Effect of BABA treatment on controlling grey mould disease of postharvest strawberries and transcriptomic analysis

Abstract:【Objective】β-aminobutyric acid (BABA), known as a representative non-protein amino acid, has emerged as an effective elicitor that primes defense in various agronomic fruits. It is commonly be- lieved that the burst of reactive oxygen species is a critical process after pathogen infection. However, a few studies employing transgenic Arabidopsis have found that a striking alteration of the redox poten- tial, which directly responds to a rapid production of ROS, is a prominent feature in case of different stress tolerances. Thus, In order to elaborate the mechanism involved in generation of the BABA-in- duced resistance in postharvest strawberries, the links between the responses of the BABA- treated strawberries to disease stress and intracellular redox state were researched by following the methods of transcriptome sequencing.【Methods】The‘Fengxiang’strawberries with uniform size and colour and without visual mechanical damage were randomly divided into four groups, surface- sterilized with 75% (V/V) alcohol, air-dried for 1 hour, and then punched two uniform wounds with 1.5-mm-deep and 1.5-mm-diameter holes around the equatorial zone with dissecting needles. The first group of strawber- ries was injected with sterile distilled water alone, the second group was injected with 10 mmol·L-1 BABA alone, the third group was inoculated with B. cinerea spore suspensions alone and the fourth group was  pre-injectedwith10mmol·L-1 BABAandtheninoculatedwithB.cinerea.Afterwards,allthestrawber- ries were sealed in polyethylene (60- μm thickness)- lined plastic boxes and stored at (20 ± 1) °C with 80%-90% R.H. for 5 days. During the inoculation, the disease incidence, lesion diameter, storage quali- ties, expressions of PR genes as well as contents of reductive substances were measured daily. In addi- tion, the transcription responses of strawberries with or without BABA elicitation or B. cinerea inocula- tion for 1 day were analyzed by RNA sequencing approach. Read counts were calculated using HTSeq and normalized to the FPKM (fragments per kilobase of transcript per million mapped reads) values to obtain the relative expression levels of Unigenes. Furthermore, the edgeR package was employed to fil- ter the differentially expressed genes (DEGs) with |log2FC| ≥1 and P-value ≤ 0.05 in the pairwise com- parisons of BABA- vs- control and BABA + inoculation- vsinoculation, and Venn diagram was per- formed to discriminate the co- expressed DEGs between the two pairwise comparisons. Additionally, Blast2GO was employed to analyze the annotation of DEGs to obtain the Gene ontology (GO) classifi- cations based on biological process, cellular component and molecular function ontologies. DEGs werefurther mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and significantlyenriched terms were filtered in comparison with the genome background.【Results】The results showed that10mmol·L-1 BABAtreatmentonlyinducedthediseaseresistanceinstrawberriesduringthelastpe- riod of the incubation, while BABA treatment and subsequent B. cinerea inoculation primed the straw- berries for the enhanced disease resistance as demonstrated by the clearly elevated expression levels of pathogenesis-related (PR) genes (FaNPR1, FaPR1, FaPAL and FaCHI) accompanied by the lower lev- els of disease incidence and lesion diameter than those in the strawberries inoculated solely with B. cine- rea. Furthermore, the results of RNA sequencing showed that 706 or 1 348 DEGs were relatively up- or down-regulated in the pairwise comparison of BABA-vs-control and 744 or 1 750 DEGs were relative- ly up- or down- regulated in 10 mmol · L-1 BABA pretreatment and subsequent B. cinerea inoculated strawberries compared with those inoculated solely with B. cinerea. GO and KEGG pathway analysis of the DEGs identified in the two pairwise comparisons of BABA-vs-control and BABA + inoculation-vs- inoculation revealed that BABA elicitation and subsequent B. cinerea inoculation induced a large num- ber of differentially expressed genes and those DEGs mainly enriched in“plant hormone signal trans- duction”,“glutathione metabolism”,“ascorbate metabolism”and“biosynthesis of secondary metabo- lites”. Meanwhile, BABA elicitation and subsequent B. cinerea inoculation enhanced the transcriptional levels of the critical genes in AsA-GSH cycle (GRs, DHARs, MDHARs and APXs), reactive oxygen spe- cies metabolism (SODs, PODs, CATs and APXs), glutathione synthesis pathway (GSS), PPP (G6PDHs,6PGDH, PGLSs, TKT and TALs) and GPXs accompanied by the accumulations of NADPH, GSH and AsA.【Conclusion】The data presented in this study indicated that the reduction of grey mould decay caused by B. cinerea on BABA-treated strawberries could be attributed to a priming defense, because the fruit expressing disease resistance showed a more rapid and intensive activation of defensive re- sponses upon the pathogen inoculation or infection. In addition, the enhancement of the synthesis of the reductive substances and the increase on intracellular redox state in strawberries by BABA elicitation can be associated with the promotion on transcription levels of GPXs and a series of critical genes in ascorbate-glutathione cycle (AsA-GSH cycle), reactive oxygen species metabolism, glutathione synthe- sis pathway and pentose phosphate pathway. Thus, it is reasonable to infer that BABA can prohibit the oxidation of the redox state necessary to induce a priming response in strawberry against biotic stresses. Further research is merited to explore more important redoxdependent immune regulators in horticultur- al crops and to analyze their functions in different modes of defense responses.