【作者】 刘玉凤；

【导师】 李天来；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2011， 博士

【摘要】 设施蔬菜的生产已成为我国现代农业重要的组成部分,“十二五期间”还将继续加大设施农业的发展力度,确保百姓“菜篮子”安全、充足,而发展“冬季菜篮子”工程成为保障全国冬季蔬菜的供给,满足老百姓需求的重大举措。然而,在我国北方的早春季节栽培中,番茄的设施栽培极易遭受到低夜温,导致光合作用下降,严重影响其产量与品质。因此,探究低夜温对设施番茄光合作用的限制部位,有助于我们及时进行大工调控,消除光合作用的限制因素,减轻低温对植物造成的伤害,达到高产优质栽培的目的,是现阶段可持续设施农业亟待解决的重要问题。本文以番茄品种“辽园多丽”为试材,研究了番茄光合作用对低夜温及其恢复的响应机理,全面系统的分析了低夜温限制番茄叶片光合作用的因素。在此基础上,研究了番茄叶片光合作用和水-水循环对低夜温响应的钙素调控,为化学调控植物代谢,缓解低夜温逆境障碍提供理论依据。主要研究结果如下：1.明确了不同低夜温显著降低番茄叶片光合作用的气孔因素。在9d的9℃和6℃低夜温条件下,伴随着净光合速率(Pn)不可逆的降低,气孔导度(Gs)、胞间C02浓度(Ci)和蒸腾速率(Tr)将同步大幅度降低,气孔限制值(Ls)显著的高于对照；且导致有转录水平调控的Rubisco活性的降低；以上说明气孔限制和Rubisco活性的下降是9℃和6℃低夜温导致番茄叶片净光合速率降低的因素；在恢复阶段,主要是气孔因素影响了番茄光合作用的恢复速度和程度。2.明确了不同低夜温导致番茄叶片光合产物反馈抑制。9℃和6℃低夜温处理9d导致番茄叶片蔗糖的大量积累,且降低蔗糖磷酸合成酶(SPS)活性；通过RT-PCR分析,发现低夜温对番茄叶片中蔗糖代谢合成酶活性的影响是通过：mRNA转录水平上进行的调控。从蔗糖和淀粉含量分别与净光合速率(Pn)和SPS活性呈极显著负相关关系的结果看,低夜温导致的番茄光合效率的降低可以用光合产物反馈抑制假说来解释；在恢复阶段,9℃和6℃低夜温胁迫后光合作用的后继影响并不是碳水化合物反馈抑制的结果。3.明确了不同低夜温对番茄叶片中PSⅡ光抑制及光化学活性的影响。9℃和6℃低夜温处理9d内导致叶片PSⅡ最大光化学量子产量(Fv/Fm)的可逆性降低、PSⅡ潜在的活性(Fv/F。)、实际光化学量子效率(ΦPSⅡ)、PSⅡ的电子传递速率(ETR)、光化学猝灭系数(qP)均下降、非光化学猝灭系数(NpQ)提高；15℃适温恢复9d时,叶绿素荧光参数均能恢复到对照的85%以上。上述结果说明：低夜温导致番茄叶片PSII的可逆光抑制的发生和光化学活性的下调。过剩光能的耗散主要是通过增加类囊体膜两侧的质子动力势；天线色素上的能量耗散(D)和经色素去激发过程的能量耗散(Ex),其中天线色素耗散的形式占主要地位；及状态转换等措施来保护光合机构。4.明确了不同低夜温对番茄叶片中PSⅠ光抑制的影响。9℃和6℃低夜温降低了番茄叶片由供体侧限制引起的PSⅠ处非光化学能量耗散的量子产量Y(ND),增加了P700-还原态、由受体侧限制引起的PSⅠ处非光化学能量耗散的量子产量Y(NA)和PSI有效的光化学效率Y(I)。表明低夜温导致PSⅠ受体测受到限制,光抑制发生,其影响是可逆的。5.明确了不同低夜温对番茄叶片活性氧代谢机制的影响。9℃和6℃低夜温导致用于碳同化的电子流[Je(PCR)]的比例减少主要伴随着用于依赖于氧的电子流[Ja(O2-dependent)]的比例增加；诱导了番茄叶片中活性氧(02-和H202)的积累,同时抗氧化酶(SOD、APX、DHAR和GR)的活性及抗氧化剂(AsA和GSH)的含量高于对照；适温恢复过程中,我们发现活性氧(02-和H202)降低达到对照水平,同时抗氧化酶活性及抗氧化剂含量都能恢复到对照。低夜温条件下通过活性氧的积累看出,说明增加的番茄叶片中SOD活性和AsA-GSH循环清除活性氧的能力并未与氧还原的速率一致；活性氧的清除需要抗氧化酶及抗氧化剂整个防御系统的协调作用。6.明确了短时低夜温对番茄叶片光合作用的影响。6℃低夜温胁迫1h导致番茄叶片Pn。降低9.8%；同时Gs、Ci和Tr分别同步下降19%、8.9%和12%；我们通过扫描电子显微镜观察,6℃低夜温胁迫1h导致气孔的开张度和开张率都大幅度降低,其气孔的开张率仅为44.31%,而对照植物的气孔开张率为65.16%；胁迫3h后引起羧化效率(CE)的降低。由此可见,气孔的开闭对低夜温非常敏感。6℃低夜温处理11 h导致叶绿体的形状变的稍圆,淀粉粒的数量增多,所占叶绿体的比例增大,基粒片层结构变少,嗜锇颗粒降低。7.明确了短时低夜温对番茄叶片蔗糖代谢内源规律性的变化。短时6℃低夜温处理1h就导致蔗糖(31.2%)的大量积累,且3h后的积累是打破了原有的规律性变化导致的；同时AI、NI和SPS活性出现节律性滞后2h的现象,且这种节律性变化并不是在转录水平上发生的。说明短时低夜温11 h内并不存在着光合产物的反馈抑制的表面机制。8.明确了短时低夜温对番茄叶片PSⅡ和PSⅠ的影响。短时6℃低夜温处理11 h内Fv/Fm轻微下降,差异不显著,且一直保持在正常植株所具有的范围之内(0.8-0.83)；同时Y(NPQ)和Y(NO)一直低于对照,伴随着它们的下降,Y(I)高于对照水平,表明低夜温11h内并没有引起PSⅡ的光能过剩及光损伤,未导致PSⅡ光抑制的发生；同时,我们发现6℃低夜温处理11h内导致PSⅠ的Y(ND)和Y(NA)下降及Y(I)升高,说明单纯的低夜温胁迫11h内并未引起PSI供体侧和受体侧的限制。9.明确了短时低夜温对番茄叶片叶绿体中水-水循环的影响。短时6℃低夜温处理3h后导致叶绿体中O2·-和H202的积累,但在整个处理时间内并未引起MDA含量的增加。伴随着活性氧的积累,叶绿体中SOD和DR活性及AsA和GSH含量增加。因此,叶绿体中水-水循环的启动对于匹配氧的还原速率至关重要。而且叶绿体中活性氧的累积滞后于碳同化的降低。10.明确了钙素对低夜温下番茄叶片光合作用的调控。Ca2+通过增加6℃低夜温下番茄叶片气孔的宽度和开张度,提高了低夜温下番茄叶片的Gs,进而增加其Ci,提高了低夜温下番茄叶片的Pn；同时增加了低夜温下叶绿体宽度和面积,降低了淀粉粒的数量,导致淀粉粒所占叶绿体内的比例大幅度降低,促进光能吸收,有利于光合作用的顺利进行。因此,Ca2+预处理调控了气孔的状态及叶绿体的面积来缓解低夜温对番茄叶片光合作用的不利影响。11.明确了钙素对低夜温下番茄叶片叶绿体中水-水循环的调控。Ca2+预处理后通过进一步激活了低夜温下叶绿体中抗氧化酶(SOD、APX、DHAR和GR)活性及抗氧化剂(AsA和GSH)含量,参与了AsA-GSH循环的调控,增强了植株对活性氧的清除能力,降低了低夜温下叶绿体中活性氧(O2·-和H202)的积累及MDA的含量。说明Ca2+在逆境中通过激活叶绿体中保护酶的活性及抗氧化剂的含量以清除低夜温胁迫过程中所产生的氧自由基,从而达到使植物免受伤害的目的。更多还原

【Abstract】 Facilities vegetables have become an important part in China’s modern agricultural production, and china will continue to increase agricultural facilities, ensure people "basket " safe and adequate. " winter basket" project has become the important drives that guarantees the supply of national winter vegetables and meets people the demand in" 12 5"period. However, early spring cultivation in China, tomato cultivation under glasshourse easily suffers low night temperature, which resulting in drop of photosynthesis and the serious influence on its yield and quality.Therefore, It is important to study how the low night temperature affect on photosynthetic function of tomato leaves, which help us artificial regulation to eliminate the limiting factor of photosynthesis, reduce the harm on plants caused by low night temperature, and achieve high yield and high quality cultivation purposes.The effects of low night temperature at 9℃and 6℃then recover for 9 d at optional temperature 15℃simulated by climate chambers on the mechanism of photosynthesis and regulation of calcium in tomato leaves were studied. It is important both in scientific and practical fields. The main research results are as follows:1.It was clear that different low night temperature (LNT, i.e.,9℃and 6℃) stress led to the decrease of photosynthesis. LNT led to irreversible inhibition of photosynthesis mainly accompanying by the decrease in Gs, Ci and the increase in Ls, and the lower Rubisco activity occurred in transcription level.So stomatal limitation and the lower Rubisco activity were factors causing the net photosynthetic rate reduce under LNT at 9℃and 6℃for 9 d.In recovery process at 15℃for 9 d after LNT, stomatal limitation was mainly the reason that affected recovery speed and degree of photosynthesis.2.It was clear that different LNT at 9℃and 6℃caused carbohydrates feedback inhibition phenomenon in tomato leaves.There were abundant accumulations of sucroses; LNT inhibited the activities of sucrose phosphate synthase (SPS),which occurred in mRNA transcription level by analyzing RT-PCR.There were significantly negative linear correlations between sucrose and starch content and Pn, as well as sucrose content and SPS activity. Reduced photosynthesis under LNT could be explained by the hypothesis of carbohydrates feedback inhibition; the following influence of photosynthesis was not the result of feedback inhibition in recovery process after LNT.3. Effects of different LNT on photoinhibition and photochemical activity of PSⅡin tomato leaves were studied.It was shown that the maximum photochemical efficiency(Fv/Fm), potential activities of PSII(Fv/Fo), the actual photochemical efficiency of PSⅡreaction center (ΦPSⅡ), rate of electronic transmission of photosynthesis (ETR), photochemical quenching coefficient (qP) of leaves deceased at 9℃and 6℃for 9 d, while the non-photochemical quenching coefficient (NpQ) increased. During recovery, Fv/Fm could completely come back to control, other chlorophyll fluorescence parameters could reached more than 85% of control. LNT caused the reversible of PSII photoinhibition, the reduce in PSII photochemical activity played the major role to protect photosynthetic institutions.Excessive light energy consumed by the increase in protons power potential of both sides on thylakoid membranes, the allocation of absorbed light to thermal dissipation (D), excessive energy (Ex) and the state conversion to protect photosynthetic institutions. D was predominant among.4.Effects of different LNT on PSI photoinhibition in tomato leaves were studied.LNT increased the photochemical yield of PSI Y(I), quantum yield of non-photochemical energy dissipation in PS I due to acceptor side limitationY(NA) and the reduced of P700. LNT led to the limitation of the acceptor side of PSI,which caused PSI photoinhibition.5.The effects of different LNT on the active oxygen metabolism and Ascorbate-Glutathione cycle of tomato leaves were studied.The results showed that LNT caused the reduce in the rate of [Je(PCR)] mainly accompanying by the increase in [Ja(O2-dependent)].We found that LNT increased the active oxygen (O2- and H2O2) and MDA contents; improved the activities of antioxidant enzymes (SOD, APX, DHAR, GR) and the contents of antioxidant (AsA,GSH). In addition, the decrease in O2- production rate and H2O2 and MDA under recover condition at 15℃for 9 d after LNT for 9 d; at the same time, SOD, APX, DHAR and GR activities as well as AsA contents,AsA/DHA and GSH/GSSG were all decline. It indicated that the increase in SOD activitie and scavenging activity of Ascorbate-Glutathione cycle did not keep pace with the rate of oxygen reduction since the increase in O2-, H2O2 and MDA levels were observed,especially in 6℃treated plants.Reactive oxygen removal needed the coordination function between antioxidant enzymes and the whole antioxidant defense system.6.It was clear that short-term LNT at 6℃led to the decrease of photosynthesis in tomato leaves. Pn declined by 9.8% when plants were treated 1 h at 6℃, with the synchronical decline in Gs,Ci and Tr (up to 19%,8.9% and 12%). We found that there was a drastical reduce in the area and the stomatal open percentage through the scanning electron microscope (SEM) at 1 h of 6℃.The stomatal open percentage reached 44.31%,while the ratio of that control plants was 65.16%. The reduce in CE after 3 h at 6℃. Stomatal was very sensitive to LNT. LNT at 6℃for 11 h caused chloroplasts shape became relatively slightly rounded,the increase in the number of starch grains and the ratio between chloroplast and starch grains, and the reduce in eosinophilic granular osmium tetroxide.So the primary factors of the decline in Pn caused by short-term LNT was stomatal close.7. The effect of short-term LNT at 6℃on the sucrose metabolis internal regularity of tomato leaves. There were abundant accumulations of sucrose(31.2%) at 6℃for 1h, and sucrose accumulation caused by sucrose internal regularity beening broke after 3 h. And LNT at 6℃for 11h caused rhythmic lag phenomenon in AI, NI and SPS, which were 2 h behind compared with controls and did not occur in mRNA transcription level by analyzing RT-PCR.8.The effects of short-term LNT at 6℃on photosystemsⅠandⅡof tomato leaves were studied. We investigated direct information on PSI and PSII in vivo tomato plants using the Dual-PAM-100 fluorometer. The results showed that LNT led to the slight decrease in Fv/Fm,and the ratio of Fv/Fm maintained the normal the scope(0.79-0.83) under 6℃for 11h. The Y(NPQ)and Y(NO) was always lower than that of control plants, and the photochemical yield Y(II) of PSII was higher, Which indicated that 11 h of LNT did not cause photoinhibition and photo-damage of PSII. At the same time, LNT increased the photochemical yield of PSI Y(I) and decreased the donor side limitation of PSI Y(ND) and acceptor side limitation of PSI Y(NA).9. The effects of short-term LNT at 6℃for 11h simulated by climate chambers on the Water-water cycle in chloroplasts of tomato leaves were studied.The results showed that LNT increased ROS(O2- and H2O2), but did not increased MDA contents; improved the activities of antioxidant enzymes (SOD,DHAR) and the contents of antioxidant (AsA, GSH) after 3 h.So, it was very important to start up water-water cycle in chloroplast as for matching oxygen reduction rate.ROS accumulation in the chloroplasts occured behind the decrease in carbon assimilation.10. Regulation of calcium on the photosynthesis parameters in tomato leaves under LNT at 6℃for 7d. Pretreatment with 0.3% CaCl2 increased widen and area of the stomata by SEM, alleviated the reduce of Gs, improved Ci and the Pn of tomato seedlings under LNT; Ca2+ increased widen and area of chloroplasts under LNT and the area of starch grains,but decreased the amount more, so led to the great decrease in the ratio between starch grains and chloroplast, which promoted the absorb of the light energy and photosynthesis.11. Regulation of calcium on the water-water cycle in chloroplasts of tomato leaves under LNT. Pretreatment with CaCl2 further improved the activities of antioxidant enzymes (SOD, APX, DHAR, GR) and the contents of antioxidant (AsA, GSH) in the chloroplasts, So Ca2+ improved scavenging ability to reactive oxygen species by regulating Ascorbate-Glutathione cycle and decreased the accumulation of reactive oxygen species (O2- and H2O2) in the chloroplasts.It indicated that Ca2+ mainly activated protective enzyme activity and antioxidants contents to eliminate the reactive oxygen species under LNT, so as to protect the plant from harm.更多还原