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.

The U-box protein family of ubiquitin ligases is important in the biological processes of plant growth, development, and biotic and abiotic stress responses. Plants in the genus Zoysia are recognized as excellent warm-season turfgrass species with drought, wear and salt tolerance. In this study, we conducted the genome-wide identification of plant U-box (PUB) genes in Zoysia japonica based on U-box domain searching. In total, 71 ZjPUB genes were identified, and a protein tree was constructed of AtPUBs, OsPUBs, and ZjPUBs, clustered into five groups. The gene structures, characteristics, cis-elements and protein interaction prediction network were analyzed. There were mainly ABRE, ERE, MYB and MYC cis-elements distributed in the promoter regions of ZjPUBs. ZjPUBs were predicted to interact with PDR1 and EXO70B1, related to the abscisic acid signaling pathway. To better understand the roles of ZjPUBs under salt stress, the expression levels of 18 ZjPUBs under salt stress were detected using transcriptome data and qRT-PCR analysis, revealing that 16 ZjPUBs were upregulated in the roots under salt treatment. This indicates that ZjPUBs might participate in the Z. japonica salt stress response. This research provides insight into the Z. japonica PUB gene family and may support the genetic improvement in the molecular breeding of salt-tolerant zoysiagrass varieties.

Dollar spot is reported to be caused by multiple Clarireedia species and is a serious problem on many turfgrasses around the world. To our knowledge, the distribution of different Clarireedia species and their sensitivity profiles to fungicides remains unknown. In this study, a total of 275 isolates were characterized by ITS sequence. Amounts of 124, 59 and 75 isolates were identified as C. jacksonii, C. monteithiana and C. paspali, respectively, while each species of C. homoeocarpa and C. bennettii had only five isolates. Four and three isolates were identified as two potential new species, which remained to be further characterized. C. jacksonii and C. monteithiana were distributed worldwide, while C. paspali was restricted to China. Of the isolates with host information, 81% (93/115) and 19% (22/115) of C. jacksonii isolates were collected from C3 and C4 plants, respectively, 97% (56/58) of the C. monteithiana isolates were collected from C4 plants and all C. paspali isolates were collected from C4 plants. The coexistence of different Clarireedia species on the same C4 host type in the same locales was found in Shanghai (Paspalum vaginatum), Jiangsu (Paspalum vaginatum) and Florida (Cynodon dactylon). The study revealed that differential fungicide sensitivity patterns were observed in different species in Clarireedia for the first time. Similar differential sensitivity profiles were also found in the locales with coexistence of at least two species. The findings from this study suggest that the adjacent coexistence of different Clarireedia species and the differential fungicide sensitivity profiles of different species will complicate dollar spot disease control.

Salinity and drought stress are major abiotic stresses negatively affecting the growth and productivity of alfalfa (Medicago sativa L.). Exploration of genes exhibiting superior tolerance to salinity and drought stress in alfalfa will help aid target molecular breeding and developing tolerant forages. In this study, we adopted a high-efficient yeast FOX hunting system for the identification of salinity and drought tolerant genes in alfalfa. Based on the Gateway-compatible vector system, a high-quality expression library was constructed, containing 1.3 × 10⁷ clones with an average size of 1.44 kb. Through heterologous transformation of mixed library plasmid into salt or drought sensitive yeast mutants, monoclonal resistant strains were screened and tolerant genes were captured. Eighteen salinity-tolerance genes were obtained which were involved in several pathways, containing GRAS and zinc finger transcriptional factors, PP2A interaction module, ERVT vesicle transporter and LETM transmembrane protein. Twelve drought tolerance genes were separated, including ERF and SCL transcriptional factors, CIPK and BSK protein kinases, TGL-type ligase, cPGM and cPDL protease. The mRNA transcription levels of these tolerant genes were inducible or suppressible for response to salt or drought stress conditions following quantitative PCR detection, respectively. Furthermore, heterologous transformation of ERVT and CIPK11 can improve the salt and drought stress tolerance in Arabidopsis, which indicates the conservative function of the screening gene in yeast and Arabidopsis. Obtaining these candidate genes can provide new insights for future research with respect to plant salt and drought tolerance.

Cadmium (Cd) is considered to be one of the most toxic metals, causing serious harm to plants’ growth and humans’ health. Therefore, it is necessary to study simple, practical, and environmentally friendly methods to reduce its toxicity. Until now, people have applied zinc sulfate to improve the Cd tolerance of plants. However, related studies have mainly focused on physiological and biochemical aspects, with a lack of in-depth molecular mechanism research. In this study, we sprayed high (40 mM) and low (2.5 mM) concentrations of zinc sulfate on seashore paspalum (Paspalum vaginatum Swartz) plants under 0.5 mM Cd stress. Transcriptome sequencing and physiological indicators were used to reveal the mechanism of Cd tolerance. Compared with the control treatment, we found that zinc sulfate decreased the content of Cd²⁺ by 57.03–73.39%, and that the transfer coefficient of Cd decreased by 58.91–75.25% in different parts of plants. In addition, our results indicate that the antioxidant capacity of plants was improved, with marked increases in the glutathione content and the activity levels of glutathione reductase (GR), glutathione S-transferase (GST), and other enzymes. Transcriptome sequencing showed that the differentially expressed genes in both the 0.5 Zn and 40 Zn treatments were mainly genes encoding GST. This study suggests that genes encoding GST in the glutathione pathway may play an important role in regulating the Cd tolerance of seashore paspalum. Furthermore, the present study provides a theoretical reference for the regulation mechanism caused by zinc sulfate spraying to improve plants’ Cd tolerance.