Studies On Screening Of Highly Active Oligosaccharide And Its Physiological Mechanisms Of Plant Growth Promotion

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 photosystemâ…¡(PHâ…¡). 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. PHâ…¡fluorescence emission intensity and Hill reaction activity were also improved. ADOs improved the minimal fluorescence (Fo), the maximal fluorescence (Fm), the variable fluorescence (Fv), PHâ…¡maximal 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.