Study On Transcriptional Regulation Of Chlorophyll Degradation Related Genes In Banana Fruit During Ripening At High Temperature

Banana is one of the important fruits in tropical and subtropical regions.Most of banana cultivars in the world are evolved in self-bred or hybrid progeny between wild diploid banana species(Musa acuminata,A genome,2n = 2x =22;Musa balbisiana,B genome,2n = 2x=22).Banana widely cultivated in China include three types: Xiangyajiao(AAA group),Fenjiao(ABB group)and Dajiao(ABB group),and a small number of excellent types such as Longyajiao(AAB group).The main cultivated banana in China is Baxijiao(Musa acuminata AAA Cavendish subgroup cv.‘Baxi’,commonly known as banana,the ‘banana’ in the present study refer specifically to Baxijiao.)which belongs to the type of Xiangyajiao.The phenomenon of ‘green ripe’ formed during ripening at temperatures above 24 °C severely restricts the yellowing and transportation(from south to north)of banana(Musa acuminata AAA Cavendish subgroup)produced in south China in hot seasons,leading to great losses in the economic value of the fruit.Therefore,it is of great significance to explore the effect of high temperatures(30 °C)on the appearance quality of banana fruit,and to clarify the formation mechanism of‘green ripe’ banana.Numerous studies have suggested that the ‘green ripe’ banana mainly result from the repression of chlorophyll(Chl)degradation,especially the repression of the expression of Chl catabolic enzymes(CCEs)by high temperatures,while the fundamental mechanism has remained unclear.Fenjiao(Musa ABB Pisang Awak subgroup cv.‘Guang Fen No.1’),Dajiao(Musa ABB Bluggoe subgroup cv.‘Zhongshan Dajiao’)and Longyajiao(Musa AAB Mysore subgroup cv.‘Pisang Ceylon’)which belong to the genus Musa with genotypes different from Cavendish banana,exhibit faster fruit yellowing at higher temperatures.Unlike the ‘green ripe’Cavendish banana,the expression of CCEs was promoted instead of repressed by high temperatures in Fenjiao,Dajiao and Longyajiao.It is still unclear whether the repressed/induced expression of CCEs at high temperatures are related to the genotypes.In this study,we analysed the effect of high temperatures on the appearance quality of banana fruit from the transcriptome level,and searched for transcription factors regulating the expression of CCEs by RNA-seq.To clarify how high temperatures repress the expression of CCEs in Cavendish banana,we analysed the transcription regulation of CCEs from the perspective of interaction between promoter and transcription factor.To clarify why Fenjiao,Dajiao and Longyajiao have no ‘green ripe’ phenomenon similar to Cavendish banana,we compared the CCEs of bananas with different genotypes,analy sed their possible source and sequence characteristics,Chl degradation function,and their responses to high temperatures during fruit ripening.The results are as follows.1.By constructing differential transcriptomic profiles of Cavendish banana fruit at 20 °C and 30 °C,we analysed the differential expression of genes related to Chl degradation and wax biosynthesis.The differential expression and its role in affecting the appearance quality of banana fruit at high temperatures was discussed.The high temperature at 30 °C significantly down-regulated the expression of Chl degradation-related genes(SGR1,NYC1,PPH),wax biosynthesis-related genes(CER3/WAX2)and transcription factors(NAC,MYC,ERF,MYB).The transcription factors NAC,MYC,ERF and MYB,which are repressed by high temperatures at 30 °C,may affect the appearance quality of banana fruit by regulating the expression of key genes in Chl degradation and wax biosynthesis pathway.In particular,the down-regulation of NAC at 30 °C may play an important role in regulating the expression of CCEs.2.The promoter activity of temperature-sensitive CCEs of banana and their potential cis-elements were analysed by introducing the relevant banana promoters in Arabidopsis.The results showed that the Ma SGR1,Ma NYC1 and Ma PPH promoters showed substantial activity in Arabidopsis leaves.The Ma SGR1 promoter was sensitive to high temperatures in which the activity at 30 °C was significantly lower than that at 20 °C.However,Ma NYC1 and Ma PPH promoters were not sensitive to high temperatures in which there was no significant difference in activity at 20 °C and 30 °C.The heat stress element(HSE)was not a core element that resulted in the high temperature sensitivity of Ma SGR1 promoter.There may be other high temperature-sensitive cis-elements in Ma SGR1 promoter and is closely related to the enriched ACG triplet.3.We performed yeast one-hybrid screening using a transcription factor c DNA library to search for transcription factors controlling the expression of Chl catabolic genes.Five NAC(NAM,ATAF1-2 and CUC2)transcription factors were obtained in which 4 Ma NAC29 and 1Ma NAC68 were all targeted to nucleus.The 4 Ma NAC29 were repressed by high temperatures that the expression at 30 °C was significantly lower than that at 20 °C.Both Ma NAC29-a and Ma NAC29-c could bind to the Ma SGR1 promoter and activate its expression.These results indicate that Ma NAC29 may regulate the Chl degradation of ‘green ripe’ banana by interacting with the Ma SGR1 promoter.Furthermore,to further analyze whether the high temperatures sensitivity of the Ma SGR1 promoter is dependent on Ma NAC29,we analyzed the activity of the Ma SGR1 promoter in the Arabidopsis nac29 mutant and found that the Ma SGR1 promoter was unable to activate its downstream gene in the nac29 mutant.At NAC29,which is homologous to Ma NAC29,is targeted to nucleus.At NAC29 could also activate Ma SGR1,and the expression of At NAC29 at 30 °C is significantly lower than that at 20 °C in Arabidopsis leaves.These results indicate that the high temperatures sensitivity of the Ma SGR1 promoter is dependent on Ma NAC29.4.By cloning and sequencing,A-SGR1 and B-SGR1 were both identified in Longyajiao(AAB),Fenjiao(ABB)and Dajiao(ABB)while only A-SGR1 was identified in Cavendish banana.And H-SGR1,a recombinant of A-SGR1 and B-SGR1,was identified in Longyajiao.Further analysis showed that A/B/H-SGR1 were all targeted to chloroplasts,and B/H-SGR1 had higher Chl degradation capacity than A-SGR1.Allele-specific expression analysis showed that Dajiao transcribed only B-SGR1,while Fenjiao and Longyajiao expressed B/H-and A/B-SGR1,respectively,and higher B-SGR1 transcription levels were detected at 30 °C than at 20 °C.In summary,the repressed expression of A-SGR1 resulted from its weak promoter activity regulated by high temperature-down-regulated Ma NAC29,along with the weak Chl degradation capacity,is responsible for the high temperatures induced ‘green ripe’ of Cavendish banana.In contrast to the repressed expression of A-SGR1 in Cavendish banana at high temperatures,allele-specific expression favoring B-SGR1 and expression that are maintained at high levels at high temperatures are responsible for the ability of the hybrids(Fenjiao,Dajiao and longyajiao)to avoid green-ripening.