Physiological And Molecular Mechanisms Of Pre-harvest Granulation In Late-ripening Navel Orange And Its Regulation Techniques

Late navel orange,as one kind of late-maturing orange cultivars with good quality,such as lanelate navel orange?Citrus sinensis Osbeck‘Lanelate'Navel?,is suitable for cultivation in Three Gorges Reservoir area in China.It plays an important role in optimization of citrus varieties structure and extension of citrus fresh fruit supply.However,in recent years,serious fruit ganulation arose in this cultivar during fruit ripen period.Juice sac granulation is one kind of wide-spread physiological diseases in citrus,which seriously hinders eating quality and commodity value of lanelate orange fruit.Therefore,the research on the cause and mechanism of juice sacs granulation has become a key problem in the production of late maturing navel orange.In this study,the late maturing navel orange varieties‘Lanelate navel orange'and‘Cara Cara navel orange'?Citrus sinensis Osbeck‘Cara Cara'Navel?was used as experimental material to study the effects of low temperature and altitude in winter on the granulation and quality of late maturing navel orange fruits,and to clarify the causes of granulation of late maturing navel orange fruits in the Three Gorges Reservoir area of Hubei Province.To study the physiological changes and gene expression related to fruit juice sacs granulation.To screen the differentially expressed genes and target genes related to fruit granulation through RNA-seq sequencing,and changes of gene expression related to juice sacs granulation were analyzed to elucidate the physiological and molecular mechanism of preharvest granulation.The study on the planting area layout of late maturing navel orange was carried out,and the effects of bagging and plastic film mulching in winter on the granulation and quality of late maturing navel orange were also studied,which provided theoretical guidance for the cultivation and control technology of preharvest fruit granulation in Hubei Three Gorges Reservoir area.The main results of this study are as follows:1. Effects of low temperature and altitude in winter on pre harvest granulation and quality of late maturing navel orangeThe fruit granulation of late maturing navel orange in the Three Gorges Reservoir area of Hubei Province is closely related to the low temperature in winter,and low temperature in winter induced fruit granulation.In the Three Gorges Reservoir area of Hubei Province,the fruit quality at high altitude?520 m?was significantly lower than that at middle and low altitude?380-220 m?.The results showed that the soluble solid content,solid/acid ratio,water content and fruit juice ratio were low,and the fruit surface coloring was poor.With the extension of harvest time,the water content of the fruit decreased gradually,resulting in granulation.The proportion of granulation in the flowering period?late April?and the new shoot growth period?early May?was 50.0%and 38.8%respectively.At the end of spring shoot growth,the total water content of fruit pulp decreased by about 10.0%.In addition,the granulation index increased,and the degree of granulation gradually increased.while the occurrence of granulated fruit was not observed in the middle and low altitudes during the experiment.2. Physiological mechanism of pre harvest fruit granulation in late maturing navel orangeThe observation of cell tissue structure showed that the granulation of fruit juice cell caused the cell wall cellulose,wall thickening,cell content decreasing,and the cell gradually shrunk and hollow.The results showed that the granulation rate and degree of fruit juice increased significantly at flowering stage,and the water content of juice cell and fruit juice rate decreased significantly.The respiration rate of fruit first increased and then decreased rapidly with the occurrence and development of granulation.At the same time,the soluble solids?TSS?and titratable acid?TA?contents decreased significantly,and the color of juice cells became lighter.The content of pectin,cellulose and lignin increased with the increase of granulation level of juice cell.The activities of pectin methylesterase?PME?,peroxidase?POD?,superoxide dismutase?SOD?and catalase?CAT?increased,while the activities of polygalacturonase?PG?and cellulase?CL?decreased.At the same time,juice sacs granulation also led to significant changes of endogenous hormones.Auxin and GA₃ decreased significantly with the aggravation of granulation,but ABA kept high content in granulation.The changes of these substance contents caused the imbalance of endogenous hormones in fruit,which became one of the important reasons for accelerating fruit granulation.3. Study on the molecular mechanism of pre harvest granulation in late maturing navel orangeA total of 903 differentially expressed genes were identified in the preharvest granulated fruit of late maturing navel orange,most of which were significantly enriched in nine metabolic pathways.QRT-PCR results showed that the juice sac granulation was closely related to cell wall metabolism.In addition,PME gene related to pectin decomposition was up-regulated,PG gene was down-regulated;phenylalanine enzyme?PAL?,cinnamyl alcohol dehydrogenase?CAD?and POD gene related to lignin synthesis were up-regulated,while CL gene,which was involved in cellulose decomposition,was down regulated.The expression patterns of these genes were consistent with the expression patterns of low temperature treatment revealed by QRT-PCR,which further confirmed that low temperature in winter was related to the formation of granulation in overwintering fruits of late maturing navel orange.Therefore,low temperature could aggravate the fruit granulation by affecting cell wall metabolism of late maturing navel orange.At the same time,136 known mi RNAs and 6 Novell mi RNAs were identified,among which 24mi RNAs were differentially expressed in granulated fruits.Functional enrichment analysis and metabolic pathway analysis showed that the functions of target genes were mainly concentrated in three pathways.Phytohormone signal transduction played an important role in the molecular signal pathway of late maturing navel orange fruit responding to granulation,which was consistent with the results of metabolic pathway analysis in m RNA transcription sequencing.Mi RNA involved in the regulation of disease resistance related genes may improve the disease resistance of late maturing navel orange by mediating the expression of disease resistance genes.In conclusion,the intracellular cells of late maturing navel orange fruit juice are regulated by endogenous hormones,cell wall related physiological metabolism,secondary metabolism biosynthesis,hormone signal transduction and other related genes,as well as mi RNA target genes involved in stress and stimulus response,resulting in a series of physiological and biochemical changes,such as sugar and acid metabolism,nutrient consumption and continuous transfer and consumption of juice cell water and inclusions.Finally,the fruit juice cell collapse and granulation occurred.The physiological and molecular mechanism of preharvest fruit granulation induced by low temperature in winter was preliminarily formed.4. Study on the cultivation techniques of prevention and control of fruit granulation before harvest of late maturing navel orangeThe cultivation techniques of late maturing navel orange in Hubei Three Gorges Reservoir area were discussed,and the planting the regional distribution of late maturing navel orange was studied.The results showed that in the three Gorges Reservoir area of Hubei,the suitable regional planting area of late maturing navel orange in south of the Yangtze River was below 360 meters,and that of north of the Yangtze River was below350 meters.Outside the suitable range,with the increase of altitude,the quality of late maturing navel orange fruit decreased,the edible rate and juice rate decreased,the acid decreased rapidly,the slagging ability was poor,and the fruit granulation aggravated.Secondly,the production technology of late maturing navel orange fruit granulation prevention and control was studied.The effect of bagging experiment in winter was good.The fruit granulation rate was controlled below 5%,which was significantly lower than that of the control fruit?50%?.It can effectively reduce and prevent the late maturing navel orange fruit from granulation,and the bagged fruit has good appearance and good quality.The effect of film mulching technology is the second,the fruit granulation ratio of fruit can be controlled below 20%,and the technology is simple,labor is small and the cost is low,so it can be applied in production.