【作者】 赵小英；

【导师】 刘选明； 林辰涛；

【作者基本信息】 湖南大学 ， 分析化学， 2006， 博士

【摘要】 隐花素(Cryptochrome)是植物感受蓝光的蓝光受体,它介导蓝光调节植物的光形态建成反应,如抑制下胚轴伸长、促进子叶伸展及调节开花等。但是,关于蓝光调节植物光形态建成反应如抑制下胚轴伸长的生化机制还不明确,蓝光是否影响种子萌发也没有报道。因此,本论文以模式植物拟南芥野生型和蓝光受体突变体为材料系统地研究了蓝光抑制拟南芥下胚轴伸长的生化机制,以及初步研究了蓝光对拟南芥种子萌发的影响及其生化机制。论文的具体研究结果如下:(1)通过测量培养在不同光照条件下拟南芥幼苗下胚轴长度发现,T-DNA插入突变体scc7-D(suppressors of cry1cry2 7-dominant)对光特别敏感,蓝光、红光或远红光下其下胚轴比母本cry1cry2或野生型的要短,但是暗培养中scc7-D突变体幼苗下胚轴长度与母本cry1cry2或野生型的相比则无差异。采用RT-PCR分析发现,scc7-D突变体中T-DNA插入位点附近的GA2ox8基因的表达量比母本中的表达量明显增加,此基因在拟南芥中编码一种GA2ox代谢酶,该酶在拟南芥赤霉素(Gibberellin,GA)生物合成途径中将有生物活性的GA4或其前体代谢为无生物活性的GAs。施用具有生物活性的外源GA3时,scc7-D突变体幼苗的表型得到恢复。GA2ox8基因过表达株系(35S:GFP-GA2ox8-1和35S:GFP- GA2ox8-8)对蓝光也特别敏感,施用具有生物活性的外源GA3也可使其表型恢复。说明不同光照条件下,光对scc7-D突变体下胚轴伸长的抑制是由于突变体内GA2ox8基因表达增加,幼苗体内有生物活性的GA含量下降所致。(2)通过比较野生型和cry1cry2、cry1、cry2突变体的下胚轴对外源有生物活性的GA3、GA4以及GA生物合成抑制剂多效唑(paclobutrazol)和嘧啶醇(ancymidol)的反应时发现:野生型和cry1cry2、cry1、cry2突变体下胚轴对外源GA3或GA4均无明显反应。但高光照强度(20-100μmolm-2 s-1)蓝光下,当多效唑和嘧啶醇浓度分别为10-2和10-1μM时,cry1和cry1cry2突变体下胚轴的伸长明显受到抑制,而对cry2突变体下胚轴伸长的抑制程度较小,最后均和野生型的下胚轴一样长。施用多效唑(10-2μM)或嘧啶醇(10-1μM)的同时施加GA3,野生型和cry1cry2、cry1、cry2突变体的下胚轴又各自恢复到原有表型。结果表明,蓝光下cry1和cry1cry2突变体的长下胚轴表型可能是由于其幼苗体内有生物活性的GA含量增加以及GA信号传导增强所致。此外,红光下phyB突变体和远红光下phyA突变体对外源GA3和GA生物合成抑制剂多效唑的反应与蓝光下cry1和cry1cry2突变体对外源GA3、GA生物合成抑制剂多效唑的反应相似。说明红光下phyB突变体和远红光下phyA突变体的长下胚轴表型也可能是由于其幼苗体内有生物活性的GA含量增加以及GA信号传导增强所致。(3)通过与澳大利亚James Reid实验室合作,采用气质联用(GC-MS)方法检测了蓝光下拟南芥野生型和cry1cry2突变体幼苗体内有生物活性的GA4含量。结果发现,将拟南芥黄化幼苗转入蓝光4h时,野生型幼苗体内GA4含量下降3倍,而cry1cry2突变体幼苗体内有生物活性的GA4含量则无变化。说明隐花素介导蓝光通过降低幼苗体内GA4含量,而抑制拟南芥下胚轴的伸长。(4)采用RT-PCR分析方法检测了不同蓝光条件处理时,拟南芥黄化幼苗中GA2ox家族基因的表达情况。GA2ox家族基因总共有8个(GA2ox1-8),除了GA2ox3和GA2ox5基因因表达量太低而无法检测出来外,其它6个基因的表达均不同程度地受蓝光诱导。与野生型相比,cry1突变体中4个GA2ox家族基因(GA2ox1, GA2ox2, GA2ox6, GA2ox8),尤其是GA2ox1和GA2ox8基因的表达受蓝光诱导的程度降低,而cry1cry2突变体中这几个基因的表达受蓝光诱导的程度更低,而在cry1cry2phyA突变体中GA2ox1基因的表达几乎不受蓝光诱导。说明phyA协同隐花素部分通过不同程度地诱导大多数GA2ox家族基因的表达,促进GA4或其前体代谢而介导蓝光抑制拟南芥下胚轴的伸长。此外,红光也不同程度地诱导大多数GA2ox家族基因的表达,但是红光的诱导效应可能由phyB和其它光敏素协同介导。(5)GA20ox家族基因有5个(GA20ox1-5),采用RT-PCR分析发现,cry1、cry2和phyA协同介导蓝光不同程度地抑制GA20ox家族基因中GA20ox1、GA20ox2、GA20ox3基因的表达。其它2个基因因表达量低而无法检测。蓝光对GA20ox1基因表达的抑制作用最强,其次是GA20ox2和GA20ox3。说明phyA协同隐花素部分通过不同程度地抑制GA20ox家族基因的表达,减少GA4合成而介导蓝光抑制拟南芥下胚轴的伸长。此外,GA20ox1、GA20ox2、GA20ox3基因的表达均不同程度地受红光和远红光抑制,phyB主要介导红光抑制GA20ox3基因的表达,phyA则主要介导远红光抑制GA20ox1基因的表达,而红光和远红光对GA20ox2基因的抑制可能由phyA、phyB与其它光敏素协同介导。(6)GA2ox8基因表达增加反馈抑制光对其它GA2ox家族基因成员表达的诱导,同时则反馈促进大多数GA20ox家族基因的表达。说明拟南芥幼苗中的这种不同家族基因之间以及本家族基因成员之间的反馈调节可以更加精确的控制植物体内有生物活性的GA4含量的动态平衡,从而增强植物适应光和其它环境条件的变化。(7)长日和短日条件下GA2ox1和GA2ox2基因的表达明显受生物钟调节,生物钟节律表达的最大值与最小值的分别出现在光周期培养的光培养时期和暗培养时期。GA2ox4和GA2ox6基因的表达也表现出生物钟节律现象,但是其振幅相对GA2ox1和GA2ox2基因来说则比较小。其它两个GA2ox基因如GA2ox7和GA2ox8的表达在光周期培养过程中则表现出依赖光调的节律现象。光周期培养条件下, cry1cry2突变体中GA2ox1基因的表达不受白光诱导,说明隐花素在介导白光诱导GA2ox1基因的表达中也具有重要的作用。上述结果表明,生物钟可能通过诱导GA2ox家族基因的表达而介导光抑制拟南芥幼苗下胚轴的伸长。(8)GA20ox1和GA20ox3基因受生物钟调节。GA20ox2基因的表达在长日条件下受生物钟调节,但在短日条件下则表现出受光调节的节律。光周期培养条件下,隐花素在介导白光抑制GA20ox1基因的表达中也具有重要作用。(9)蓝光诱导拟南芥种子萌发。隐花素主要介导蓝光诱导拟南芥种子的早期(蓝光培养1-3天)萌发,并且与光照强度有关,较低光照强度(0.1-10μmolm-2 s-1)下隐花素的作用比较明显。通过施用GA生物合成抑制剂多效唑或嘧啶醇发现,相同浓度的抑制剂对cry1cry2突变体种子萌发的抑制作用比对野生型的要强,并且解除抑制剂对cry1cry2突变体种子萌发的抑制作用,所需的外源有生物活性的GA3量也较野生型的多。这些试验结果初步证实了,隐花素可能通过增加萌发种子中有生物活性的GA合成而介导蓝光诱导种子萌发。更多还原

【Abstract】 Cryptochromes are blue light receptors that regulate various photomophogenic responses in plant, including inhibition of hypocotyl elongation, stimulation of cotyledon expansion, and regulation of flowering time. It is not clear about how cryptochromes mediate blue light regulating photomophogenic responses, such as inhibition of hypocotyl elongation. To understand the underling mechanism, the wild-type and some blue light receptor mutants of Arabidopsis were used in this study, and they all are in the col background. In addition, there is no report about regulation of seed germination by blue light, so the effect of blue light on seed germination was also studied in this research. The results of this study were listed as follows:(1) We measured the hypocotyl of one T-DNA insertion mutant scc7-D(suppressors of cry1cry2 7-dominant)which grown under different fluence rates of blue, red, or far-red light. The results showed that scc7-D mutant is hypersensitive to not only blue light but also to red and far-red light, and scc7-D mutant suppressed the long-hypocotyl phenotype of the cry1cry2 parent when grown under blue light, and also suppressed the phenotype of wild-type when grown under red or far red light, but it showed a normally elongated hypocotyl when grown in the dark. Analysis of RT-PCR demonstrated that mRNA expression of GA2ox8 gene flanking the T-DNA insert was significantly increased. It has been shown that GA2ox8 encodes a GA 2β-hydroxidase that catalyzes inactivation of bioactive GA4 or its precursor. The short-hypocotyl phenotype of the scc7-D mutant can be completely reversed by including a bioactive GA (GA3) in the growth medium when grown under blue, red, or far-red light. GA2ox8 overexpressing lines (35S: GFP-GA2ox8-1 and 35S: GFP- GA2ox8-8) also showed a GA-rescuable hypersensitivity to light. We concluded that the short-hypocotyl phenotype of scc7-D mutant under different wavelengths of light was caused by the increased expression of GA2ox8 and reduced accumulation of bioactive GA4.(2) Response of hypocotyl of wild-type, and cry1cry2, cry1, cry2 mutant to bioactive GA3, GA4 or GA biosynthesis inhibitors, pacolobutrazol or ancymidol was tested in this study. The results showed that GA3 or GA4 failed to promote significant hypocotyl elongation in wild-type, and cry1cry2, cry1, cry2 seedlings. However, application of GA biosynthesis inhibitors, pacolobutrazol (10-2μM) or ancymidol (10-1μM) can significantly inhibit hypocotyls elongation of cry1cry2 and cry1, and hypocotyl elongation of cry2 was also inhibited slightly, and cry1cry2, cry1, cry2 seedlings exhibited short hypocotyls phenotype similar to wild-type grown under high fluence rate(20-100μmolm-2 s-1) of blue light . The growth inhibition by the GA inhibitors can be reversed by GA3, such that the long hypocotyl phenotype was restored by GA3 in the presence of GA inhibitor, but long hypocotyl phenotype was not observed in wild-type. These results demonstrated that long hypocotyl phenotype of cry1and cry1cry2 might be due to both elevated level of bioactive GA and enhanced GA transduction. In addition, response of phyB grown under red light and phyA mutants grown under far-red light to GA3, or GA biosynthesis inhibitors, pacolobutrazol, or pacolobutrazol plus GA3 at different concentration was similar to that of cry1 and cry1cry2 grown under blue light. This also demonstrated that long hypocotyl phenotype of phyB and phyA might be also due to both elevated level of bioactive GA and enhanced GA transduction.(3)We cooperated with James Reid’s laboratory in Australia to measure GA4 content of wild-type and cry1cry2 mutant using GC-MS method. The results showed that the bioactive GA4 level in wild-type seedlings fall by three folds when it transferred from dark to blue light for 4 h. In contrast, there was no reduction of GA4 in cry1cry2 mutant seedlings. These results demonstrated that cryptochrome-mediated blue light-reduction of bioactive GA4 is responsible for cryptochrome–dependent hypocotyl inhibition in response to blue light(4) Expression of GA2ox genes in wild-type, cry1, cry1cry2 and cry1cry2phyA mutant were analyzed using RT-PCR. Up to eight GA2ox genes in Arabidopsis, and expression of six genes of them were induced variously by blue light treatment except that little transcripts of GA2ox3 and GA2ox5 have been detected in this study. Blue light induced expression of all four GA2ox genes(GA2ox1, GA2ox2, GA2ox6, GA2ox8), especially GA2ox1 and GA2ox8 slightly decreased in cry1 mutant seedlings, but more significantly impaired in the cry1cry2 mutant, and the blue light-induced expression of GA2ox1 was almost completely abolished in the cry1cry2phyA mutant. These results demonstrated that cry1, cry2, and phyA act redundantly to activate expression of most of GA2ox genes to reduce GA4 accumulation. Therefore, cryptochrome-mediated blue light-induction of GA2ox gene expression is at least partially responsible for cryptochrome–dependent hypocotyl inhibition in response to blue light. In addition, red light also variously induced expression of most of members of GA2ox gene family, and phyB and other phytochromes might act redundantly to mediate red light-induced expression of GA2ox genes.(5) There are five GA20ox genes(GA20ox1-5)in Arabidopsis. cry1, cry2, and phyA act redundantly mediating blue light inhibition of GA20ox1-3 genes expression variously. The other two genes can not be detected in this study because of their little expression in Arabidopsis. Expression of GA20ox1 was more significantly inhibited in response to blue light than that of GA20ox2 and GA20ox3. Therefore, cryptochrome-mediated blue light-reduction of GA20ox gene expression is also at least partially responsible for cryptochrome–dependent hypocotyl inhibition in response to blue light. In addition, expression of GA20ox1, GA20ox2and GA20ox3 were also inhibited variously by red light and far-red light treatment. phyB mainly mediated red light-reduced expression of GA20ox3, and phyA mainly mediated far-red light-reduced expression of GA20ox1, and phyB, phyA and other phytochromes might act redundantly to mediate red/far-red light-reduced expression of GA20ox2.(6) Overexpression of GA2ox8 gene suppressed light-induced gene expression of other GA2ox genes, and stimulated light induction of some GA20ox genes expression. Such interactions of expression between members of GA2ox gene family and between GA2ox genes and GA20ox genes may allow a more precise and subtle control of the homeostasis of bioactive GAs in response to light and other environmental fluctuations.(7) GA2ox1 and GA2ox2 genes expression was regulated by clock. The circadian rhythms of its expression peak were in the light (or subjective light) phase and the troughs of the rhythms were in the dark (or subjective dark) phase. The expression of GA2ox4 and GA2ox6 also showed circadian rhythm, but the oscillation was smaller than that of GA2ox1. The other two GA2ox genes, GA2ox7 and GA2ox8, showed a light-dependent diurnal rhythm. The amplitude of GA2ox1 expression is significantly diminished in the cry1cry2 mutant. It demonstrated that cryptochrome alone does play an important function in the regulation of GA2ox1 in seedlings grown in a white-light illuminated photoperiodic condition. These results also showed that circadian rhythmic expression might contribute to the transient light induction of the expression of most of the GA2ox genes to mediate light inhibition of hypocotyl elongation.(8)expression of GA20ox1 and GA20ox3 genes also showed circadian rhythm. Expression of GA20ox2 was regulated by clock when seedlings were grown under white light with long day (16h light/8h dark), but showed a light-dependent diurnal rhythm when seedlings were grown under white light with short day (8h light/16h dark). Cryptochrome mediated white light inhibition of GA20ox1 gene expression. (9) The seed germination of Arabidopsis was induced by blue light, and Cryptochrome mediated blue light induction of early seed germination in which seed germination rate were calculated when seeds were grown under blue light for less than three days. Cryptochrome-mediated blue light induction of seed germination was low fluence rates(0.1-10μmolm-2 s-1)-dependent. Response of wild-type and cry1cry2 mutant seed germination to GA inhibitor pacolobutrazol and ancymidol was investigated. Cry1cry2 mutant was more sensitive to GA inhibitor, and more GA3 was needed to recover inhibition of cry1cry2 by GA inhibitor. We concluded that cryptochrome-mediated increase of GAs synthesis might responsible for cryptochrome–dependent induction of seed germination in response to blue light.更多还原