MaMADS2 repression in banana fruits modifies hormone synthesis and signalling pathways prior to climacteric stage

Abstract

Background: While the role of ethylene in fruit ripening has been widely studied, the contributions of additional plant hormones are less clear. Here we examined the interactions between the transcription factor MaMADS2-box which plays a major role in banana fruit ripening and hormonal regulation. Specifically, we used MaMADS2 repressed lines in transcriptome and hormonal analyses throughout ripening and assessed hormone and gene expression perturbations as compared to wild-type (WT) control fruit.

Results: Our analyses revealed major differences in hormones levels and in expression of hormone synthesis and signaling genes mediated by MaMADS2 especially in preclimacteric pulp. Genes encoding ethylene biosynthesis enzymes had lower expression in the pulp of the repressed lines, consistent with reduced ethylene production. Generally, the expression of other hormone (auxin, gibberellins, abscisic acid, jasmonic acid and salicylic acid) response pathway genes were down regulated in the WT pulp prior to ripening, but remained high in MaMADS2 repressed lines. Hormone levels of abscisic acid were also higher, however, active gibberellin levels were lower and auxin levels were similar with MaMADS2 repression as compared to WT. Although abscisic level was higher in MaMADS2 repression, exogenous abscisic acid shortened the time to ethylene production and increased MaMADS2 mRNA accumulation in WT. Exogenous ethylene did not influence abscisic acid level. CRE - a cytokinin receptor, increased its expression during maturation in WT and was lower especially at prebreaker in the repressed line and zeatin level was lower at mature green of the repressed line in comparison to WT.

Conclusions: In addition to previously reported effects of MaMADS2 on ethylene, this transcription factor also influences other plant hormones, particularly at the pre-climacteric stage. The cytokinin pathway may play a previously unanticipated role via MaMADS2 in banana ripening. Finally, abscisic acid enhances MaMADS2 expression to promote ripening, but the transcription factor in turn auto inhibits ABA synthesis and signaling. Together, these results demonstrate a complex interaction of plant hormones and banana fruit ripening mediated by MaMADS2.

Keywords: Abscisic acid; Auxin; Gibberellic acid; Hormones; Jasmonic acid; Salicylic acid.

Fig. 1

Ethylene production, respiration rate, color and total soluble solids (TSS) in WT and MaMADS2 repressed banana fruits after harvest. Ethylene production rate (a), CO₂ production rate (b), color expressed as hue angle (o) (c) and total soluble solids expressed as brix (%) (d) were measured in fruits of the second hand of WT, MaMADS2 RNAi line (#32) and MaMADS2 antisense line (#44) during ripening. For each time point at least three fruits were measured and average and standard error plotted. Arrow in panel A indicate the samples taken as PB in each of the lines

Fig. 2

Transcriptome differences between WT and MaMADS2 repressed banana fruits during ripening. a. Heatmap of the normalized gene expression averages of all samples and hierarchical clustering of all samples. On right the different samples clustered into three groups (I, II and III). The analysis was performed on samples taken from peel (P) and pulp (U) of WT (WT) and repressed lines MaMADS2 antisense (#44) and MaMADS2 RNAi (#32) at harvest/mature green (H), prebreaker (PB), breaker (B) and ripe (R). the color scale shows the distances between different conditions; red show low distance while blue larger distance; b. Number of genes significantly changed (FDR < 0.05) between #32 or #44 lines and WT fruits in peel and pulp in the developmental stages of MG, PB, B and R

Fig. 3

Ethylene biosynthesis and signaling pathway in WT and MaMADS2 repressed fruits. a. Heat map of the genes changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; b. The ethylene biosynthesis and signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or #44 fruits in comparison to WT at PB. Yellow color indicates no change. Detailed description of genes is provided at [47]

Fig. 4

ABA biosynthesis and signaling pathway and ABA level in WT and MaMADS2 repressed fruits. a. Heat map of the genes changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; b. ABA level in WT and #32 pulps; c. The average of MaMADS2 expression levels in WT after ABA treatment (**: p < 0.01 standard error is presented); d. The ABA biosynthesis and signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or #44 fruits in comparison to WT at PB. Yellow color indicates no change. Detailed description of genes is provided by [50, 51]; e. Effect of ethylene treatment on ABA content in banana slices; f. A suggested model for the interactions of ABA with MaMADS2 and ethylene: rectangular: transcript, oval: protein, X: interaction that was rejected based on our results

Fig. 5

CK signaling pathway and zeatin level in WT and MaMADS2 repressed fruits. a. Heat map of the genes changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; b. Zeatin level in WT and #32 pulps; c. The CK signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or #44 fruits in comparison to WT at PB. Yellow color indicates no change. Detailed description of genes is provided at [55]

Fig. 6

IAA signaling pathway and IAA level in WT and MaMADS2 repressed fruits. a. Heat map of the genes changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; b. IAA level in WT and #32 pulps; c. The IAA signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or #44 fruits in comparison to WT at PB. Yellow color indicates no change. Detailed description of genes is provided at [58, 59]

Fig. 7

GA biosynthesis and signaling pathway and GA levels in WT and MaMADS2 repressed fruits. a. Heat map of the genes changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; b. GA24 and GA4 levels in WT and #32 pulps; c. The GA biosynthesis and signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or 44 fruits in comparison to WT at PB. Yellow color indicates no change. Detailed description of genes is provided at [60, 61]

Fig. 8

JA and SA signaling pathway and JA and SA levels in WT and MaMADS2 repressed fruits. a. Heat map of the genes in JA signaling pathway changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; b. JA level in WT and #32 pulps; c. The JA signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or #44 fruits in comparison to WT at PB. Yellow color indicates no change; d. Heat map of the genes in SA signaling pathway changed in PB between WT and the MaMADS2 repressed lines. Each gene is normalized to the expression level in WT at MG; e. SA level in WT and #32 pulps; f. The SA signaling pathway scheme. The genes’ expression significantly higher (red) or lower (blue) in #32 or #44 fruits in comparison to WT at PB. Yellow color indicates no change. Detailed description of genes is provided at [62] for JA and at [63] for SA

Fig. 9

Difference in hormonal footprint between MaMADS2 repressed lines and WT fruits. Hormonometer analysis of the “Arabidopsis homologs gene pool” (see methods) expressed in MaMADS2 repressed lines in comparison to WT. Dark brown indicates strong hormone footprint in MaMADS2 repressed line and blue indicates strong hormone footprint in WT.