D-type cyclin (cyclin D, CYCD), combined with cyclin-dependent kinases (CDKs), participates in the regulation of cell cycle G1/S transition and plays an important role in cell division and proliferation. CYCD could affect the growth and development of herbaceous plants, such as Arabidopsis thaliana, by regulating the cell cycle process. However, its research in wood plants (e.g., poplar) is poor. Phylogenetic analysis showed that in Populus trichocarpa, CYCD3 genes expanded to six members, namely PtCYCD3;1–6. P. tomentosa CYCD3 genes were amplified based on the CDS region of P. trichocarpa CYCD3 genes. PtoCYCD3;3 showed the highest expression in the shoot tip, and the higher expression in young leaves among all members. Therefore, this gene was selected for further study. The overexpression of PtoCYCD3;3 in plants demonstrated obvious morphological changes during the observation period. The leaves became enlarged and wrinkled, the stems thickened and elongated, and multiple branches were formed by the plants. Anatomical study showed that in addition to promoting the differentiation of cambium tissues and the expansion of stem vessel cells, PtoCYCD3;3 facilitated the division of leaf adaxial epidermal cells and palisade tissue cells. Yeast two-hybrid experiment exhibited that 12 PtoCDK proteins could interact with PtoCYCD3;3, of which the strongest interaction strength was PtoCDKE;2, whereas the weakest was PtoCDKG;3. Molecular docking experiments further verified the force strength of PtoCDKE;2 and PtoCDKG;3 with PtoCYCD3;3. In summary, these results indicated that the overexpression of PtoCYCD3;3 significantly promoted the vegetative growth of Populus, and PtoCYCD3;3 may interact with different types of CDK proteins to regulate cell cycle processes.

Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants. Although the GSTs gene family has been reported in many land plants, our knowledge of the evolution and function of the willow GSTs is still limited. In this study, 22 full-length GST genes were cloned from Salix babylonica and divided into three classes based on the conserved domain analysis, phylogenetic tree and gene structure: tau, phi and DHAR. The tissue-specific expression patterns were substantially different among the tau and phi GSTs. The Salix GST proteins showed functional divergences in the substrate specificities, substrate activities and kinetic characteristics. The site-directed mutagenesis studies revealed that a single amino acid mutation (Ile/Val53→Thr53) resulted in the lowest activity of SbGSTU7 among the Salix GSTs. These results suggest that non-synonymous substitution of an amino acid at the putative glutathione-binding site may play an important role in the divergence of enzymatic functions of Salix GST family.

Cobalt (Co) is an essential component of several enzymes and coenzymes in living organisms. Excess Co is highly toxic to plants. The knowledge of molecular response mechanisms to cobalt stress in plants is still limited, especially in woody plants. The responses of weeping willow (Salix babylonica) seedlings to Co stress were studied using morphological and physiochemical measurements and RNA-seq analysis. The physiological and biochemical indexes such as growth rate, the content of chlorophyll and soluble sugar, photosynthesis and peroxidase activity were all changed in willow seedlings under Co stress. The metal ion concentrations in willow including Cu, Zn and Mg were disturbed due to excess Co. Of 2002 differentially expressed genes (DEGs), 1165 were root-specific DEGs and 837 were stem and leaf-specific DEGs. Further analysis of DEGs showed there were multiple complex cascades in the response network at the transcriptome level under Co stress. Detailed elucidation of responses to oxidative stress, phytohormone signaling-related genes and transcription factors (TFs), and detoxification of excess cellular Co ion indicated the various defense mechanisms in plants respond to cobalt stress. Our findings provide new and comprehensive insights into the plant tolerance to excess Co stress.

Aconitase (ACO) is a key enzyme that catalyzes the isomerization of citrate to isocitrate in the tricarboxylic acid (TCA) and glyoxylate cycles. The function of ACOs has been well studied in model plants, such as Arabidopsis. In contrast, the evolutionary patterns of the ACO family in land plants are poorly understood. In this study, we systematically examined the molecular evolution and expression divergence of the ACO gene family in 12 land plant species. Thirty-six ACO genes were identified from the 12 land plant species representing the four major land plant lineages: bryophytes, lycophytes, gymnosperms, and angiosperms. All of these ACOs belong to the cytosolic isoform. Three gene duplication events contributed to the expansion of the ACO family in angiosperms. The ancestor of angiosperms may have contained only one ACO gene. One gene duplication event split angiosperm ACOs into two distinct clades. Two clades showed a divergence in selective pressure and gene expression patterns. The cis-acting elements that function in light responsiveness were most abundant in the promoter region of the ACO genes, indicating that plant ACO genes might participate in light regulatory pathways. Our findings provide comprehensive insights into the ACO gene family in land plants.

Cyclin-dependent kinases (CDKs) control the progression of the cell cycle. D-type cyclin (CYCD) is generally believed to form a complex with CDK and control the G1/S transition. In plants, CYCD and CDK gene families can be divided into 6 (D1–D7) and 7 (CDKA–CDKG) subclasses, respectively. Different subclasses in the CYCD and CDK families have different numbers, structures and functions. In some heterologous woody plants, the functions of these subclass family members remain unclear. In this study, 43 CYCD and 27 CDK gene family members were identified in the allodiploid Populus tomentosa Carr. Phylogenetic analysis suggested that these CYCDs and CDKs were divided into 6 and 7 subclasses, respectively, which were the same as other species. The analysis of protein properties, gene structure, motifs, domains, cis-acting elements and tissue-specific expression of all members of these CYCDs and CDKs showed that the differences between members of different subclasses varied widely, but members of the same subclass especially in the CDK gene family were very similar. These findings also demonstrated a strong correlation between CYCD and CDK gene family members in response to hormones and specific expression. The collinear analysis of P. tomentosa, Populus trichocarpa and Arabidopsis thaliana showed that the expansion patterns of CYCD and CDK gene families were predominantly whole genome duplications (WGD). The protein interaction prediction results of different subclasses of CYCD and CDKs showed that the interaction between different subclasses of CYCD and CDKs was significantly different. Our previous study found that transgenic PtoCYCD2;1 and PtoCYCD3;3 poplars exhibited opposite phenotypes. Y2H and BIFC results showed that the interaction between PtoCYCD2;1 and PtoCYCD3;3 was significantly different with CDKs. This finding might suggest that the functional differences of different CYCD subclasses in plant growth and development were closely related to the different interactions between CYCD and CDK. Our results provide a good idea and direction for the functional study of CYCD and CDK proteins in woody plants.