柑橘作為全球第一大水果品種，也是我國南方最重要的經(jīng)濟果樹之一，其果實主要用于鮮果市場，提升果實品質(zhì)是柑橘品種改良的核心目標(biāo)。檸檬酸作為柑橘果實中最主要的有機酸，占據(jù)柑橘果實有機酸總量90%以上，顯著影響果實風(fēng)味。深入探討柑橘果實中檸檬酸積累機制，對于揭示其分子基礎(chǔ)和推動柑橘品質(zhì)改良具有重要的科學(xué)意義。
為深入解析檸檬酸積累的分子機制，團隊利用早期以二倍體HB柚為母本與異源四倍體體細胞雜種“甜橙+紅橘”（細胞融合技術(shù)創(chuàng)制）為父本倍性雜交創(chuàng)制的三倍體有性雜交群體為材料，經(jīng)過多年品質(zhì)評價，篩選獲得了1對檸檬酸差異積累的三倍體，為解析柑橘檸檬酸代謝調(diào)控機理研究提供了理想材料。
通過對兩個三倍體不同發(fā)育期果肉的比較轉(zhuǎn)錄組分析，鑒定獲得了1個具有R3結(jié)構(gòu)域的MYB轉(zhuǎn)錄因子—CAPRICE（CsCPC）。通過CsCPC基因功能驗證（包括在柑橘愈傷組織和金柑果實中過表達以及在檸檬葉片中的RNAi沉默），發(fā)現(xiàn)CsCPC轉(zhuǎn)錄因子通過抑制液泡膜質(zhì)子泵相關(guān)基因CsPH1和CsPH5的表達負向調(diào)控檸檬酸積累；生化互作實驗表明，CsCPC可與CsPH4競爭性結(jié)合CsAN1蛋白，抑制CsPH4-CsAN1轉(zhuǎn)錄復(fù)合體的形成，從而通過抑制CsPH5表達使得果實中檸檬酸含量降低。而CsPH4可反饋激活CsCPC表達，形成負反饋調(diào)節(jié)環(huán)，防止檸檬酸過量積累。綜上所述，該研究揭示了CsCPC和CsPH4通過構(gòu)建一個“激活-抑制”的反饋回路，協(xié)同控制檸檬酸積累的機理，為柑橘果實風(fēng)味改良提供了理論依據(jù)。
華中農(nóng)業(yè)大學(xué)2023級果樹學(xué)博士研究生王婷婷和已畢業(yè)博士生宋鑫為該論文共同第一作者，解凱東副教授為通訊作者，郭文武教授和伍小萌教授等參與了研究工作。該研究依托華中農(nóng)業(yè)大學(xué)果蔬園藝作物種質(zhì)創(chuàng)新與利用全國重點實驗室平臺，受到國家重點研發(fā)計劃課題、國家自然科學(xué)基金和現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系資助。
【英文摘要】
The citric acid accumulation during fruit ripening determines the quality of fleshy fruits. However, the molecular mechanism underlying citric acid accumulation is not clearly understood yet in citrus due to the scarcity of paired germplasm that exhibits significant difference in organic acid accumulation. Two citrus triploid hybrids with distinct citric acid content in their mature fruits were herein identified from a previously conducted interploidy cross in our group, providing an ideal paired material for studying acid accumulation in citrus. Through a comparative transcriptome analysis of the pulps of the above two triploid hybrids, an R3-MYB transcription factor, CAPRICE （CsCPC）， was identified to be a regulator of citric acid accumulation in citrus fruits. Through transgenic experiments involving overexpression （in callus and kumquat fruits） and RNAi （in lemon leaves）， we demonstrated that CsCPC suppresses citric acid accumulation by negatively regulating the expression of CsPH1 and CsPH5. Moreover, CsCPC competed with an R2R3-MYB CsPH4 for binding to ANTHOCYANIN1 （CsAN1） and thus disturbed the activation of CsPH1 and CsPH5 that encode vacuolar P-ATPase, which eventually led to a decrease in citric acid content. CsPH4 activated the expression of CsCPC and thus formed an activator–repressor feedback loop, which ultimately inhibited citric acid accumulation in citrus fruit. In summary, this study reveals a new regulatory mechanism of CsCPC-mediated inhibition of citric acid accumulation in citrus fruits, which would support the improvement of citrus fruit quality.
