華中農(nóng)業(yè)大學(xué)學(xué)者在柑橘果實(shí)葉綠素降解和類胡蘿卜素合成分子機(jī)制研究中有新發(fā)現(xiàn)

　　近日，華中農(nóng)業(yè)大學(xué)園藝林學(xué)學(xué)院/果蔬園藝作物種質(zhì)創(chuàng)新與利用全國(guó)重點(diǎn)實(shí)驗(yàn)室鄧秀新院士團(tuán)隊(duì)在Plant Physiology在線發(fā)表了題為“Transcription factor CrWRKY42 coregulates chlorophyll degradation and carotenoid biosynthesis in citrus”的研究論文。該研究解析了轉(zhuǎn)錄因子CrWRKY42通過(guò)協(xié)同調(diào)控葉綠素降解和類胡蘿卜素生物合成途徑促進(jìn)柑橘果實(shí)著色的分子機(jī)制，該研究結(jié)果為柑橘果實(shí)色澤品質(zhì)遺傳改良提供了理論依據(jù)和重要基因資源。

 

　　柑橘是世界上最重要的果樹(shù)之一，具有巨大的產(chǎn)業(yè)和經(jīng)濟(jì)價(jià)值。近年來(lái)我國(guó)柑橘產(chǎn)業(yè)快速發(fā)展，種植面積和產(chǎn)量均居世界首位，產(chǎn)量占到世界的1/3左右。色澤是柑橘果實(shí)的重要品質(zhì)之一，葉綠素和類胡蘿卜素組成及含量決定了其外觀色澤。葉綠素降解和類胡蘿卜素生物合成兩條代謝途徑十分復(fù)雜，受多基因多節(jié)點(diǎn)多因素調(diào)控。在柑橘果實(shí)成熟過(guò)程中，葉綠素降解和類胡蘿卜素生物合成幾乎同時(shí)發(fā)生，然而這兩個(gè)代謝過(guò)程在轉(zhuǎn)錄水平上的協(xié)同調(diào)控尚不完全明晰。

 

　　本研究以“不知火”及其黃色果實(shí)突變體“金樂(lè)柑”為材料挖掘到一個(gè)WRKY家族轉(zhuǎn)錄因子CrWRKY42參與柑橘果實(shí)轉(zhuǎn)色進(jìn)程。在柑橘愈傷組織中超量表達(dá)CrWRKY42，八氫番茄紅素脫氫酶（CrPDS）、番茄紅素β-環(huán)化酶（CrLCYB2）以及β-胡蘿卜素羥化酶（CrBCH1）等多個(gè)類胡蘿卜素生物合成基因受到誘導(dǎo)顯著上調(diào)表達(dá)，類胡蘿卜素含量顯著增加。在柑橘愈傷組織中干涉CrWRKY42，總類胡蘿卜素含量顯著降低，多個(gè)類胡蘿卜素合成相關(guān)基因的表達(dá)水平受到抑制。此外，在柑橘果實(shí)中瞬時(shí)超量表達(dá)和干涉CrWRKY42基因，發(fā)現(xiàn)CrWRKY42能夠同時(shí)激活葉綠素降解和類胡蘿卜素生物合成關(guān)鍵基因的表達(dá)，協(xié)同調(diào)控兩條代謝途徑，進(jìn)而促進(jìn)柑橘果實(shí)轉(zhuǎn)色。生化實(shí)驗(yàn)結(jié)果表明，CrWRKY42能夠直接與CrBCH1啟動(dòng)子上的W-box元件結(jié)合并激活其表達(dá)。進(jìn)一步研究發(fā)現(xiàn)CrWRKY42還能夠直接結(jié)合并激活多個(gè)葉綠素降解和類胡蘿卜素生物合成基因的啟動(dòng)子，包括NonYELLOW COLORING（CrNYC）、STAY-GREEN（CrSGR）、CrPDS和CrLCYB2。

 

　　以上結(jié)果表明，CrWRKY42作為葉綠素降解和類胡蘿卜素生物合成的直接正調(diào)節(jié)因子，在柑橘果實(shí)著色過(guò)程中發(fā)揮重要作用。該研究結(jié)果有助于深入認(rèn)識(shí)果實(shí)成熟過(guò)程中葉綠素和類胡蘿卜素代謝的復(fù)雜轉(zhuǎn)錄調(diào)控機(jī)制，對(duì)于果實(shí)色澤品質(zhì)改良及調(diào)控具有指導(dǎo)意義。

 

　　華中農(nóng)業(yè)大學(xué)園藝林學(xué)學(xué)院鄧秀新院士為該論文的通訊作者，博士生陳紅艷為該論文的第一作者。華中農(nóng)業(yè)大學(xué)碩士生紀(jì)惠宇、博士生黃文鎧和張哲惠、博士后朱凱杰、葉俊麗副研究員和柴利軍教授以及中國(guó)農(nóng)業(yè)科學(xué)院柑桔研究所朱世平副研究員也參與了此項(xiàng)研究。該研究在國(guó)家重點(diǎn)研發(fā)計(jì)劃和國(guó)家自然科學(xué)基金的資助下完成。

 

　　英文摘要：

 

　　Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for the coloration and nutritional value of fruits. However, the synergistic regulation of these two processes at the transcriptional level remains largely unknown. Here, we identified a WRKY transcription factor, CrWRKY42, from the transcriptome data of the yellowish bud mutant 'Jinlegan' [（Citrus unshiu×C. sinensis）×C. reticulata] tangor （MT） and its wild type 'Shiranuhi' tangor （WT）， which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 directly bound to the promoter of β-carotene hydroxylase 1 （CrBCH1） and activated its expression. Overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in carotenoid biosynthesis, including phytoene desaturase （CrPDS） and lycopene β-cyclase 2 （CrLCYB2）。 In addition, CrWRKY42 could bind to the promoters of NonYELLOW COLORING （CrNYC） and STAY-GREEN （CrSGR） and activate their expression, thus promoting chlorophyll degradation. Overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expression of genes involved in both pathways. Our data revealed that CrWRKY42 acts as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening.