Effect of long-term high temperatures on seed maturation and seed coat integrity in Brassica napus

Cultivation of canola at temperatures above the optimum growth temperature of 21°C for prolonged periods, especially during the flowering stage, resulted in several adverse effects, including rapid vegetative growth, reduced viability of female gametophytes, increased seed abortion rate, accelerated embryo development, and a reduction in seed oil composition (Young et al., 2004; Mácová et al., 2022; Secchi et al., 2023). One of the distinctive phenotypes observed during the seed development of certain canola cultivars subjected to prolonged heat stress affecting the seed yield is the occurrence of the pre-harvest sprouting phenotype (PHS) (Mácová et al., 2022). Misregulation of seed dormancy by abscisic acid and dormancy-related genes is thought to be the primary cause of PHS in many cereal crops (Benech-Arnold & Rodríguez, 2018; Tai et al., 2021). This phenotype is associated with seed coat rupture (SCR), observed in seeds during the early stages of maturation. In this study, we employed a multi-methodological approach to investigate the occurrence of SCR phenotype in seeds of Brassica napus cv. Topas. The results demonstrate that SCR occurs in seeds around 20 days after pollination (20DAP) when the plants are cultivated in elevated temperatures over an extended period. The unrestricted embryonic growth exerts pressure on the seed coat, as evidenced by a reduction in the thickness of the seed coat cell layers. This results in an early alteration to the cell wall composition, with an increased proportion of demethylesterified pectin, which is likely to stiffen the seed coat, thereby rendering it more susceptible to rupture. The precise mechanism by which accelerated embryo development influences heat stress-mediated seed development in canola plants has yet to be elucidated. To investigate the impact of high temperature on seed coat rupture, we collected seeds of Brassica napus cv. Topas at 14 days after pollination (DAP), 18 DAP, and 20DAP from plants grown in control conditions (TCT) (21oC day / 18oC night, long-day) and high-temperature conditions (THT) (32oC day / 18 oC night, long-day). Plants were moved to THT at the start of bolting, and kept in THT until ripenning. Samples of 20DAP seeds at THT with SCR phenotype were also collected. We performed gene expression profiling analysis using data obtained from RNA-seq for these 3 samples at TCT and 4 samples at THT at 3 time points, in four biological replicates.

当甘蓝型油菜（Brassica napus）在高于其21℃最适生长温度的环境中长期种植时，尤其是在开花期，会产生多种不利影响，包括营养生长过速、雌配子体活力降低、种子败育率上升、胚胎发育加速以及种子油脂组分改变（Young等，2004；Mácová等，2022；Secchi等，2023）。在部分遭受长期热胁迫、进而影响种子产量的甘蓝型油菜品种的种子发育过程中，所观察到的典型表型之一为收获前发芽表型（pre-harvest sprouting phenotype, PHS）（Mácová等，2022）。对于多数谷类作物而言，脱落酸（abscisic acid）及休眠相关基因对种子休眠的调控失常被认为是引发PHS的主要原因（Benech-Arnold & Rodríguez，2018；Tai等，2021）。该表型与种子成熟早期观察到的种皮破裂（seed coat rupture, SCR）相关。本研究采用多方法学研究策略，探究甘蓝型油菜品种Topas种子的种皮破裂表型发生情况。结果显示，当植株长期处于高温环境时，授粉后约20天（days after pollination, DAP）的种子会出现SCR现象。不受调控的胚胎生长会对种皮施加压力，这一点可通过种皮细胞层厚度的降低得到证实；同时该过程会提前改变细胞壁组成，使去甲酯化果胶的比例升高，这一变化可能会硬化种皮，进而使其更易发生破裂。目前，胚胎发育加速如何介导甘蓝型油菜在热胁迫下的种子发育，其具体机制仍有待阐明。为探究高温对种皮破裂的影响，本研究从处于对照条件（TCT：昼温21℃/夜温18℃，长日照）与高温条件（THT：昼温32℃/夜温18℃，长日照）下种植的植株中，分别采集了授粉后14天（DAP）、18DAP及20DAP的种子。植株于抽薹初期被转移至THT环境，并持续培养至成熟。此外，还采集了THT环境下20DAP且呈现SCR表型的种子样本。本研究针对上述样本开展基于RNA测序（RNA-seq）的基因表达谱分析：对照条件下共3个样本，高温条件下3个时间点共4个样本，均设置4次生物学重复。