| Abscission, in plant sciences it most commonly refers to the process by which a plant drops one or more of its parts, such as leaves, fruits, flowers or seeds. A plant will abscise a part either to discard a member that is no longer necessary, such as a leaf during autumn, or a means of plant defense, or a flower following fertilisation, or for the purposes of reproduction, however, in agricultural production the physiology disorder and bio-stress caused flower and fruit dropping resulted in seriously yield and economic loss. Study of the abscission may have great value in understanding the theory of abscission and provide proper guidance for preventing flower abscission. This paper reports the tomato pedicel abscise-relative genes screened by Agilent microarrays, on basis of this mainly study the character of key abscission gene polygalacturonase and its distribution in cell level during abscission by immunolocalization. The results are as follows:1. Application of Agilent 4x44K microarrays constructed gene expression profiles of tomato pedicel ethylene induced and natural abscission, screening 17469 differentially expressed probe and clustered into 8 groups according to expression patterns.2. Ethylene biosynthesis and signal pathway cluster:SAM, ACO, EGY3, EIN2, ER1, ER33, ER6, ERF1, ERF7 and ETR2 were up-regulated expression, while ETR4, ER49, ER66, EGY1, ERF11, ERF9 and ERF3 significant down-regulated. Ethylene treatment up-regulated the expression of RTE1, TCTR2, ERF4, Never-ripe, ETR1, ETR6, ERS1, ER24, and ERF1-1, but down-regulated ER43, ERF2, TCTRlv, ATEBP, ER21, ATERF-1, ERF5 and ER5.3. Auxin signal pathway cluster:AFB5, ARF10, ARF3, ARF6, AUX1, IAA14, LAX3, PIN3 and TIR1were up-regulated, while ARF1, Axil, IAA8, PIN4 and SAR3 down-regulated. Ethylene treatment up-regulated the expression of SAUR-like protein, ARGOS and IAA1, but down-regulated ARF4, ARF8, ATAUX2-11, CTD1, IAA16, IAA29, IAA3, IAA7, IAA9 and SARI.4. Cell wall-degrading enzymes and proteins cluster: PG (polygalacturonase), CEL (cellulose), PE (pectin esterase), CHIT (chitinase), XET (xyloglucan endotransglycosylase), EXT (extension) and EXP (expansin). Most of them show up-regulated during separated process which implicated play important roles in abscission.5. Before tomato flower pedicel separation, PG mainly distributed in cortex of AZ (abscission zone), little observed in and central parenchymatous region. As the abscission initial, PG showed abundant accumulative in cortical tissue and transmitted into central parenchymatous region. PG was significant increased in cell, especially in cell wall and membrane systems. In the late stage of abscission, PG showed abundant accumulative in whole AZ region. Moreover, there is no observed of PG appearance in distal and proximal part throughout abscission.6. Before tomato flower pedicel separation, PG mainly distributed in AZ (abscission zone) cell wall, cell membrane and cytoplasm of adjacent parts, no observed in nucleus and the vacuole. As the abscission initial, PG was significant increased in cell, especially in cell wall and membrane systems. In the late stage of abscission, PG showed abundant accumulative in whole AZ region, besides in cell wall and membrane systems, cytoplasm, nucleus and the vacuole was also great amount observed.7. The optimum extraction condition for polygalacturonase from the crude enzyme extracts of tomato pecdicel is 50 mmol·L-1 acetate buffer solution (pH5.5) with 0.1 M NaCl and 1 mM DTT. Then enzyme extracts which concentrated in low temperature are purified by Sephadex G-75 column equilibrated with flow rate of 0.2 mL·min-1 and sample volume of 3.5 mL. Then the active fraction of polygalacturonase which concentrated in low temperature are further absorbed onto a CM sepharose CL-6B ionic exchange chromatographic with acetate buffer at pH5.5 and flow rate of 0.3 mL·min-1. The purified polygalacturonase of tomato pecdicel molecular mass is 30.2 kD,30.85-fold purification and 52.58% recovery.8. The pH optimum for activity of purified polygalacturonase is determined to be 5.0, temperature optima to be 40℃, and Km 26.14 mg·ml-1. The enzyme activity was inhibited by 1 mmol·L-1 Ca2+, Gu2+, Ba2+, Co2+ and Mn2+ and activated by 1 mmol·L-1Mg2+, K+, Fe2+ Zn2+ and Fe3+.9. The effect of plant phytohormones on the activity of polygalacturonase was also investigated. The result indicated that GA and IAA could inhibit the activity of PG with the optimum concentration of 0.3 mg·mL-1 and 0.1 mg·mL-1 respectively. ABA could activate the activity of PG with the optimum concentration of 0.3 mg·mL-1. Low concentration of ZT could activate the activity of PG, however, high concentration ZT inhibit the activity of PG.According to systematic analysis of gene expression profiles, summarized the molecular mechanism of ethylene induced abscission. On basis of this, screened the key abscission gene polygalacturonase and made out PG distribution first accumulative in cortical tissue and transmitted into central parenchymatous region. Moreover, there is no observed of PG appearance in distal and proximal part throughout abscission. We also purified polygalacturonaseis, its enzyme character. The results will be helpful for understanding the theory of abscission and provide proper guidance for preventing flower abscission. |
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