Mutagenesis is an important tool for breeding and genomic research. In this study, the germinated seeds and isolated microspores of a double haploid line ‘FT’ were treated with EMS, respectively, with the aim of comparing the effects of the two approaches on generating mutants in Chinese cabbage. For microspore EMS mutagenesis, the isolated microspores were treated with 0.12% EMS for 20 min, a total of 1268 plantlets were obtained, and 15 M₁ mutants were screened with a mutation frequency of 1.2%. For seed EMS mutagenesis, 7800 germinated seeds were treated with 0.8% EMS for 12 h, and a total of 701 M₂ mutants were screened, with a mutation frequency of 18.78%. In total, 716 mutants with heritable morphological variation including leaf color, leaf shape, leafy head, bolting, and fertility, were obtained from the EMS mutagenesis experiments. Homozygous mutant plants could be screened from M₁ lines by microspore mutagenesis, and M₂ lines by seed mutagenesis. The mutation frequency was higher in seed mutagenesis than in microspore mutagenesis. Based on these results, we propose that seed EMS mutagenesis is more suitable to generate a large-scale mutant library, and the microspore EMS mutagenesis is conducive to rapidly obtaining homozygous mutants.

Actin filaments are essential for plant adaptation to high temperatures. However, the molecular mechanisms of actin filaments in plant thermal adaptation remain unclear. Here, we found that the expression of Arabidopsis actin depolymerization factor 1 (AtADF1) was repressed by high temperatures. Compared with wild-type seedlings (WT), the mutation of AtADF1 and the overexpression of AtADF1 led to promoted and inhibited plant growth under high temperature conditions, respectively. Further, high temperatures induced the stability of actin filaments in plants. Compared with WT, Atadf1-1 mutant seedlings showed more stability of actin filaments under normal and high temperature conditions, while the AtADF1 overexpression seedlings showed the opposite results. Additionally, AtMYB30 directly bound to the promoter of AtADF1 at a known AtMYB30 binding site, AACAAAC, and promoted the transcription of AtADF1 under high temperature treatments. Genetic analysis further indicated that AtMYB30 regulated AtADF1 under high temperature treatments. Chinese cabbage ADF1 (BrADF1) was highly homologous with AtADF1. The expression of BrADF1 was inhibited by high temperatures. BrADF1 overexpression inhibited plant growth and reduced the percentage of actin cable and the average length of actin filaments in Arabidopsis, which were similar to those of AtADF1 overexpression seedlings. AtADF1 and BrADF1 also affected the expression of some key heat response genes. In conclusion, our results indicate that ADF1 plays an important role in plant thermal adaptation by blocking the high-temperature-induced stability of actin filaments and is directly regulated by MYB30.

Actin depolymerizing factors (ADFs), as the important actin-binding proteins (ABPs) with depolymerizing/severing actin filaments, play a critical role in plant growth and development, and in response to biotic and abiotic stresses. However, the information and function of the ADF family in melon remains unclear. In this study, 9 melon ADF genes (CmADFs) were identified, distributed in 4 subfamilies, and located on 6 chromosomes respectively. Promoter analysis revealed that the CmADFs contained a large number of cis-acting elements related to hormones and stresses. The similarity of CmADFs with their Arabidopsis homologue AtADFs in sequence, structure, important sites and tissue expression confirmed that ADFs were conserved. Gene expression analysis showed that CmADFs responded to low and high temperature stresses, as well as ABA and SA signals. In particular, CmADF1 was significantly up-regulated under above all stress and hormone treatments, indicating that CmADF1 plays a key role in stress and hormone signaling responses, so CmADF1 was selected to further study the mechanism in plant tolerance low temperature. Under low temperature, virus-induced gene silencing (VIGS) of CmADF1 in oriental melon plants showed increased sensitivity to low temperature stress. Consistently, the stable genetic overexpression of CmADF1 in Arabidopsis improved their low temperature tolerance, possibly due to the role of CmADF1 in the depolymerization of actin filaments. Overall, our findings indicated that CmADF genes, especially CmADF1, function in response to abiotic stresses in melon.

Potassium (K) is an essential nutrient for the growth and development of plants. Root hairs are the main parts of plants that absorb K⁺. The regulation of plant root hair growth in response to a wide range of environmental stresses is crucially associated with the dynamics of actin filaments, and the thick actin bundles at the apical and sub-apical regions are essential for terminating the rapid elongation of root hair cells. However, the dynamics and roles of actin filaments in root hair growth in plants’ response to low K⁺ stress are not fully understood. Here, we revealed that root hairs grow faster and longer under low K⁺ stress than the control conditions. Compared to control conditions, the actin filaments in the sub-apex of fast-growing wild-type root hairs were longer and more parallel under low K⁺ stress, which correlates with an increased root hair growth rate under low K⁺ stress; the finer actin filaments in the sub-apex of the early fully grown Col-0 root hairs under low K⁺ stress, which is associated with low K⁺ stress-induced root hair growth time. Further, Arabidopsis thaliana actin bundling protein Villin1 (VLN1) and Villin4 (VLN4) was inhibited and induced under low K⁺ stress, respectively. Low K⁺ stress-inhibited VLN1 led to decreased bundling rate and thick bundle formation in the early fully grown phase. Low K⁺ stress-induced VLN4 functioned in keeping long filaments in the fast-growing phase. Furthermore, the analysis of genetics pointed out the involvement of VLN1 and VLN4 in the growth of root hairs under the stress of low potassium levels in plants. Our results provide a basis for the dynamics of actin filaments and their molecular regulation mechanisms in root hair growth in response to low K⁺ stress.

Considering that the presence of a leafy head is a key morphological characteristic that determines the yield and quality of Chinese cabbage, identifying the major genes regulating the formation of a leafy head is crucial for variety improvement. A heading-related gene, BrKS, was previously predicted from a non-heading mutant, nhm1, derived from a heading variety, which encodes a key enzyme involved in gibberellin biosynthesis. Here, another mutant, nhm2, was identified from the same EMS-mutagenized population, and the phenotype of nhm2 was consistent with that of nhm1. We crossed mutants nhm1 and nhm2, and their F₁ plants exhibited the mutant phenotype, which indicated that their mutant genes were allelic. A single non-synonymous mutation in the fourth exon of BrKS in mutant nhm1 and another single non-synonymous mutation in the tenth exon of BrKS in mutant nhm2. The same gene mutation in mutants nhm1 and nhm2 produced a similar non-heading phenotype, which confirmed the role of BrKS in the leafy head formation of Chinese cabbage. RNA-Seq analysis indicated that a transcription factor gene, BrERF1A, which is associated with leaf development, significantly down-regulated expression in mutant nhm1, and after the mutant was treated with GA₃, the expression level of BrERF1A was recovered, which indicated that BrKS might be involved in leafy head formation through regulating the expression level of BrERF1A. Our findings provide important clues for revealing the molecular mechanism of leafy head formation in Chinese cabbage.