萌芽期苹果花芽中糖含量及相关酶活性的变化

A study on carbon metabolism in apple flower bud during budbreak

【目的】探讨萌芽期花芽中碳水化合物和与之相关的代谢酶活性的变化,了解苹果花芽在此期间的糖代谢规律及果树贮藏营养物质再利用的机制。【方法】以9 a生矮化‘富士’和‘嘎拉’苹果为材料,分析了花芽中碳水化合物的含量及其相关酶活性的变化。【结果】‘富士’、‘嘎拉’花芽中可溶性总糖、还原糖和蔗糖含量在此期间的整体变化趋势一致,都呈现下降趋势;淀粉含量在萌芽初期(3月8—20日)上升,进入盛期(3月20日—4月8日)后迅速下降。萌芽期间‘富士’和‘嘎拉’花芽中酸性转化酶(acid invertase,AI)与蔗糖、还原糖、总糖含量都呈显著负相关(相关系数分别为-0.871*、-0.904*、-0.877**和-0.957**、-0.908*、-0.857**);淀粉含量与淀粉酶活性呈显著负相关(相关系数分别为-0.908*和-0.861*)。【结论】萌芽期苹果花芽中调控代谢的关键酶是AI和淀粉酶,且树体中贮藏的营养物质被大量消耗用于萌芽。

[Objective] Perennial apple trees need to use stored nutrients for sprouting. Starch and soluble sugars, as the main carbon nutrition, undergo complex transformation processes during budbreak. This paper discussed changes in sugars and related enzymes and explored the mechanism of reutilizing the stored carbohydrates during budbreak in order to enrich the understanding of carbon metabolism in apple flower bud. [Methods] Flower buds used in the experiment were all from 9-year-old dwarfed ＂Fuji＂ and ＇Gala＇ apple trees growing in an orchard at Baishui. Sugars in buds were extracted and determined ac- cording to a modified procedure of GAO Junfeng. 0.25 g of dried sample was extracted with 80% ethanol in a centrifuge tube for three times. The extract was used for determination of soluble carbohydrates, and the residue was used to extract starch by perchloric acid. Total soluble sugars and starch were determined with anthrone colorimetry. 3,5-dinitrosalicylic acid was applied to measure reducing sugar and the resor- cinol method was used for determining sucrose. For enzyme extraction, 0.5 g fresh tissue was ground with quartz sand into homogenate with 7 mL of 50 retool·L^-1 HEPES-NaOH （pH 7.5）, 10 mmol·L^-1 MgCI2, 1 retool. L-1EDTA, 2.5 mmol·L^-1 DTT, 0.05% （V/V）Triton-100 and 0. 1% （m/V） BSA, and the homogenate was centrifuged at 10 000 r· min^-1 for 20 rain at 2 ℃. The supernatant was dialyzed for 12 h at 2 ℃ in Hepes buffer （10 fold dilution of the extraction solution without Triton- 100）. Sucrose-phosphate synthase （SPS） and sucrose synthase （SS） activities were determined by measurement of fructose-6-P- and fruc- tose- dependent formation of sucrose from UDP-glucose, respectively. The SS assay mixtures （140 txL） contained 3 mmol·L^-1 UDPG, 3 mmol·L^-1 fructose, 5 mmol·L^-1 MgC12, 50 mmol·L^-1 HEPES-NaOH （pH 7.5） and 80 μL extract. Reactions were stopped after 40 min at 37 ℃ by addition of 70 μL 1 mol·L^-1 NaOH and reaction products were determined by the resorcinol method. Blanks contained reaction mix-tures （without UDPG） added with NaOH before reaction, with water as reference. For SPS assay, 100 mmol·L^-1 HEPES-NaOH （pH 7.5） and 4 mmol. L-1 F6P replaced 50 mmol·L^-1 HEPES-NaOH （pH 7.5） and 3 retool·L^-1fructose, respectively, in the reaction mixture. Acid invertase （AI） and neutral invertase （NI） were assayed in a reaction mixture （final volume, 1 mL） containing 0.1 tool·L^-1 Na-acetate （pH 4.8 for AI） or 0.1 mol·L^-1 K2HPO4-0.1 mol·L^-1 Na-citrate （pH 7.2 for NI）, 0.1 mol·L^-1 sucrose and 0.2 mL ex- tract. After incubation for 40 min at 37 ℃, the reactions were stopped by addition of 1 mL 3,5-dinitrosal- icylic acid reagent. Amylase activity was assayed in a reaction mixture （1 mL） containing 0.1 mol. L-1 Na- acetate （pH 6.5）, 1.5 mmol·L^-1 NaF, 5 mmol·L^-1 Ca（NO3）2, 0.5% soluble starch and 0.1 mL extract. Af- ter 40 min at 37 ℃ the reaction was terminated, and the release of reducing groups was determined as in the invertase assay. In both the invertase and amylase assays, boiled extract was used for blanks and wa- ter as reference. Enzyme activities was calculated by the equation： Enzyme activity （μmol·L^-1·h^-1·g^-1）=（C× Vtxl 100）/（FWxtxVsXW）, C： sucrose or glucose content calculated from the standard curve （rag）; Vt： total volume of extract （mL） ; t： reaction time （h） ; Vs： sampling volume of extract （mL） ; M： molar mass fraction of sucrose or glucose. [Results] During budbreak stage, contents of total soluble sugars, reducing sugars and sucrose in apple flower buds all showed a falling trend, and the reduction amplitude of total soluble sugars and sucrose was higher than 30 mg·g^-l, but that of reducing sugars was within 10 mg·g^-l. Starch content increased during the initial stage of budbreak （March 8th-March 20th）, and decreased rap- idly after entering the full break stage （March 20th-April 8th）. The results showed that sucrose might be broken down into simple sugars initially. Some of the sucrose was consumed for budbreak, some allocated to the synthesis of starch （in preparation for the full budbreak）. The decline of sucrose and starch with the entrance to the full break stage indicated metabolism in flower buds was enhanced at this stage, and that sucrose and starch were decomposed to produce more readily used nutrients. The contents of total soluble sugars, reducing sugars and sucrose showed no correlation with starch content in flower bud, which sup- ports the above view. In ＇ Gala＇ flower buds, the contents of total soluble sugars, reducing sugars and su- crose during this period were slightly higher than those in ＇ Fuji＇, and the starch content displayed an op- posite pattern. It seemed that metabolism in ＇Gala＇ flower buds which broke earlier, were more active than that in ＇Fuji＇ flower buds, and the higher soluble sugar content satisfied the nutrient demand for budbreak. The overall change trends of AI and NI in ＇ Fuji＇ and ＇ Gala＇ flower buds were similar. At the initial stage they were relatively stable, and then rose rapidly later. SS began to rise in the later period （from April 1）. SPS in ＇ Fuji＇ flower buds reduced rapidly initially but rapidly increased later. The turning point in ＇Gala＇ was a week earlier than ＇Fuji＇. Amylase displayed a falling-rising trend in both culti- vars, and the increase in ＇Fuji＇ was faster than in ＇Gala＇. AI activity and the contents of total soluble sugars, reducing sugars and sucrose in ＇Fuji＇ and ＇Gala＇ flower buds had significant negative correla- tions （correlation coefficients were - 0.871＊, - 0.904＊ and - 0.877＊＊ , and - 0.957＊＊, - 0.908＊ and -0.857＊＊ in ＇Fuji＇ and ＇Gala＇, respectively）. The results showed that with increased AI activity su- crose was decomposed for synthesis of starch, which could explain the reduced sucrose and elevated starch contents in the initial stage. Starch content and amylase activity were significantly and negatively correlated （correlation coefficient were- 0.908＊ and-0.861＊）. Lower amylase activity and higher starch content were found in the initial stage, and with the commence of full budbreak, the content of starch was decreased by increased amylase activity. [Conclusion] Soluble sugars in apple floral buds were the main energy source at initial stage of budbreak. During the full break stage, the degradation of stored starch played a vital role in sprouting because of increased material and energy requirements. The key regulating enzymes were AI and amylase, and the nutrient reserve were largely consumed during budbreak.