Seashore paspalum (Paspalum vaginatum) is among the most salt- and cadmium-tolerant warm-season perennial grass species widely used as turf or forage. The objective of this study was to select stable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of seashore paspalum in response to four abiotic stresses. The stability of 12 potential reference genes was evaluated by four programs (geNorm, NormFinder, BestKeeper, and RefFinder). U2AF combined with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed stable expression in Cd-treated leaves and cold-treated roots. U2AF and FBOX were the most stable reference genes in Cd-treated roots and cold-treated leaves. In Polyethylene Glycol (PEG)- or salt-treated roots, the reference gene U2AF paired with either ACT or CYP were stable. SAND and CACS exhibited the most stability in salt-treated leaves, and combining UPL, PP2A, and EF1a was most suitable for PEG-treated leaves. The stability of U2AF and instability of UPL and TUB was validated by analyzing the expression levels of four target genes (MT2a, VP1, PIP1, and Cor413), and were shown to be capable of detecting subtle changes in expression levels of the target genes in seashore paspalum. This study demonstrated that FBOX, U2AF, and PP2A could be used in future molecular studies that aim to understand the mechanisms of abiotic stress tolerance in seashore paspalum.

Cadmium (Cd) is a non-essential heavy metal and high concentrations in plants causes toxicity of their edible parts and acts as a carcinogen to humans and animals. Paspalum vaginatum is widely cultivating as turfgrass due to its higher abiotic stress tolerance ability. However, there is no clear evidence to elucidate the mechanism for heavy metal tolerance, including Cd. In this study, an RNA sequencing technique was employed to investigate the key genes associated with Cd stress tolerance and accumulation in P. vaginatum. The results revealed that antioxidant enzyme activities catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione S-transferase GST) were significantly higher at 24 h than in other treatments. A total of 6820 (4457/2363, up-/down-regulated), 14,038 (9894/4144, up-/down-regulated) and 17,327 (7956/9371, up-/down-regulated) differentially expressed genes (DEGs) between the Cd1 vs. Cd0, Cd4 vs. Cd0, and Cd24 vs. Cd0, respectively, were identified. The GO analysis and the KEGG pathway enrichment analysis showed that DEGs participated in many significant pathways in response to Cd stress. The response to abiotic stimulus, the metal transport mechanism, glutathione metabolism, and the consistency of transcription factor activity were among the most enriched pathways. The validation of gene expression by qRT-PCR results showed that heavy metal transporters and signaling response genes were significantly enriched with increasing sampling intervals, presenting consistency to the transcriptome data. Furthermore, over-expression of PvSnRK2.7 can positively regulate Cd-tolerance in Arabidopsis. In conclusion, our results provided a novel molecular mechanism of the Cd stress tolerance of P. vaginatum and will lay the foundation for target breeding of Cd tolerance in turfgrass.