Unraveling Cadmium Toxicity in Trifolium repens L. Seedling: Insight into Regulatory Mechanisms Using Comparative Transcriptomics Combined with Physiological Analyses.

Trifolium repens (T. repens) can accumulate significant amounts of heavy metal ions, and has strong adaptability to wide environmental conditions, and relatively large biomass, which is considered a potential plant for phytoremediation. However, the molecular mechanisms of T. repens involved in Cd tolerance have not yet been studied in detail. This study was conducted to examine the integrative responses of T. repens exposed to a high-level CdCl2 by investigating the physiological and transcriptomic analyses. The results suggested that T. repens seedlings had a high degree of tolerance to Cd treatment. The roots accumulated higher Cd concentration than leaves and were mainly distributed in the cell wall. The content of MDA, soluble protein, the relative electrolyte leakage, and three antioxidant enzymes (POD, SOD, and APX) was increased with the Cd treatment time increasing, but the CAT enzymes contents were decreased in roots. Furthermore, the transcriptome analysis demonstrated that the differentially expressed genes (DEGs) mainly enriched in the glutathione (GSH) metabolism pathway and the phenylpropanoid biosynthesis in the roots. Overexpressed genes in the lignin biosynthesis in the roots might improve Cd accumulation in cell walls. Moreover, the DEGs were also enriched in photosynthesis in the leaves, transferase activity, oxidoreductase activity, and ABA signal transduction, which might also play roles in reducing Cd toxicity in the plants. All the above, clearly suggest that T. repens employ several different mechanisms to protect itself against Cd stress, while the cell wall biosynthesis and GSH metabolism could be considered the most important specific mechanisms for Cd retention in the roots of T. repens.

R2R3-MYB gene family: Genome-wide identification provides insight to improve the content of proanthocyanidins in Trifolium repens.

The R2R3-MYB family is one of largest transcription factor families in plants playing significant roles in regulating anthocyanin and proanthocyanidin biosynthesis. Proanthocyanidins are one of major objectives to improve the quality of white clover (Trifolium repens L.), which have a beneficial effect on ruminant to prevent the lethal pasture bloat. A total of 133 TrR2R3-MYB genes were identified and distributed on all 16 chromosomes based on the whole genome information of white clover. Also, by exploring the gene structure, motifs and duplication events of TrR2R3-MYBs, as well as the evolutionary relationship with TrR2R3-MYB genes of other species, 10 TrR2R3-MYB genes with the potential to regulate the anthocyanins and proanthocyanidins biosynthesis were screened. These TrR2R3-MYB genes responded significantly to low temperature in white clover. In addition, they have different expression patterns in leaves, petioles and inflorescences of white clover. Importantly, TrMYB116 and TrMYB118 may positively regulate anthocyanin accumulation and low temperature response in white clover. TrMYB118 may also be associated with anthocyanin pigmentation pattern in Purple leaves. This study provides a basis for verifying the function of TrR2R3-MYB and breeding white clover cultivars with high proanthocyanidins.