Background: Low temperature limits the growth and development and geographical distribution of plants. Poa pratensis is a cool-season turfgrass mainly grown in urban areas. However, low winter temperature or cold events in spring and autumn may cause P.pratensis mortality, affecting the appearance of lawns. P.pratensis var. anceps cv. Qinghai (PQ) is widely distributed in the Qinghai-Tibet Plateau above 3000 m. PQ has greater cold tolerance than the commercially cultivated P.pratensis varieties. However, existing studies on the response mechanism of PQ to low temperatures have mainly focused on physiological and biochemical perspectives, while changes in the PQ transcriptome during the response to cold stress have not been reported. Results: To investigate the molecular mechanism of the PQ cold response and identify genes to improve the low-temperature tolerance of P.pratensis, we analyzed and compared the transcriptomes of PQ and the cold-sensitive P.pratensis cv. ‘Baron’ (PB) under cold stress using RNA sequencing. We identified 5996 and 3285 differentially expressed genes (DEGs) between the treatment vs control comparison of PQ and PB, respectively, with 5612 DEGs specific to PQ. Based on the DEGs, important Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as “starch and sucrose metabolism”, “protein processing in endoplasmic reticulum”, “phenylalanine metabolism” and “glycolysis/gluconeogenesis” were significantly enriched in PQ, and “starch and sucrose metabolism”, “phenylpropanoid biosynthesis”, “galactose metabolism” and “glutathione metabolism” were significantly enriched in PB. In addition, the “glycolysis” and “citrate cycle (TCA cycle)” pathways were identified as involved in cold tolerance of P.pratensis. Conclusions: As we know, this is the first study to explore the transcriptome of P.pratensis var. anceps cv. Qinghai. Our study not noly provides important insights into the molecular mechanisms of P.pratensis var. anceps cv. Qinghai responds to cold stress, but also systematically reveals the changes of key genes and products of glycolysis and TCA cycle in response to cold stress, which is conductive to the breeding of cold-tolerance P.pratensis genotype.

Kentucky bluegrass (Poa pratensis L.) belong to Gramineae and is widely used in lawns, golf courses, landscapes and sport fields as a prominent cool-season grass. Gene expression patterns during different stages of plant development can provide clues towards the understanding of its biological functions. The selection and validation of reference genes are the first steps in any real-time quantitative PCR gene expression study. Therefore, suitable reference genes are necessary for obtaining reliable results in real-time quantitative PCR analyses of Kentucky bluegrass. In the present study, 9 candidate reference genes were chosen, and their expression stability in the leaves and roots of Kentucky bluegrass under different stresses (drought, salt, heat, and cold) were evaluated using the GeNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the experimental conditions. The combination of SAM with GAPDH was the most stable in leaves under salt stress and cold stress, while TUB combined with ACT or GAPDH was stable in roots under salt or cold stress, respectively. ACT and SAM maintained stable expression in drought-treated leaves, and GAPDH combined with ACT was stable in drought-treated roots. SAM and TUB exhibited stable expression in heat-treated leaves. ACT and RPL were stable in heat-treated roots. In addition, the expression patterns of PpFEH in response to drought and cold stress were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause erroneous results. This work is the first study on the expression stability of reference genes in Kentucky bluegrass and will be particularly useful in the selection of stress-tolerance genes and the identification of the molecular mechanisms conferring stress tolerance in this species.

Drought stress is a major limiting factor that affects forage yields, and understanding the drought resistance mechanism of plants is crucial for improving crop yields in arid areas. Alfalfa (Medicago sativa L.) is the most important legume plant, mainly planted in arid and semi-arid areas. However, the adaptability of alfalfa to drought stress and its physiological and molecular mechanisms of drought resistance remains unclear. In this study, we analyzed the physiological and transcriptome responses of alfalfa cultivars with different drought resistances (drought-sensitive Gannong No. 3 (G3), drought-resistant Gannong No. 8 (G8), and strong drought-resistant Longdong (LD)) under drought stress at 0, 6, 12, and 24 h. LD had higher catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities and a higher soluble protein content, lower malondialdehyde (MDA) content, a lower O2·− production rate, and a lower H2O2 content than G8 and G3 (P < 0.05). The functional enrichment analysis, temporal expression pattern analysis, and weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes (DEGs) showed phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, glutathione metabolism, and biosynthesis of amino acid responses to drought stress in alfalfa. The differential expression of genes during phenylpropanoid biosynthesis, starch and sucrose metabolism, and the glutathione metabolism pathway was further studied, and it was speculated that PAL, COMT, 4CL, CCR, CAD, HXK, INV, SUS, WAXY, AGP, GST, and APX1 played important roles in the alfalfa drought stress response. The aim of this study was to enhance alfalfa drought resistance by overexpressing positively regulated genes and knocking out negatively regulated genes, providing genetic resources for the subsequent molecular-assisted breeding of drought-resistant alfalfa crops.