLH7 was suppressed by the interaction with L. japonicus JASMONATE-ZIM DOMAIN protein LjJAZ4. Based on these results, we proposed that LjbHLH7 acts as an activator and LjJAZ4 acts as a repressor of JAs-induced regulation of cyanogenic glucosides biosynthesis in L. japonicus.

The common vetch (Vicia sativa) is often used as feed for livestock because of its high nutritional value. However, drought stress reduces forage production through plant damage. Here, we studied the transcriptional profiles of common vetch exposed to drought in order to understand the molecular mechanisms of drought tolerance in this species. The genome of the common vetch has not been sequenced, therefore we used Illumina sequencing to generate de novo transcriptomes. Nearly 500 million clean reads were used to generate 174,636 transcripts, including 122,299 unigenes. In addition, 5313 transcription factors were identified and these transcription factors were classified into 79 different gene families. We also identified 11,181 SSR loci from di- to hexa-nucleotides whose repeat number was greater than five. On the basis of differentially expressed genes, Gene Ontology analysis identified many drought-relevant categories, including “oxidation-reduction process”, “lipid metabolic process” and “oxidoreductase activity”. In addition to these, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified pathways, such as “Plant hormone signal transduction”, “Glycolysis/Gluconeogenesis” and “Phenylpropanoid biosynthesis”, as differentially expressed in the plants exposed to drought. The expression results in this study will be useful for further extending our knowledge on the drought tolerance of common vetch.

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