採後光照、離層酸及褪黑激素處理調控青花菜黑暗誘導老化與相關基因表現之研究

Abstract

青花菜(Brassica oleracea var. italica)富營養且經濟價值高是臺灣重要蔬菜作物。青花菜於花苞未開放前採收，呼吸與代謝作用旺盛，採收後易發生黃化與老化現象。植物於持續黑暗環境下開啟黑暗誘導老化(dark-induced senescence, DIS)途徑，透過phytochrome-interacting factor (PIFs)、ABA INSENSITIVE 5 (ABI5)、ETHYLENE INSENSITIVE 3 (EIN3)等轉錄因子誘導PHEOPHORBIDE A OXYGENASE (PAO)、STAY-GREEN 1 (SGR1)與NON-YELLOW COLORING 1 (NYC1)等基因使葉綠素降解；光照則透過維持CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)、LATE ELONGATED HYPOCOTYL (LHY)等基因表現抑制PIF以延緩植株老化。然而，目前對於青花菜採收後老化期間DIS扮演之角色並不清楚。本研究探討光照處理延緩青花菜貯藏期間DIS的效果，進而釐清DIS對青花菜老化之影響。結果顯示以100 μmol·m-2·s-1進行2小時光照處理可延緩於8℃貯藏之青花菜。其中100 μmol·m-2·s-1光照處理組之青花菜中BoCCA1表現量於貯藏5天後高於對照組約4倍；BoPIF5、BoEIN3、BoNYC1表現量則較對照組分別低約1.3、4.8、1.8倍，總葉綠素含量則高於對照組約1.4倍。這些結果顯示青花菜採後DIS過程中BoPIF5、BoEIN3、BoNYC1表現量提升，並導致葉綠素含量降低及花球黃化；而較高的BoCCA1表現則降低BoPIF5、BoABI5、BoPAO等DIS相關基因表現，間接延緩青花菜花球黃化現象。本研究亦發現光照會導致氣孔開啟較對照組的孔徑增加約1.5倍，導致失重率於貯藏12天後會高於對照組約2倍。因此嘗試以離層酸(ABA)抑制氣孔開啟與失重。然而於青花菜貯藏前噴施ABA無法延緩失重，因此再測試褪黑激素抑制氣孔開啟與失重之效果。以100 μM褪黑激素浸泡處理可使青花菜氣孔關閉並延緩失重。然而，若於100 μM褪黑激素浸泡處理後再以100 μmol·m-2·s-1進行光照處理則無法抑制氣孔開啟或延緩失重。本研究結果顯示採收後青花菜貯藏期間以光照處理可延緩DIS，但會使氣孔開啟導致失重率增加，且無法藉由離層酸或褪黑激素抑制氣孔開啟。此成果可作為未來對採收後青花菜DIS相關研究及開發延緩老化技術之基礎。

Broccoli (Brassica oleracea var. italica), renowned for its nutritional richness and high economic value, is an important vegetable crop in Taiwan. Broccoli is harvested before the flower buds open, during which its respiration and metabolic activities are vigorous. However, it is prone to yellowing and senescence after harvest. Under dark conditions, plants initiate dark-induced senescence (DIS), mediated by phytochrome-interacting factors (PIFs), which induce the expression of genes such as ABSCISIC ACID INSENSITIVE 5 (ABI5), ETHYLENE INSENSITIVE 3 (EIN3), leading to the expression of PHEOPHORBIDE A OXYGENASE (PAO), STAY-GREEN 1 (SGR1), NON-YELLOW COLORING 1 (NYC1), resulting in chlorophyll degradation. Conversely, under light conditions, genes like CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) reduce PIF expression, thereby inhibiting DIS and delaying plant senescence. However, the role of DIS in the senescence process of harvested broccoli remains unclear. Therefore, this study aimed to investigate the effect of light treatment on delaying DIS during broccoli storage, thereby understanding its impact on broccoli senescence. Results indicated that storing broccoli at 8°C with 2 hours of light exposure at 100 μmol·m-2·s-1 delayed yellowing. Expression of BoCCA1 in broccoli treated with 100 μmol·m-2·s-1 was approximately 4-fold higher after 5 days of storage compared to the control. Conversely, the expression levels of BoPIF5, BoEIN3, and BoNYC1 were approximately 1.3-, 4.8-, and 1.8-fold lower, respectively, in the treated groups compared to the control, with a corresponding approximately 1.4-fold increase in total chlorophyll content. These results indicate that during DIS, increased expression of genes such as BoPIF5, BoEIN3, and BoNYC1 led to reduced chlorophyll content and yellowing. However, higher BoCCA1 expression delayed broccoli head yellowing through downregulating DIS-related gene expression such as BoPIF5、BoABI5、BoPAO. Additionally, we found that light exposure caused stomatal opening, increasing stomatal aperture by approximately 1.5 times compared to the control, resulting in increased weight loss. Attempts were made to use abscisic acid (ABA) to inhibit stomatal opening and reduce weight loss. However, spraying ABA before broccoli storage did not delay weight loss. Therefore, we tested the effect of melatonin to inhibit stomatal opening and reduce weight loss. Pre-soaking broccoli in 100 μM melatonin before storage closed the stomata and delayed weight loss. Then, a combined experiment was conducted using 100 μM melatonin and light treatment. However, the combined treatment did not suppress stomatal opening or delay weight loss. In conclusion, we demonstrated that light treatment during post-harvest storage delayed DIS in broccoli, but at the expense of increased stomatal opening and weight loss. Strategies to optimize stomatal closure under light treatment could improve the effectiveness of this approach in extending the storage life of broccoli. These findings provide insights for future research on pathways related to DIS in post-harvest broccoli.