【作者】 刘婧；

【导师】 王宝山；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2011， 博士

【摘要】 对高等植物而言,光不仅是光合作用的能量来源,也是调节植物生长发育最基本的信号。高等植物利用光敏色素（phytochrome,phy）感受红光/远红光,参与调节植物从种子萌发到果实成熟的多个发育过程。水稻光敏色素基因家族包括3个成员,PHYA、PHYB和PHYC。在对水稻光敏色素突变体的研究中,我们发现水稻phyB缺陷型突变体（phyB）具有较强的干旱胁迫耐性,因此可被用做一种重要的、耐旱新种质资源。本研究用phyB突变体和野生型水稻作为研究材料,围绕根系水分吸收能力和地上部分水分散失两个方面,在生理学和分子生物学水平上分析了与水稻干旱胁迫耐性相关的生理变化和相关基因,旨在揭示水稻phyB调控植物干旱胁迫耐性的机制。主要结果如下:1.水稻phyB突变体具有较强的干旱胁迫耐性将在正常条件下生长的水稻野生型和phyB突变体（六叶期）停止浇水13天,发现大部分野生型水稻叶片萎蔫,而phyB突变体叶片仍保持展开。停止浇水16天后复水12天,45.7%的野生型水稻恢复生长,而90%的phyB突变体恢复生长。这说明phyB突变体具有较强的干旱胁迫耐性。采用15%PEG溶液处理野生型和phyB突变体1～5天,发现phyB突变体在正常和干旱胁迫条件下的脯氨酸和可溶性糖含量均高于野生型,说明phyB突变体具有较高的渗透调节能力。2. phyB突变体与野生型植株的根系没有明显差异将水稻野生型和phyB突变体种子根部避光在0.5%的琼脂培养基中培养至两叶期。结果显示,phyB突变体的主根长度明显大于野生型,而根/冠比无显著差别。当野生型和phyB突变体根部避光在蛭石中培养至六叶期时,野生型和phyB突变体的根长也无显著差别。利用根系分析仪进一步测定发现,野生型和phyB突变体的根系长度、表面积以及直径也均没有显著性差异。这说明根系生长及表面积不是影响phyB突变体较强干旱胁迫耐性的主要因素。3. phyB突变体表现出较低的失水速率和蒸腾速率比较野生型和3个不同phyB等位基因突变体（phyB1,phyB2,phyB5）离体叶片的失水速率。结果表明,phyB突变体的失水速率明显小于野生型。进一步利用美国便携式光合仪（LI-COR LI-6400）在强制光源(1500μmol m^-2s^-1)下测定野生型和phyB突变体的光合速率、气孔导度、蒸腾速率,发现phyB突变体的光合速率、气孔导度、蒸腾速率均低于野生型,而水分利用率在phyB突变体和野生型之间没有显著差异。此外我们还发现phyB突变体植株的总叶面积明显小于野生型。这些结果说明,在同样生长条件下,与野生型相比phyB突变体就可能具有较低的蒸腾量。这些特征可能是决定phyB突变体具有较强干旱胁迫耐性的关键因素。4. phyB正调控气孔密度通过印迹法比较了野生型和phyB突变体六叶期发育完全的第三叶、第四叶和第五叶的气孔数目和大小。结果表明,phyB突变体单位面积上的气孔数目和气孔器长度均显著小于野生型。气孔密度的降低将减少植物与外界环境之间水分或二氧化碳的交换,导致光合速率和蒸腾速率降低、失水速率降低。5. phyB负调控气孔发育相关基因ERECTA的表达为了分析phyB调控气孔密度的分子机制,我们利用实时荧光定量PCR分析了水稻气孔发育相关基因在野生型和phyB突变体中的表达模式。结果表明,无论在水稻幼苗时期的分生组织和叶原基（种子萌发2天和4天）,还是六叶期植株的成熟叶,ERECTA（ER）家族基因（分别命名为ER、ER1/2和ERL）在phyB突变体中的表达量均显著高于野生型。已有报道表明,ERECTA基因负调控拟南芥气孔发育。据此推测,phyB可能通过负调控ER基因的表达而影响水稻叶片气孔密度。6. ERECTA基因负调节转基因烟草叶片的水分蒸腾为了进一步研究ERECTA基因对水分代谢的影响,我们构建了植物过量表达载体pCAMBIA1390-Ubi-OsERL,转化烟草。结果发现,当停止浇水18天时,野生型植株的叶片出现萎蔫,颜色较黄,而转基因株系仍可以维持一定膨压;各转基因株系植株离体叶片的失水速率显著低于野生型;通过便携式光合仪（LI-COR LI-6400）在强制光源(1500μmol m^-2s^-1)下测定野生型和转基因烟草植株的光合速率、气孔导度和蒸腾速率,发现野生型和转基因株系植株的光合速率没有显著差异,而转基因株系植株的蒸腾速率低于野生型,水分利用率高于野生型。以上说明,OsERL基因的过量表达可能通过降低植株的失水速率和蒸腾速率来调节植株的耐旱性。观测野生型和转基因烟草植株第六片完全展开叶中段位置上的气孔分布,结果表明各转基因株系植株单位面积上的气孔数目均少于野生型,表皮细胞明显大于野生型,而气孔发育形态没有明显差异。这些结果表明,水稻ERL基因在烟草中表达引起表皮细胞增大,从而导致气孔密度减小、失水速率和蒸腾速率降低。7. phyB介导的红光信号抑制ERECTA基因的表达phyB是如何影响ERECTA基因的表达呢?利用荧光定量PCR,我们比较了野生型和phyB突变体在黑暗和红光条件下的基因表达水平。结果表明,在野生型中,ERECTA基因在红光下的表达水平低于黑暗条件下的水平,这表明红光抑制ERECTA基因的表达。然而在phyB突变体中,红光对ERECTA基因的抑制效果明显减弱。据此推测phyB感受红光抑制ERECTA基因的表达。8. phyB突变体的耐旱性与ABA途径有关ABA是一种重要的胁迫激素。为了分析phyB突变体较强的干旱胁迫耐性是否与ABA途径有关,我们利用种子萌发实验比较了野生型和phyB突变体对外源ABA的敏感性。结果表明,phyB突变体对ABA更为敏感。利用实时荧光定量PCR,我们比较了ABA代谢相关基因在野生型和phyB突变体中的表达水平。结果表明,ABA合成基因在phyB突变体中的表达水平高于野生型,而降解基因低于野生型。据此推测,phyB负调控ABA合成基因的表达,正调控ABA降解基因的表达。我们比较了野生型和phyB突变体干旱处理不同时间后的基因表达图谱,并通过半定量PCR进一步验证其中与ABA途径相关基因的表达模式。结果表明,多个ABA途径调控的、与干旱胁迫耐性相关的基因如脱水素基因、蛋白磷酸酶基因、bZIP基因和胁迫诱导基因等,在phyB突变体中的表达显著高于野生型,据此推测ABA依赖途径与phyB突变体较强的干旱胁迫耐性有关。总之,本实验通过研究正常和干旱胁迫条件下,水稻phyB突变体与野生型的相关生理指标、气孔发育相关基因表达及功能、以及ABA途径相关基因的表达模式探讨光敏色素参与耐旱的机理。我们初步认为,一方面,phyB负调控ERECTA基因的表达,影响单位面积气孔数目,导致蒸腾速率降低;另一方面,phyB通过影响ABA代谢基因和ABA信号途径基因的表达而负调控水稻对ABA依赖的胁迫途径,从而影响ABA依赖的干旱胁迫耐性。这些因素共同决定着phyB突变体具有较强的干旱胁迫耐性。更多还原

【Abstract】 For the higher plants, light is not only a source of energy for photosynthesis, but also a key environmental factor that regulates plant growth and development. Plants use phytochromes to perceive and respond to the red （R） and far-red （FR） light which is involved in controlling multiple responses in the plant life cycle from seed germination to fruit ripening. Phytochromes in higher plants are encoded by a small gene family. In rice （Oryza sativa）, the phytochrome gene family is composed of three members, PHYA, PHYB, and PHYC. The characterization of rice phytochrome mutants has revealed that phyB mutants （phyB） exhibited improved drought tolerance. Therefore, the phyB mutants can be regarded as an important germplasm resource with improved drought tolerance.In this study, to explore underlying mechanism by which phyB regulate the drought tolerance, the physiological characters such as water uptake and water loss of the roots and shoot of the phyB mutants and wild type and genes related to drought tolerance were examined. The main results were as follows.1. phyB mutants of rice exhibited improved drought toleranceMost of WT plants were wilting, while the phyB mutants were not after the phyB1 mutant and WT plants at the six-leaf stage were water-withheld for 13 days. After being water-withheld for 16 days and then being re-watered for 12 days, 90% of phyB mutant plants regrew and produced new leaves, while only 47.5% for the WT plants. These results clearly demonstrated improved drought tolerance in the phyB-deficient mutants. We measured the proline contents and soluble sugar content in wild type and phyB mutants grown either under the normal condition or 15% PEG4000-treated condition. It was shown that both proline and sugar contents were higher in phyB mutants than in wild type under both the normal and PEG-treated condition. Therefore, it indicated that the phyB mutants have higher osmotic adjustment capacity than wild type to cope with drought stress.2. phyB mutants and WT have similar root charecteristicsThe length of seminal roots and ratios of root to shoot in WT and phyB mutant seedlings grown in 0.5% agar medium at the two-leaf stage were measured. It was revealed that phyB mutants exhibited longer seminal roots than WT, whereas ratios of root to shoot were not significantly different. We further analyzed the root system in WT and phyB mutants grown in nutrient soil at the six-leaf stage. Root lengths of phyB mutants were not statistically different from that of wild type. Investagation on root system of phyB mutants and wild type at the six-leaf stage by root system analyzer （WinRHIZO） revealved that root length, suface root area and root diameter were similar in the WT and phyB mutant. Therefore, it is believed that growth and uptake surface area of roots were probably not a major factor contributing to drought tolerance in the phyB mutants.3. phyB mutants exhibited reduced water loss rate and transpiration rateThe fifth detached leaves of the 3 phyB mutants （phyB1,phyB2,phyB5） and WT plants at the six-leaf stage were used to compare water loss rate. Leaves of phyB plants showed an obviously slower rate of water loss than WT leaves. Net photosynthesis rate, stomatal conductance and transpiration rates of WT and the phyB mutants were futher determined using potable phtosynthesis analyzer （LI-6400）, and the results showed that both photosynthesis rate and the transpiration rate were lower in the phyB1 mutant as compared to those in WT, which is consistent with the phenotypes of stomatal density and length. However, WUE of the phyB mutant and WT was not significantly different. It suggseted that reduced water loss from leaves is likely one of the major factors contributing to drought tolerance in the phyB mutants. In addition, the total leaf area of phyB mutants was obviously smaller than that of wild type. These results suggest that phyB mutants have the lower transpiration rate than WT under the same growth condition, which is possibly the key factor for phyB mutants have higher drought-tolerance.4. phyB positively regulates stomatal densityThe number and size of stomata in the third, fourth, and fifth leaves of WT and the phyB1 mutant at the six-leaf stage were measured. Stomatal density of phyB mutant was significantly lower and stomatal length was markedly smaller that those of WT based on the imprint images of adaxial epidermis. These results suggest that the deficiency of phyB causes a decreased stomatal density and size, which will reduce photosynthesis, transpiration and water loss rate.5. phyB down-regulate the expression of putative ER family genes controling stomata developmentTo explore the molecular mechanisms of the reduced stomatal density and stomatal size in the phyB mutants, we examined the expression profiles of genes related to the stomatal development in primordial leaves and mature leaves of WT and phyB mutants at the stage of 2^nd and 4^th day of seed germination and 6th leaf by real -time PCR. Results showed that three genes （ER、ER1/2 and ERL） homologous to members of the Arabidopsis ER family always expressed higher in phyB mutants than in wild type, suggesting that phyB down-regulates the expression of putative ER family genes in rice. ER family was reported to be negative regulators of stomatal development in Arabidopsis. Therefore, we hyphothesize that phyB regulates the stomatal density and size by regulating the expression of ER family gene in rice.6. ERECTA negatively regulate the transpiration in transgenic tobaccoTo further access the role of rice ER family gene on the transpiration rate, plant transformation vector pCAMBIA1390-Ubi-OsERL was constructed and transferred into tobacco. After water was withheld for 18 days, several independent transgenic lines were shown to keep turgor while wild type was obviously wilt, and water loss of the detached loeaves of transgenic lines was significantlt lower thant that of wild type. Net photosynthesis rate, stomatal conductance and transpiration rates of WT and the phyB mutants were futher determined using potable phtosynthesis analyzer （LI-6400）, and the results showed that transgenic lines exhibited lower transpiration rate and higher and water use effiency （WUE） than wild type while photosynthesis rate was not significantly different. These results suggest that overexpression of OsERL confers drought tolerance possibly via reducing transpiration rate and water loss rate. After analyzing the stomata number and epidermal cell size on the 6th expanded leaves, it was observed that transgenic lines have lower stomatal density and lager epidermal cell compared towild types, but stomatal morphogenesis was similar. These results suggest that ERECTA increased epidermal cell size and reduced stomatal density to reduce the transpiration rate.7. phyB–mediated red light signal transduction represses expression of ERECTA genesTo analyze how phyB regulates the expression of ERECTA genes, the expression levels of three ER family genes in wild type and phyB mutants grown either under dark condition or under continous red light were dermined. For the wild type, expressions of ER genes were obviously repressed by red light. However, the repression effect triggered by red light was weakened in phyB mutants. These results suggested that phyB–mediated red light signal transduction represses the expression of ER family genes in rice.8. ABA-dependent pathway is involved in the improved drought tolerance of phyB mutantsABA is an important stress hormone. To investigate the involvement of ABA in phyB-regulated drought responses, we compared the sensitivity of wild type and phyB mutant to exogenous ABA based on the seed germination assay. It was revealed that phyB mutant is hypersensitive to ABA. The expression of ABA metabolic genes in wild type and phyB mutants were analyzed using real-time PCR. ABA biosynthesis genes （ZEP and NCED） expressed higher in the phyB mutant than in wild type, whereas the ABA degradation genes expressed lower in phyB mutant than in wild type. These results suggest that phyB down-regulates the expression of ABA biosynthesis genes and up-regulates the expression of ABA degradation genes.The ABA metabolic gene profiles of wild type and phyB mutants of drought for different times were compared, it was found that expression levels of several genes involved in ABA signal pathway were significantly higher phyB mutants than in wild type, such as osmotin, PP2C family gene, bZIP transcriptional factor genes and stress-induced genes. These results suggest that ABA-dependent pathway is probably involved in the regulation of improved drought tolerance of phyB mutant.In summary, phyB-mediated mechanism of drought-tolerance has been invesrigated at different levels of the physiological characters such as root growth, water loss and genes related to stomata development and ABA metabolism. It concluded that on one hand phyB down-regulates ERECTA expression leading to increase of epidermal cell size, decrease of stomata density and transpiration. On the other hand, phyB up-regulates ABA-synthesis gene expression and down regulates ABA-catabilism gene expression leading to improvement of drought tolerance via ABA-dependent pathway. Taken together, we hypothesize that improved drought tolerance in phyB mutants is, on one hand, attributed to the up-regulated expression of the negative regulators （ER family genes） involved in stomata development. On the other hand, phyB also affects ABA-dependent pthway via regulating the expression gene related to ABA metabolism and ABA-dependent pathway.更多还原