Color variation in plant leaves has a significant impact on their photosynthesis and plant growth. Camellia chekiangoleosa yellow-leaf mutants are ideal materials for studying the mechanisms of pigment synthesis and photosynthesis, but their mechanism of leaf variation is not clear. We systematically elucidated the intrinsic causes of leaf yellowing in the new Camellia chekiangoleosa variety ‘Diecui Liuji’ in terms of changes in its cell structure, pigment content, and transcript levels. This study indicates that the incomplete structure of chloroplast-like vesicles, the decrease in blue-green chlorophyll a, and the increase in yellow-green chlorophyll b in yellowing leaves are the direct causes of yellowing-leaf formation. The high expression of genes that catalyze the degradation of chlorophyll a (PAO and RCCR) and its conversion to chlorophyll b (CAO) in yellowing leaves leads to a decrease in the chlorophyll a content, while the low expression of CLH genes is the main reason for the increase in the chlorophyll b content. We also found transcription factors such as ERF, E2F, WRKY, MYB, TPC, TGA, and NFYC may regulate their expression. RT-qPCR assays of 12 DEGs confirm the RNA-seq results. This study will provide a foundation for investigating the transcriptional and regulatory mechanisms of leaf color changes.

The WRKY gene family is essential for controlling a variety of plant physiological functions, yet the involvement of specific WRKY members in pigment biosynthesis and accumulation in Camellia chekiangoleosa remains unexplored, particularly in anthocyanins and carotenoids, which play crucial roles in the pigmentation of C. chekiangoleosa. This study systematically identified 87 WRKY genes across 15 chromosomes in C. chekiangoleosa through bioinformatic approaches. Further structural and phylogenetic analyses of these TFs enabled their classification into six different subgroups. WRKY family expansion was shown to be mostly driven by tandem duplication. W-box elements, which can be binding sites for WRKY transcription factors, were present in a number of biosynthetic genes in the pigment production pathway. Yeast one-hybrid assay confirmed that five WRKY transcription factors (CchWRKY15/24/33/47/76) directly bind to the promoter regions of two key biosynthetic genes, CchPSY1 and Cch4CL1. Intriguingly, among the five WRKYs tested, the expression levels of CchWRKY15, CchWRKY33, and CchWRKY47 showed the strongest positive associations with flavonoid accumulation (p < 0.05, Pearson correlation analysis).These findings provide novel insights into the evolutionary patterns, transcriptional regulation, and functional characteristics of CchWRKYs, while elucidating their possible regulatory mechanisms in the fruit coloration of C. chekiangoleosa.

The provincial-level registered superior cultivar Camellia chekiangoleosa ‘Ganhongyou 1’ boasts superior economic traits coupled with significant ornamental value, driving demand for an efficient propagation system. Consequently, this study aimed to develop a rapid micropropagation protocol by investigating culture conditions using semi-woody nodal segments with axillary buds as explants on Hyponex basal medium supplemented with varying combinations of plant growth regulators. Contamination was effectively minimized to 18% by a combined approach of surface sterilization (75% ethanol, 0.1% HgCl₂, and 20% NaClO) and incorporating 1 mL/L bactericide into the induction medium. For bud induction, the optimal medium was 2 g/L Hyponex supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA) and 0.2 mg/L indole-3-acetic acid (IAA), achieving an 86.67% induction rate. The best proliferation was achieved on the medium containing 2 g/L Hyponex, 1.0 mg/L 6-BA, 0.15 mg/L 3-indolebutyric acid (IBA), and 0.5 mg/L gibberellic acid (GA₃), yielding a proliferation coefficient of 6.53. A combined strategy, integrating in vitro pre-culture with ex vitro treatment, proved most effective for rooting and acclimatization: shoots were first pre-cultured for 20 days on 1/2 strength Hyponex medium supplemented with 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 2.0 mg/L IBA, followed by ex vitro base treatment with 1.0 g/L ABT (a rooting powder complex) solution before transplantation into seedling bags. This approach resulted in an 88% survival rate. Furthermore, anatomical analysis revealed the origin of adventitious root primordia from phloem parenchyma cells, thereby confirming a phloem-rooting pattern for this species. In conclusion, this study establishes a practical and efficient micropropagation protocol for ‘Ganhongyou 1’, providing a reliable technical foundation for its commercial-scale seedling production.

Plant seedling morphogenesis is considerably related to photosynthesis, pigment synthesis, and circadian periodicity during seedling development. We identified and cloned a maize zebra or crossbanding leaves mutant wk3735, which produces pale white kernels and was identified and plays a role in the equilibrium of the Redox state the in/out of ETC by active oxygen scavenging. Interestingly, it produces the zebra leaves during the production of the first seven leaves, which is apparently different from the mutation of homologs AtPTOX in Arabidopsis. It is intriguing to investigate how and why yellow crossbands (zebra leaf phenotype) emerge on leaves. As expected, chlorophyll concentration and photosynthetic efficiency both significantly declined in the yellow sector of wk3735 leaves. Meanwhile, we observed the circadian expression pattern of ZmPTOX1, which was further validated by protein interaction assays of the circadian clock protein TIM1 and ZmPTOX1. The transcriptome data of yellow (muW) and green (muG) sectors of knock-out lines and normal leaves of overexpression lines (OE) at the 5th-leaf seedling stage were analyzed. Zebra leaf etiolated sections exhibit a marked defect in the expression of genes involved in the circadian rhythm and rhythmic stress (light and cold stress) responses than green sections. According to the analysis of co-DEGs of muW vs. OE and muG vs. OE, terms linked to cell repair function were upregulated while those linked to environmental adaptability and stress response were downregulated due to the mutation of ZmPTOX1. Further gene expression level analyses of reactive oxygen species (ROS) scavenging enzymes and detection of ROS deposition indicated that ZmPTOX1 played an essential role in plant stress resistance and ROS homeostasis. The pleiotropic roles of ZmPTOX1 in plant ROS homeostasis maintenance, stress response, and circadian rhythm character may collectively explain the phenotype of zebra leaves during wk3735 seedling development.