Proteome-wide and lysine crotonylation profiling reveals the importance of crotonylation in chrysanthemum (Dendranthema grandiforum) under low-temperature.

BACKGROUND: Low-temperature severely affects the growth and development of chrysanthemum which is one kind of ornamental plant well-known and widely used in the world. Lysine crotonylation is a recently identified post-translational modification (PTM) with multiple cellular functions. However, lysine crotonylation under low-temperature stress has not been studied. RESULTS: Proteome-wide and lysine crotonylation of chrysanthemum at low-temperature was analyzed using TMT (Tandem Mass Tag) labeling, sensitive immuno-precipitation, and high-resolution LC-MS/MS. The results showed that 2017 crotonylation sites were identified in 1199 proteins. Treatment at 4 °C for 24 h and - 4 °C for 4 h resulted in 393 upregulated proteins and 500 downregulated proteins (1.2-fold threshold and P < 0.05). Analysis of biological information showed that lysine crotonylation was involved in photosynthesis, ribosomes, and antioxidant systems. The crotonylated proteins and motifs in chrysanthemum were compared with other plants to obtain orthologous proteins and conserved motifs. To further understand how lysine crotonylation at K136 affected APX (ascorbate peroxidase), we performed a site-directed mutation at K136 in APX. Site-directed crotonylation showed that lysine decrotonylation at K136 reduced APX activity, and lysine complete crotonylation at K136 increased APX activity. CONCLUSION: In summary, our study comparatively analyzed proteome-wide and crotonylation in chrysanthemum under low-temperature stress and provided insights into the mechanisms of crotonylation in positively regulated APX activity to reduce the oxidative damage caused by low-temperature stress. These data provided an important basis for studying crotonylation to regulate antioxidant enzyme activity in response to low-temperature stress and a new research ideas for chilling-tolerance and freezing-tolerance chrysanthemum molecular breeding.

Transcriptomic Analysis of Verbena bonariensis Leaves Under Low-Temperature Stress.

Verbena bonariensis is a valuable plant for both ornament and flower border. As a major constraint, low temperature affects the growing development and survival of V. bonariensis. However, there are few systematic studies in terms of molecular mechanism on the tolerance of low temperature in V. bonariensis. In this study, Illumina sequencing technology was applied to analyze the cold resistance mechanism of plants. Six cDNA libraries were obtained from two samples of two groups, the cold-treated group and the control group. A total of 271,920 unigenes were produced from 406,641 assembled transcripts. Among these, 19,003 differentially expressed genes (DEGs) (corrected p-value <0.01, |log2(fold change) | >3) were obtained, including 9852 upregulated and 9151 downregulated genes. The antioxidant enzyme system, photosynthesis, plant hormone signal transduction, fatty acid metabolism, starch and sucrose metabolism pathway, and transcription factors were analyzed. Based on these results, series of candidate genes related to cold stress were screened out and discussed. The physiological indexes related to response mechanism of low temperature were tested. Eleven upregulated DEGs were validated by Quantitative Real-time PCR. In this study, we provided the transcriptome sequence resource of V. bonariensis and used these data to realize its molecular mechanism under cold stress. The results contributed to valuable clues for genetic studies and helped to screen candidate genes for cold-resistance breeding.

The Overexpression of a Transcription Factor Gene VbWRKY32 Enhances the Cold Tolerance in Verbena bonariensis.

Cold stress poses a serious threat to the survival and bloom of Verbena bonariensis. The enhancement of the cold tolerance of V. bonariensis is the central concern of our research. The WRKY transcription factor (TF) family was paid great attention to in the field of abiotic stress. The VbWRKY32 gene was obtained from V. bonariensis. The VbWRKY32 predicted protein contained two typical WRKY domains and two C2H2 zinc-finger motifs. Under cold stress, VbWRKY32 in leaves was more greatly induced than that in stems and roots. The overexpression (OE) in V. bonariensis increased cold tolerance compared with wild-type (WT). Under cold stress, the OE lines possessed showed greater recovery after cold-treatment restoration ratios, proline content, soluble sugar content, and activities of antioxidant enzymes than WT; the relative electrolyte conductivity (EL), the accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2 -) are lower in OE lines than that in WT. In addition, a series of cold-response genes of OE lines were compared with WT. The results revealed that VbWRKY32 worked as a positive regulator by up-regulating transcription levels of cold-responsive genes. The genes above can contribute to the elevation of antioxidant activities, maintain the membrane stability, and raise osmotic regulation ability, leading to the enhancement of the survival capacity under cold stress. According to this work, VbWRKY32 could serve as an essential gene to confer enhanced cold tolerance in plants.