A study on carbon metabolism in apple flower bud during budbreak

Abstract:【Objective】Perennial apple trees need to use stored nutrients for sprouting. Starch and solublesugars,as the main carbon nutrition,undergo complex transformation processes during budbreak. Thispaper discussed changes in sugars and related enzymes and explored the mechanism of reutilizing thestored carbohydrates during budbreak in order to enrich the understanding of carbon metabolism in appleflower 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% ethanolin a centrifuge tube for three times. The extract was used for determination of soluble carbohydrates,andthe residue was used to extract starch by perchloric acid. Total soluble sugars and starch were determinedwith 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 withquartz sand into homogenate with 7 m L of 50 mmol·L-1HEPES-Na OH(p H 7.5),10 mmol·L-1Mg Cl2,1mmol·L-1EDTA,2.5 mmol·L-1DTT,0.05%(V/V)Triton-100 and 0.1%(m/V) BSA,and the homogenatewas centrifuged at 10 000 r·min- 1for 20 min at 2 ℃. The supernatant was dialyzed for 12 h at 2 ℃ inHepes 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 μL)contained 3 mmol·L-1UDPG,3 mmol·L-1fructose,5 mmol·L-1Mg Cl2,50 mmol·L-1HEPES-Na OH(p H7.5) and 80 μL extract. Reactions were stopped after 40 min at 37 ℃ by addition of 70 μL 1 mol·L- 1Na OH and reaction products were determined by the resorcinol method. Blanks contained reaction mixtures(without UDPG) added with Na OH before reaction,with water as reference. For SPS assay,100mmol·L-1HEPES-Na OH(p H 7.5) and 4 mmol·L-1F6 P replaced 50 mmol·L-1HEPES-Na OH(p H 7.5)and 3 mmol·L-1fructose,respectively,in the reaction mixture. Acid invertase(AI) and neutral invertase(NI) were assayed in a reaction mixture(final volume,1 m L) containing 0.1 mol·L-1Na-acetate(p H 4.8for AI) or 0.1 mol·L-1K2HPO4-0.1 mol·L-1Na-citrate(p H 7.2 for NI),0.1 mol·L-1sucrose and 0.2 m L ex-tract. After incubation for 40 min at 37 ℃,the reactions were stopped by addition of 1 m L 3,5-dinitrosal-icylic acid reagent. Amylase activity was assayed in a reaction mixture(1 m L) containing 0.1 mol·L-1Na-acetate(p H 6.5),1.5 mmol·L-1Na F,5 mmol·L-1Ca(NO3)2,0.5% soluble starch and 0.1 m L extract. Af-ter 40 min at 37 ℃ the reaction was terminated,and the release of reducing groups was determined as inthe 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×Vt×1 000)/(FW×t×Vs×W),C: sucrose or glucose content calculated from the standard curve(mg);Vt: totalvolume of extract(m L);t: reaction time(h);Vs: sampling volume of extract(m L);M: molar mass fractionof sucrose or glucose.【Results】During budbreak stage,contents of total soluble sugars,reducing sugarsand sucrose in apple flower buds all showed a falling trend,and the reduction amplitude of total solublesugars and sucrose was higher than 30 mg · g- 1,but that of reducing sugars was within 10 mg · g- 1.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 bebroken down into simple sugars initially. Some of the sucrose was consumed for budbreak,some allocatedto the synthesis of starch(in preparation for the full budbreak). The decline of sucrose and starch with theentrance to the full break stage indicated metabolism in flower buds was enhanced at this stage,and thatsucrose and starch were decomposed to produce more readily used nutrients. The contents of total solublesugars,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 activethan that in‘Fuji'flower buds,and the higher soluble sugar content satisfied the nutrient demand forbudbreak. The overall change trends of AI and NI in‘Fuji'and‘Gala'flower buds were similar. At theinitial 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 turningpoint 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 solublesugars,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 elevatedstarch contents in the initial stage. Starch content and amylase activity were significantly and negativelycorrelated(correlation coefficient were-0.908* and-0.861*). Lower amylase activity and higher starchcontent were found in the initial stage,and with the commence of full budbreak,the content of starch wasdecreased by increased amylase activity.【Conclusion】Soluble sugars in apple floral buds were the mainenergy source at initial stage of budbreak. During the full break stage,the degradation of stored starchplayed a vital role in sprouting because of increased material and energy requirements. The key regulatingenzymes were AI and amylase,and the nutrient reserve were largely consumed during budbreak.