Table_4_Genome-wide identification, interaction of the MADS-box proteins in Zanthoxylum armatum and functional characterization of ZaMADS80 in floral development.docx

As a typical dioecious species, Zanthoxylum armatum establishes apomictic reproduction, hence only female trees are cultivated. However, male and hermaphrodite flowers have recently appeared in female plants, resulting in a dramatic yield reduction. To date, the genetic basis underlying sex determination and apomixis in Z. armatum has been largely unknown. Here, we observed abortion of the stamen or carpel prior to primordium initiation, thus corroborating the potential regulation of MADS-box in sex determination. In Z. armatum, a total of 105 MADS-box genes were identified, harboring 86 MIKC-type MADSs with lack of FLC orthologues. Transcriptome analysis revealed candidate MADSs involved in floral organ identity, including ten male-biased MADSs, represented by ZaMADS92/81/75(AP3/PI-like), and twenty-six female-specified, represented by ZaMADS80/49 (STK/AGL11-like) and ZaMADS42 (AG-like). Overexpressing ZaMADS92 resulted in earlier flowering, while ZaMADS80 overexpression triggered precocious fruit set and parthenocarpy as well as dramatic modifications in floral organs. To characterize their regulatory mechanisms, a comprehensive protein-protein interaction network of the represented MADSs was constructed based on yeast two-hybrid and bimolecular fluorescence complementation assays. Compared with model plants, the protein interaction patterns in Z. armatum exhibited both conservation and divergence. ZaMADS70 (SEP3-like) interacted with ZaMADS42 and ZaMADS48 (AP3-like) but not ZaMADS40 (AP1-like), facilitating the loss of petals in Z. armatum. The ZaMADS92/ZaMADS40 heterodimer could be responsible for accelerating flowering in ZaMADS92-OX lines. Moreover, the interactions between ZaMADS80 and ZaMADS67(AGL32-like) might contribute to apomixis. This work provides new insight into the molecular mechanisms of MADS-boxes in sex organ identity in Z. armatum.

作为典型的雌雄异株（dioecious）物种，竹叶花椒（Zanthoxylum armatum）采用无融合生殖（apomictic reproduction）策略，因此生产中仅栽培雌性植株。但近期研究发现雌性植株上出现了雄性花与两性花，导致产量大幅下降。截至目前，竹叶花椒性别决定与无融合生殖的遗传基础仍未被充分解析。本研究通过观察发现，雄蕊或心皮在花原基起始阶段即发生败育，从而佐证了MADS-box基因家族（MADS-box）在其性别决定中的潜在调控作用。本研究共在竹叶花椒中鉴定出105个MADS-box基因，其中包含86个MIKC型MADS基因，且未检测到FLC同源基因。转录组分析筛选得到参与花器官身份调控的候选MADS基因，包括10个雄性偏好型MADS基因，以ZaMADS92/81/75（AP3/PI类）为代表，以及26个雌性特异型MADS基因，以ZaMADS80/49（STK/AGL11类）和ZaMADS42（AG类）为代表。过表达ZaMADS92可导致植株提早开花，而过表达ZaMADS80则触发提前坐果与单性结实（parthenocarpy），同时引发花器官的显著修饰改变。为解析其调控机制，本研究基于酵母双杂交（yeast two-hybrid）与双分子荧光互补（bimolecular fluorescence complementation）实验，构建了代表性MADS蛋白的全面蛋白质互作网络。与模式植物相比，竹叶花椒的蛋白互作模式兼具保守性与分化性：ZaMADS70（SEP3类）可与ZaMADS42及ZaMADS48（AP3类）互作，但无法与ZaMADS40（AP1类）结合，这可能促进了竹叶花椒花瓣的丢失。ZaMADS92与ZaMADS40形成的异二聚体，可能是ZaMADS92过表达株系提早开花的分子基础。此外，ZaMADS80与ZaMADS67（AGL32类）的互作可能参与调控无融合生殖过程。本研究为解析竹叶花椒MADS-box基因家族在性器官身份决定中的分子机制提供了新的研究视角。