番茄果實中轉錄因子TDR4調控茄紅素累積之分子機制研究

Abstract

在過去的研究中對於光照處理可以誘導番茄果實中茄紅素的累積已經是廣為人知的現象，但是光是藉由光訊息傳遞下的哪些因子與這些因子是如何去影響茄紅素的累積到目前為止還不太明瞭。 本實驗室在過去利用不同品系的番茄果實照射不同光源後比較茄紅素累積的情形，並以抑制扣減式雜交(suppression subtractive hybridization)得到了一些可受到藍光誘導且可能參與調控茄紅素累積的候選基因。我們對於其中一個候選基因MADS轉錄因子TDR4感到高度興趣。並在先前得到了TDR4降低表現的轉殖株，發現當果實中TDR4表現降低時，類胡蘿蔔素生合成途徑中的關鍵酵素PSY的表現量也會有降低的趨勢，且果實會呈現茄紅素含量降低的外觀。因此我們推測TDR4可直接在轉錄層次上影響PSY的表現並正向影響茄紅素累積。而TDR4是否真的會調控PSY，以及TDR4是否有其他生理調控的功能是值得探討的。 在本次研究當中，我們利用酵母菌單雜交(Yeast one-hybrid)、膠體電泳位移分析(electrophoretic mobility shift assay)與染色質絲免疫共沉澱(Chromatin immunoprecipitation)的方法，證明TDR4可直接結合於PSY啟動子與內插子區域。此外我們也在染色質絲免疫共沉澱的實驗當中，發現TDR4除PSY之外也可結合於一些果實成熟中的重要基因，像CNR、ACS4、PG2a、PDS與TDR4本身的啟動子區域。進一步我們建立了以TDR4自身啟動子表現TDR4的大量表現轉殖株。藉由偵測野生型、TDR4大量表現與TDR4降低表現轉殖株果實中，TDR4與受TDR4調控基因的表現，我們發現TDR4可正向調控這些果實成熟與類胡蘿蔔素生合成相關基因的表現量。而TDR4大量表現轉殖株的紅熟期果實相較起野生型的紅熟期果實有著較高的茄紅素含量，且TDR4大量表現轉殖株的綠熟期果實對於藍光照射之下累積茄紅素的反應比野生型也較強烈。綜合以上實驗數據，我們認為TDR4對於照光之下番茄果實累積茄紅素為一項重要的調控因子，且TDR4很可能也參與在果實成熟的其他作用當中。

It is well known that light treatment can induce the lycopene accumulation in tomato fruit, but which factors in light signaling and how these factors regulate this process are still unknown. In our previous studies, we compared the fruits of different tomato species that were treated with different light, and identified several gene candidates by suppression subtractive hybridization assay, which may be involved in the regulation of lycopene accumulation. Among them, one candidate is MADS-box transcription factor TDR4. We generated the reduction-of-function of TDR4 transgenic plants previously, and found that the expression levels of TDR4 were decreased, leading to a reduction of the levels of PSY, a key enzyme gene of the carotenoid biosynthesis. Besides, the fruit of the reduction-of-function of TDR4 transgenic plants contains a decreased level of the lycopene. So TDR4 may positively regulate the expression of PSY transcripts to modulate the lycopene accumulation. Thus, we are wondering whether TDR4 can directly regulate the expression of PSY, and whether TDR4 has other physiological functions. In this study, we used yeast one-hybrid, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays to prove that TDR4 can directly bind to the promoter and the intron region of PSY. Besides, we also found that TDR4 can bind to the promoters of some fruit ripening related genes such as CNR, ACS4, PG2a, PDS and TDR4 itself by ChIP assay. Furthermore, we generated the TDR4 promoter-driven gain-of-function of TDR4 transgenic plants. By analyzing the expression levels of TDR4 and TDR4-regulated genes in the fruits of wild type, gain-of-function and reduction-of-function of TDR4 transgenic lines, we found that TDR4 positively regulated the ripening-related and carotenoid biosynthetic genes. Moreover, the lycopene levels in the gain-of-function of TDR4 transgenic lines are higher than wild type, and the mature green fruit of the gain-of-function of TDR4 transgenic lines is more sensitive to blue light, resulting in more lycopene accumulation. Taken together, these data reveal that TDR4 is a key component in light-induced accumulation of lycopene, and may be also involved in fruit ripening.