A Study On The Effect Mechanism Of Growth Regulators On Fruit Calyx Shedding Or Persistence In ’Dangshansuli’ Pear

There are two types of Pyrus bretschneideri cv.’Dangshansuli’ fruit:CSF (calyx-shed fruit) and CPF (calyx-persistency fruit). The fruit quality of CSF is better than that of CPF. It is therefore very important to reduce the occurrence of CPF through optimum culture technology, which can improve the fruit quality and planting benefit of ’Dangshansuli’ pear.’Dangshansuli’ was used as experiment materials in this paper, the effects of growth regulators on the fruit calyx development, fruit quality and shoot growth were researched. Differences of the endogenous hormones and the mineral element content between in CSF and CPF, effects of PP333(Paclobutrazol) and GA3(Giberellic acid) on the Fe absorption and IAA content in young fruit, and the effects of PP333and GA3on the key enzyme activities in IAA metabolic pathways were all discussed. The relatived genes clone and its expression characters in CSF and CPF were studied. The main results as follows:Ⅰ. The percentage of the CSF, the soluble solids and soluble suger contents in fruit were increased, and the organic acid contents in fruit, was decreased, but the stone cell content, flesh firmness and fruit spot diameter were respectively increased by application of the high concentrations of PBO (Mixture of paclobutrazol and6-benzyladenine), PP333. The stone cell content in fruit were1.52%and1.47%, the flesh firmness were6.34kg· cm-2and6.36kg· cm-2, and fruit spot diameter were1.97mm and1.87mm treated with the2500mg· L-1PP333and2000mg· L-1PBO respectively, they were all significant higher than the contrast. Flu (Flusilazole) and PBO have a smaller effect on the fruit quality and shoot growth comparing to PP333treatment, and the appropriate concentrations of spraying with PBO and Flu were respectively200mg· L-1and100μL·L-1at the full-bloom stage. The fruit spots were enlarged, the cells arrangement of fruit dot was compact, the cell wall was thinning, the skin suberization and cracks were increased by the treatment of2500mg· L-1 PP333.Ⅱ. No positive differences were discovered between the calyx shedding behavior of young fruit and either the different pollinators, or the different pollen germination conditions. The IAA content of fruit without calyx was lower than that of fruit with calyx, while the levels of GA3, ABA and BR did not differ significantly between the two types of fruit. The percentage of calyx-shed fruit could be significantly increased by spaying PBO and PP333in blossom, but the IAA content in young fruit decreased correspondingly. On the contrary, calyx-shedding was reduced by spraying with GA3, but the IAA content in young fruit increased as a result. In summary, we showed that fruit calyx development in ’Dangshansuli’ pear were unaffected by pollinators and pollen germination conditions, and that calyx shedding is probably due to reduced levels of endogenous IAA in young fruit, which can be brought about by the application of PBO and PP333.The hormone levels in style of ’Dangshansuli’ and ’Housi’ exhibit gradient distribution, from the top to the bottom in style morphology, the endogenous hormones ABA, IAA, ZR contents were gradually increased, while GA3was gradually decreased; The mineral elements K, Ca, Fe, Zn, B showed a gradually decreased. The elongation of pollen tube could be promoted treated with2.5mg·L-1IAA and5mg·L-16-BA; Further analysis showed that the ABA, IAA contents in style is higher than that in pollen, so the major hormones for pollen germination requirement probably come from style, because the elongation of pollen tubes could be promoted by increased hormone levels in style. The mineral elements K, Ca, Fe, Zn and B in pollen higher than the style and ovary, therefore, the required mineral elements in the process of pollen germination may come from the pollen itself, because the elongation of pollen tubes could not be significantly promoted by increased mineral levels in style.Ⅲ. The Fe content in CPF was higher than that in CSF among the tested nine mineral elements. There are3Ferritin genes were cloned, which are PbFer2、PbFer3and PbFer4, the PbFer2relative expression in CPF was higher than in CSF, and GA3could enhance PbFer2expression level in young fruits too, so the PbFer2could promote iron storage and absorption for young fruit, The PbFer3expression level in CSF were remarkably higher than in CPF, we can peculated that the environmental stress may induce the expression of PbFer3. The Fe content difference between in fruitlet and in its calyx whose Fe content was6.13times higher than that in fruitlet was the biggest among the tested nine mineral elements. During the sepal developing process, the Iron demands were obviously higher than the fruitlets but at the same time of less demands for other mineral elements. The percentage of calyx persistency fruit, Fe content in fruitlet and its calyx were all increased under the spraying of400mg· L-1GA3. On the contrary, the percentage of calyx persistency fruit and the Fe content were decreased under th treatment of2500mg· L-1PP333, otherwisely, the calyx persistency fruit percentage was increased after spraying0.3%FeSO4solution too. So it was postulated that the sufficient Fe content was the guarantees to both fruit calyx development and the fruit calyx persistency. There was an positive correlation between Fe and Chla content dynamic changes in fruitlet calyx, it indicated that the abundant of Fe was probably used for Chla synthesis.Ⅳ. The results of the cDNA-AFLP analysis showed that there were10TDFs (Transcript-derived fragments) between CSF and CPF, their homology were high with related genes by NCBI alignments. The full length of PbARF and PbMT genes were cloned by RACE (Rapid-amplification of cDNA ends) techniques. The full length cDNA of ARF gene was1280bp, including44bp of5’ untranslated region and315bp of3’untranslated region. It had a coding sequence of921bp, which encoding306amino acid residues. The ARF protein molecular weight was32.2243kD and the theoretical pI was7.73. It has two kinds of structure domain about Aux/IAA protein structure domain and the Aux/IAA-ARF-dimerisation. IAA-ARF-dimerisation is the target site of Aux/IAA-ARF dimers, which related to the function of ARF kind protein, and involved in auxin signal transduction pathways. The phylogenetic tree analysis revealed that the gene have highest similarity with Malus domestic AFR3gene amino acid sequence, therefore it was named PbARF3. The full length cDNA of MT gene was552bp, including129bp of5’untranslated region and222bp of3’untranslated region. It had a coding sequence of201bp, which encoded66amino acid residues. The protein molecular weight was6.4498kD and the theoretical pI was4.79. It contained the conserved domains of all known MTs, rich in homocysteine residual base. The phylogenetic tree analysis revealed that that the amino acids sequence of MT shared higher similarity with Malus domestica and Prunus avium, therefore, it was named PbMT2. The PbARF3expression quantity in young fruit was significantly improved by GA3, and the PbMT2gene expression quantity was significantly improved by GA3in young fruit at the beginning of calyx abscission. It concluded that PbARF3may control the fruit calyx development. The above research showed that the sufficient Fe was beneficial to fruit calyx persistency, so the regulation of PbMT2on calyx development probably was attributed to improve iron absorbing and utilizing of young fruit.V. Tryptophane (Trp) and indole aldehyde oxidase (IAAldO) in CPF were higher than that of CSF, and the Trp transaminase (TRT) activity have no obvious difference between in CPF and CSF, the TRT and IAAldO activity in young fruit were improved by GA3. The peroxidase (POD) in CPF was lower than that in CSF, and IAA oxidase (IAAO) and POD activity were increased by PP333. Therefore, the increase of IAA content treated with GA3was attributed to the related enzymes activities in IAA synthesis pathway, and the decrease of IAA content treated with PP333was attributed to the related enzymes activities in IAA degradation pathway. The main results laid a foundation to reveal the physiological mechanism of fruit calyx development of ’Dangshansuli’ pear regulated by exogenous growth regulators further more.