Gene cloning and function analysis of VcPLs genes in blueberries with different fruit firmness

Abstract: 【Objects】Fruit firmness is an significant classification indicator of fruit quality, which directly affects fruit mechanical harvesting, transportability, storage and processing. This study aimed to explore the function of the pectin lyase genes on fruit firmness between firm-flesh and soft-flesh blueberry cultivars in order to enrich the molecular mechanism of fruit firmness alteration, and to provide a reference for breeding high- firmness blueberry cultivars.【Methods】The blueberry fruits at six developmental stages from S3 (fruit setting) to S8 (maturation) of firm-flesh cultivar Star and soft-flesh cultivar O’Neal were used as materials for the study. The fruit firmness, cell wall components including cellulose, hemicellulose and soluble pectin were measured. The activity of pectin lyase were analyzed and the correlation between the fruit firmness and physiological and biochemical indicators were calculated. The fruit anatomical structures at six developmental stages were compared between two cultivars with firm and soft flesh. We screened two pectin lyase (PL) genes according to transcript abundance in the fruits at these six stages. The two PL genes were cloned and their expression patterns at six stages were studied using quantification PCR. The functions of VcPLs genes were checked through inducing overexpression in the tomato fruits.【Results】The fruit firmness of the two blueberry cultivars showed a downward trend as fruit development. They decreased rapidly from S4 to S6 stages, and then showed agentle decrease trend. Both Star and O’Neal reached the maximum value of fruit firmness at the S4 stage. The critical stage for rapid decline of the fruit firmness happened from stages S4 to S6. At the early stages of the fruit development (S3 and S4), the flesh cells closed to the fruit skin were intact and tightly arranged without structure collapse. The boundary of the flesh cells was clear. The flesh cell structures of O’Neal and Star gradually became disordered as the fruit expansion. But the changes occured earlier in the fruits of O’Neal than those of Star. The contents of cellulose and hemicellulose generally showed a downward trend during the process of the fruit firmness reduction, and the content of hemicellulose showed few changes both in O’Neal and Star. The significant differences of the hemicellulose content were observed only at the S4 and S5 stages between O’Neal and Star cultivars. The soluble pectin contents showed an increased trend as the fruit softening progress, which raised quickly from the S5 stage. The upward trend in the fruits of O’Neal was greater than those of Star, and the soluble pectin content in the mature fruits of O’Neal was significantly higher than that in the Star fruits. The alteration trend of the pectin lyase activity at S3 and S4 stages were similar to the changes pattern of the soluble pectin content. The enzyme activity started to increase from S5 stage, and upward change in the fruits of O’Neal was more obvious than those of Star. The correlation analysis results showed that there were extremely significant negative correlations between the fruit firmness, soluble pectin content and pectin lyase activity. The coding sequence lengths of the VcPL41 and VcPL65 were 1230 and 1209 base pairs, respectively. The amino acid sequences of VcPL65 in the fruits of Star and O’Neal were the same. But VcPL41 has four amino acid differences between those two cultivars, namely amino acids at positions of 3, 92, 181 and 221, of which the alteration at position of 221 occured in the conserved domain Motif 1. The period of rapid up-regulation expression of the two genes in the fruits of Star was later than those of O’Neal. The VcPL41 and VcPL65 genes were induced to express using estradiol from green stage of tomato fruits due to possible perish caused by cell wall construction failure when tomato seedlings were young. The color break time of tomato was earlier in the VcPL41 and VcPL65 overexpression tomato lines than those in the control. In addition, the sepal edges of the fruit in overexpression lines dried up, and this phenotype was more noticeable in the VcPL65 transgenic lines. The fruit firmness of the tomato overexpression lines was significantly smaller than that of the control, and the soluble pectin content was dramatically higher than that of the control.【Conclusion】The pivotal difference of fruit firmness between the firm- flesh blueberry cultivar Star and the soft- flesh cultivar O’Neal occurred at stages S4 to S6. The cell layer near the fruit skin was loose and the intercellular spaces were enlarged during the fruit softening. Both the pectin lyase enzyme activity and the soluble pectin content were increased. The physiological and biochemical indicators closely related to the decrease of fruit firmness showed significant differences between Star and O’Neal cultivars. The alteration in the softflesh cultivar O’Neal were generally earlier and more noticeable than that in the firm- flesh cultivar Star. There was a significant positive correlation between the soluble pectin content and pectin lyase activity and significant negative correlations between the fruit firmness and pectin lyase activity and soluble pectin content. Our results indicated that the VcPL41 and VcPL65 genes probably would have the function of accelerating the fruit softening and ripening process in blueberry