【作者】 张运红；

【导师】 吴礼树；

【作者基本信息】 华中农业大学 ， 植物营养学， 2011， 博士

【摘要】 施用外源物质调控植物生长发育是农业生产中的一项重要措施。生物活性寡糖作为外源激发子对植物生长及防御反应具有一定的调节功能,但目前研究多关注表观生物效应,对其在植物中信号传导过程及引发的系列生物学效应机制并不十分清楚。另外,不同种类寡糖的化学结构和生理功能也存在有差异,目前作为植物生长调节剂应用开发的并不是很多。鉴于此,本课题采用我国丰富的海藻酸钠及其寡糖、寡聚半乳糖醛酸和壳寡糖为材料,利用现代生物学和化学技术,筛选出高活性寡糖,确定其最佳施用方式；并采用水培试验,研究该寡糖对菜心光合作用、碳氮代谢、养分吸收和信号传导的影响,从而探讨其对植物的促生长生理机制。主要获得以下结论：1.采用土培和水培试验,研究了4种寡糖类物质在菜心上的生物学效应1.1产量和品质：4种寡糖叶片喷施均能一定程度促进菜心增产,改善其品质,其中壳寡糖20 mg/L和海藻酸钠寡糖40 mg/L浓度施用效果最好。海藻酸钠及其寡糖根部处理能提高菜心产量,改善其品质,且在一定浓度范围内(0-50 mg/L),产量和施用浓度间呈二次多项式变化规律；寡聚半乳糖醛酸和壳寡糖根部处理对菜心增产作用不大,高浓度时还有明显的抑制作用。1.2养分吸收：在本试验浓度范围内(0-100 mg/L),海藻酸钠及其寡糖和寡聚半乳糖醛酸低浓度处理均能显著促进菜心对N、P、Ca、Mg、B、Mn、Zn的吸收；低浓度时寡聚半乳糖醛酸对K的吸收也有一定的促进作用；壳寡糖低浓度处理能促进菜心对N、Mg、B、Cu、Zn的吸收,高浓度引起Cu的大量吸收,同时抑制N、P、K、Mg、Mn的吸收及Ca向地上部的转运。1.3根系特性：海藻酸钠及其寡糖处理对菜心根系形态及生理特性均有显著的改善效果；寡聚半乳糖醛酸10 mg/L处理对菜心根系生长也有一定的促进作用；壳寡糖10-200 mg/L处理对菜心根系形态及生理特性则有明显的抑制作用。1.4综合而言,4种寡糖类物质中,海藻酸钠寡糖对菜心生长发育具有相对最佳的促进作用,且以根部施用效果较好。2.采用水培试验,研究了海藻酸钠寡糖对植物光合碳代谢的调节及其作用机制2.1光合特性：海藻酸钠寡糖可提高菜心叶片的Pn、WUE和CE；并可通过增加LSP,降低LCP,扩大其光强可利用范围,从而促进光能的捕获和转化以及CO2的同化。2.2类囊体膜组成：海藻酸钠寡糖可增加菜心叶片类囊体膜色素及其蛋白复合体含量,并提高类囊体膜不饱和脂肪酸所占的比例,以维持膜的稳定性和流动性。同时诱导PSⅡ相关蛋白的表达；还可显著提高菜心叶绿体Mg2+-ATPase与Ca2+-ATPase活性,从而加快光合磷酸化进程。2.3类囊体膜功能：海藻酸钠寡糖处理前期促进类囊体膜对红光的吸收,后期降低对蓝光的吸收；PSⅡ荧光发射强度和希尔反应活力也有提高；海藻酸钠寡糖还可提高菜心叶片Fo、Fm、Fv、ETR和NPQ,从而促进光能的吸收和光合电子传递。2.4碳代谢：海藻酸钠寡糖可通过提高SS和SPS活性,促进菜心碳水化合物积累；并可通过诱导AI和NI活性,改变内部糖组成,从而提高其甜度。3.采用水培试验,研究了海藻酸钠寡糖对植物氮代谢的调节及其作用机制3.1正常钙水平下,海藻酸钠寡糖处理可显著提高菜心NR活性,使NH4+-N含量增加,N03--N含量降低；同时对GS、GDH和EP活性也有显著的促进作用。未施钙或低钙水平下,海藻酸钠寡糖对NR和GS的诱导作用均显著降低。说明海藻酸钠寡糖可加快植物氮代谢进程,且该效应与钙水平有关。3.2在培养液中添加钙代谢抑制剂EGTA、Vp、RR和CPZ后,海藻酸钠寡糖对菜心氨代谢的正相调节作用受到明显抑制,NR和GS活性及全氮和蛋白氮含量均有不同程度的降低,产量也有下降,表明Ca2+/CaM信号系统参与海藻酸钠寡糖对植物氮代谢的调节。3.3海藻酸钠寡糖主要通过诱导胞外Ca2+的进入,使[Ca2+]cyt浓度增加,激活植物生长相关的反应,同时胞内Ca2+库的释放也有一定的贡献；且其与钙形成的糖-钙复合物可能由于分子量过大的原因而不能通过质膜,最终在细胞壁间累积。4.采用水培试验,研究了海藻酸钠寡糖对植物内源激素代谢的影响海藻酸钠寡糖可促进菜心体内GA3的合成和运输,并能促进IAA向地下部和zR向地上部的运输,对ABA合成及其运输无显著影响,这导致叶片中ZR/ABA、ZR/GA、ZR/IAA和GA3/IAA比值的增加,从而有利于加快细胞的分裂或伸长,促进菜心植株生长。综上所述,海藻酸钠寡糖促进植物生长的可能生理机制为：被植物吸收后,在质外体空间内与Ca2+结合,形成糖-钙复合物,打破植物体内的钙稳态平衡,使[Ca2+]cyt增加,从而激活与生长发育相关的酶类,促进叶绿体对光能的吸收和转化,加快碳氮代谢进程；此外,还通过调控内源激素的水平及其平衡来加快植物细胞的分裂和伸长,促进根系的生长和养分吸收,最终表现为产量的增加和品质的改善。更多还原

【Abstract】 The application of exogenous substances to regulate plant growth is an important agronomic practice in agricultural production. Biologically active oligosaccharides act as signal molecules, influencing plant growth and development as well as defense responses. Current researches pay more attention to their apparent biological effects on plants, but the signal transduction process in plant cells and the mechanisms by which they induce a series of biological effects are still largely unknown. Moreover, physiological effects in plants differ between oligosaccharides with different chemical structure, leading to their limited application in agricultural production. In view of these, using the techniques of modern biology and chemistry, highly active oligosaccharide to plants was screened from sodium alginate and its oligosaccharide, oligoglacturonide and chitosan-oligosaccharide, and the optimal application method was determined. Moreover, solution culture experiments were conducted to study its effects on photosynthesis, carbon and nitrogen metabolism, nutrient absorption and signal transduction of flowering Chinese cabbage (Brassica campestris L.var. utilis Tsen et Lee), and then the physiological mechanisms of plant growth promotion were discussed. The main results obtained were as follows:1. Soil and solution culture experiments were to study biological effects of four oligosaccharides on flowering Chinese cabbage1.1 The yield and quality:Spraying four oligosaccharides on leaves promoted the yield and quality of flowering Chinese cabbage to some extent, and the treatments of 20 mg/L chitosan-oligosaccharide and 40 mg/L alginate-derived oligosaccharide (ADOs) had the best effect. Both yield and quality were increased after the root-treatment with sodium alginate and its oligosaccharide, and the relationships between the yield and application concentration could be described by quadratic polynomial equation in certain concentration (0-50 mg/L). The root-treatment with oligoglacturonide and chitosan-oligosaccharide did not affect on the yield with low concentration and inhibited the growth of flowering Chinese cabbage with high concentration.1.2 Nutrient absorption:Under the concentration range (0-100 mg/L) in this experiment, sodium alginate and its oligosaccharides and oligoglacturonide had promotive effects on the absorption of N, P, Ca, Mg, B, Mn, Zn in low concentration. Oligoglacturonide with low concentration also promoted slightly the K absorption. Chitosan-oligosaccharide could promote the absorption of N, Mg, B, Cu, Zn in low concentrations, but caused the luxury absorption of Cu and reduced the absorption of N, P, K, Mg, Mn and the transport of Ca from the roots to the shoots in high concentrations.1.3 Root morphology and physiology:Sodium alginate and its oligosaccharide had significant improvement effects on root morphology and physiology of flowering Chinese cabbage.10 mg/L oligoglacturonide promoted slightly the root growth. 10-200 mg/L chitosan-oligosaccharide inhibited the root growth and physiology activity of flowering Chinese cabbage.1.4 To sum up, ADOs among four oligosaccharides had the best plant growth-promoting effects, with the better application of root-treatment.2. Solution culture experiment was conducted to study the regulation of ADOs on photosynthetic carbon metabolism and its function mechanism2.1 Photosynthetic characteristics:Application of ADOs raised net photosynthetic rate (Pn), water use efficiency (WUE) and CO2 carboxylation efficiency (CE) and light saturation point (LSP) of functional leaves, and decreased the light compensation point (LCP) to expand the range of light intensity utilized by photosynthesis, indicating that ADOs promoted the light capture and transformation as well as CO2 assimilation.2.2 The composition of thylakoid membranes:ADOs increased the contents of pigments and pigment-protein compliexes as well as the proportion of unsaturated fatty acids in the thylakoid membranes to maintain the fluidity and stability of the membranes. Moreover, ADOs induced the expression of proteins associated with photosystemⅡ(PHⅡ). The activity of Mg2+-ATPase and Ca2+-ATPase in the chloroplast also was raised significantly, indicating that ADOs promoted photosphosphorylation of the chloroplas.2.3 The function of thylakoid membranes:ADOs improved the absorption of the thylakoid membrane in the red band at early period, but reduced the absorption of thylakoid membrane in the blue band at lately period. PHⅡfluorescence emission intensity and Hill reaction activity were also improved. ADOs improved the minimal fluorescence (Fo), the maximal fluorescence (Fm), the variable fluorescence (Fv), PHⅡmaximal photochemical efficiency (Fv/Fm) and its electron transfer rate (ETR) and the non-photochemical quench (NPQ), therefore improving light energy capture and electronic transmission.2.4 Carbon metabolism:ADOs increased carbohydrate accumulation by enhancing the activity of sucrose synthase (SS) and sucrose phosphate synthase (SPS), and changed sugar composition by inducing the activity of acid invertase (AI) and neutral invertase (NI) to improve the sweetness of flowering Chinese cabbage.3. Solution culture experiment was conducted to study the regulation of ADOs on plant nitrogen metabolism and its function mechanism3.1 Under normal levels of calcium, ADOs improved the activity of nitrate reductase (NR), resulting in an increase of NH4+-N content and a decrease of NO3-N content; the activity of glutamine synthetase (GS), glutamate dehydrogenase (GDH) and endpeptidase (EP) was also enhanced. When calcium was either lacking altogether or inadequate, the ADOs-induced effects on NR and GS activity were weakened. These results indicated that ADOs promoted plant nitrogen metabolism, which is related to the supply of calcium.3.2 After adding EGTA (a Ca2+ chelator), verapamil (Vp) and ruthenium red (RR) (Ca2+ -channel blockers) and chlorpromazine (CPZ) (a CaM antagonist) to culture solution, the ADOs-induced effects on nitrogen metabolism were weakened. The activity of NR and GS activity as well as the contents of total nitrogen and protein nitrogen were reduced to some extent, finally leading to a decrease in yield of flowering Chinese cabbage. There results indicated that the Ca2+/CaM signal system are involved in the regulation of nitrogen metabolism through ADOs.3.3 ADOs induced an increase in [Ca2+]cyt concentration by the release of calcium from extracellular and intracellular stores to activate plant growth responses. Calcium-alginate did not cross the plasmalemma because of their greater molecular weight, resulting in greater quantities of calcium in the cell wall than in the cytoplasm with time.4. Solution culture experiment was conducted to study the regulation of ADOs on phytohormone metabolismADOs enhanced the biosynthesis and transport of gibberellic acid (GA3), and the transport of indolacetic-3-acid (IAA) to the roots and trans-zeatin riboside (ZR) to the shoots, but did not affect abscisic acid (ABA), leading to an increase in the content ratio of ZR/ABA, ZR/GA3, ZR/IAA and GA3/IAA in leaves, which indicated that ADOs induced cell division and elongation to promote the growth of flowering Chinese cabbage. In a word, the physiological mechanisms of ADOs on promoting plant growth in flowering Chinese cabbage were:ADOs, on being absorbed by plants, form the macromolecular complexes with calcium in intercellular spaces, and thus alter calcium homeostasis to activate the enzymes associated with plant growth and development, thereby promoting the absorption and transformation of light in the chloroplast to accelerate carbon and nitrogen metabolism. Moreover, ADOs alter the levels and balance of endogenous phytohormones to accelerate the division and elongation of plant cells, thereby promoting root growth and nutrient absorption. These changes finally result in the increase in the yield and quality of flowering Chinese cabbage.更多还原