| It has been proven that abscisic acid(ABA)is involved in the regulation of climacteric fruit ripening.Nevertheless,a comprehensive investigation about the influence of ABA and ABA-ethylene interaction on ripening-related processes is still lacking.ABA also has been widely acknowledged to regulate ethylene biosynthesis and signaling during fruit ripening,but the molecular mechanism underlying the interaction between these two hormones are largely unexplored.Therefore,we treated the cherry tomato fruits at mature green stage with exogenous ABA and NDGA(an ABA biosynthesis inhibitor),and conducted a comparative analysis of the effects of exogenous ABA and NDGA on tomato fruit ripening by RNA-seq.We also explored the molecular mechanism of the regulatory effects of ABA on ethylene biosynthesis by a series of molecular biological assays.Our results not only illustrated the effects of ABA on ripening-related secondary metabolism,but also shed light on the mechanism of cross-talk between ABA and ethylene during fruit ripening.The results are as follows:1.Exogenous ABA treatment accelerated fruit ripening,including coloring,softening,carotenoids accumulation and chlorophyll catabolism.NDGA application,however,suppressed all these processes.The high throughput sequencing results showed that out of the 25728 genes expressed across all three samples,10388 were identified as significantly differently expressed genes.Exogenous ABA was found to enhance the transcription of genes involved in pigments metabolism,including carotenoids biosynthesis and chlorophyll degradation,whereas NDGA treatment inhibited these processes.The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant systems.Intriguingly,we also found that an inhibition of endogenous ABA significantly enhanced the transcriptional abundance of genes involved in photosynthesis.2.Exogenous ABA or NDGA treatment could alter the volatile compounds composition in tomato fruit.Meanwhile,ABA application promoted the accumulation of some important aroma contributors,whereas NDGA suppressed it,suggesting ABA can exert a positive effect on tomato aroma.For the synthesis of C6 volatiles,ABA enhanced the production of hexanal,3-hexenal,(E)-2-hexenal by up-regulating the expression of TomloxC,TomloxB(but not A,D),HPL and the total LOX activity.With respect to amino acid derivatives,the increased concentration of phenylethyl alcohol and 2-Isobutylthiazole is probably due to the induced expression of LeAADCs and SlBCAT1 by ABA,respectively.In addition,ABA appears to have impact on the transcription of LePAR1 and LePAR2 possibly via mediating the ripening process.6-methyl-5-hepten-2-one as a critical apocarotenoid volatiles was also positively regulated by ABA through elevating the expression of LeCCD1 B.3.Exogenous ABA treatment obviously enhanced endogenous ABA content as well as ethylene emission during tomato fruit ripening,whereas NDGA application showed the opposite biological effects.Combined RNA-seq with time-course RT-PCR analysis,our study not only helped to illustrate how ABA regulated itself at the transcription level(through LeNCED2、NCED4、UGT73B3、PYL9 etc.),but also revealed that ABA can facilitate ethylene production and response probably by regulating some crucial genes such as LeACS4,LeACS2,LeACO1,LeGR and LeETR6.In addition,investigation on the fruits treated with 1-MCP immediately after ABA exposure revealed that ethylene might be essential for the induction of ABA biosynthesis and signaling at the onset of fruit ripening.Furthermore,some specific transcription factors(TFs)known as regulators of ethylene synthesis and sensibility(e.g.MADS-RIN,TAGL1,CNR and NOR)were also observed to be ABA responsive,which implied that ABA influenced ethylene action possibly through the regulation of these TFs expression.4.SlAREB1,a downstream transcription factor in ABA signaling pathway,has been reported to mediate ABA signaling that regulates tomato ripening through induction of ethylene biosynthetic genes.NOR,a member of NAC domain family,was proved to act upstream of ethylene and essential for ripening-and ethylene-associated genes expression.Here,we found that the expression of SlAREB1 and NOR are both ABA-inducible,and SlAREB1 transcription reaches the peak level prior to NOR during the ripening process.Yeast one-hybrid(Y1H),electrophoretic mobility shift assay(EMSA)and dual luciferase assay indicated NOR as a novel direct target of SlAREB1.Transient over-expression of SlAREB1 in tomato fruits results in elevated expression of NOR as well as a number of downstream ethylene biosynthetic genes including SlACS2,SlACS4 and SlACO1,suggesting that SlAREB1 mediated ABA activation of NOR transcription would be an important molecular mechanis in ABA-ethylene crosstalks in the process of fruit ripening. |
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