Expression patterns of ethylene biosynthesis genes from bananas during fruit ripening and in relationship with finger drop

Abstract

Background and aims: Banana finger drop is defined as dislodgement of individual fruits from the hand at the pedicel rupture area. For some banana varieties, this is a major feature of the ripening process, in addition to ethylene production and sugar metabolism. The few studies devoted to assessing the physiological and molecular basis of this process revealed (i) the similarity between this process and softening, (ii) the early onset of related molecular events, between the first and fourth day after ripening induction, and (iii) the putative involvement of ethylene as a regulatory factor. This study was conducted with the aim of identifying, through a candidate gene approach, a quality-related marker that could be used as a tool in breeding programmes. Here we examined the relationship between ripening ethylene biosynthesis (EB) and finger drop in order to gain further insight into the upstream regulatory steps of the banana finger drop process and to identify putative related candidate genes.

Methods: Postharvest ripening of green banana fruit was induced by acetylene treatment and fruit taken at 1-4 days after ripening induction, and total RNA extracted from the median area [control zone (CZ)] and the pedicel rupture area [drop zone (DZ)] of peel tissue. Then the expression patterns of EB genes (MaACO1, MaACO2, MaACS1, MaACS2, MaACS3 and MaACS4) were comparatively examined in CZ and DZ via real-time quantitative polymerase chain reaction.

Principal results: Differential expression of EB gene was observed in CZ and DZ during the postharvest period examined in this study. MaACO1, MaACS2 and MaACS1 were more highly induced in DZ than in the control, while a slight induction of the MaACS4 gene was observed. No marked differences between the two zones were observed for the MaACO2 gene.

Conclusions: The finger drop process enhanced EB gene expression including developmental- and ripening-induced genes (MaACO1), specific ripening-induced genes (MaACS1) and wound-induced genes (MaACS2). Thus, this process might be associated with a specific ethylene production in DZ of the pedicel area and the result of crosstalk between developmental, ripening and wound regulatory pathways. MaACO1, MaACS1, MaACS2, and to a lesser extent MaACS4 genes, which are more highly induced in DZ than in CZ, could be considered as putative candidates of the finger drop process.

Fig. 1

Finger drop pattern during postharvest fruit ripening. Peel tissue of control (CZ) and drop (DZ) zones used in this study are illustrated in (A). Finger drop pattern observed during fruit ripening and measured via the pedicel rupture force are illustrated in (B).

Fig. 2

Ethylene biosynthesis gene expression during postharvest ripening of Cavendish bananas assessed using reverse transcription–polymerase chain reaction. RNA was extracted twice from CZ and DZ of banana peel tissues sampled at harvest and then at 1–4 days after ripening induction. The y-axis represents the relative fold difference in mRNA level and was calculated using the 2^−ΔΔCt formula (Livak and Schmittgen 2001) with actin as the reference. The mRNA fold difference was relative to that of peel tissue from the CZ of fruit sampled at harvest. Each data point is the mean of values obtained through a qPCR reaction performed in triplicate on one sample. Each sample was prepared from four fruits originating from three replicate bunches. Vertical bars indicate the standard deviation (SD). The SD was lower than the symbol when no bar is shown. The experiment was performed on two independent RNA extractions with similar results.

Fig. 3

Phylogenetic analysis of ACS (A) and ACO (B) sequences from banana and other plant species. The phylogenetic tree was constructed with the Gonnet residue weights and after a complete sequence alignment performed using the ClustalX algorithm (Jeanmougin et al. 1998). For multiple alignments, the gap opening penalty was 10, with a gap extension penalty of 0.2 and the delay divergent sequences was set at 30 %. The consensus tree was displayed using the TREEVIEW program (Page 1996). All banana sequences are indicated by underlining while those whose expression was examined in this study are in bold. ACC synthase sequences used for phylogenetic tree construction are: Musa acuminata [MaACS1 (AB021906), MaACS2 (AB021907), MACS2 (AF056162), MaACS3 (AB021908), MaACS4 (AB266314), MaACS6 (AJ223186)], Arabidopsis thaliana [AtACS2 (Q06402), AtACS4 (NP_179866), AtACS5 (AAG50098), AtACS6 (Q9SAR0), AtACS7 (AAG48754), AtACS8 (AAG50090), AtACS9 (AAG48755)], Cucumus sativum [CsACS1 (BAA33374), CsACS2 (BAA33375), CsACS3 (BAA33376)], Doritaenopsi sp. [DsACS1a (L07882), DsACS1b (L07883)], Lupinus albus [LuACS1 (AF119411), LuACS2 (AF119412), LuACS3 (AF119413), LuACS4 (AF119410), LuACS5 (AF119414)], Solanum lycopersicon [SlACS1a (AAF97614), SlACS1b (AAF97615), SlACS2(CAA41855), SlACS3(AAB48945), SlACS4(AAA03164), SlACS6(BAA34923), SlACS7(AAC32317)], Solanum tuberosum [StACS1a (Z27233), StACS1b (Z27234), StACS2 (Z27235)] and Vigna radiata [VrASC1 (CAA77688), VrACS6 (U34986), VrACS7 (U34987)]. ACC oxidase sequences used for phylogenetic tree construction are: Musa acuminata [MaACO1(AY804252), MaACO2 (X95599), MAO1B(AF030410)], Arabidopsis thaliana [AtACO1 (AEC06898), AtACO2 (AEE33960), AtACS3 (Q8H1S4), AtACS5 (Q43383), AtACS6 (AAG48754), AtACS7 (AAG50090), AtACS9 (AAG48755), AtACO10 (Q9LSW6)], Cucumus sativum [CsACO1 (AB006806), CsACO2 (AB006807)], Diospyros kaki [DkACO1 (AB073008), DkACO2 (AB073009)], Doritaenopsis sp. [DsACO1 (L37103), DsACO2(L07912)], Solanum lycopersicon [SlACO1 (X58273), SlACO4 (AB013101), SlACO5 (AJ715790)], Solanum tuberosum [StACO1 (AF384820), StACO2 (AF384821)] and Vigna radiata [VrASO1 (U06046), VrACO2(AM180697)].

Fig. 3

Phylogenetic analysis of ACS (A) and ACO (B) sequences from banana and other plant species. The phylogenetic tree was constructed with the Gonnet residue weights and after a complete sequence alignment performed using the ClustalX algorithm (Jeanmougin et al. 1998). For multiple alignments, the gap opening penalty was 10, with a gap extension penalty of 0.2 and the delay divergent sequences was set at 30 %. The consensus tree was displayed using the TREEVIEW program (Page 1996). All banana sequences are indicated by underlining while those whose expression was examined in this study are in bold. ACC synthase sequences used for phylogenetic tree construction are: Musa acuminata [MaACS1 (AB021906), MaACS2 (AB021907), MACS2 (AF056162), MaACS3 (AB021908), MaACS4 (AB266314), MaACS6 (AJ223186)], Arabidopsis thaliana [AtACS2 (Q06402), AtACS4 (NP_179866), AtACS5 (AAG50098), AtACS6 (Q9SAR0), AtACS7 (AAG48754), AtACS8 (AAG50090), AtACS9 (AAG48755)], Cucumus sativum [CsACS1 (BAA33374), CsACS2 (BAA33375), CsACS3 (BAA33376)], Doritaenopsi sp. [DsACS1a (L07882), DsACS1b (L07883)], Lupinus albus [LuACS1 (AF119411), LuACS2 (AF119412), LuACS3 (AF119413), LuACS4 (AF119410), LuACS5 (AF119414)], Solanum lycopersicon [SlACS1a (AAF97614), SlACS1b (AAF97615), SlACS2(CAA41855), SlACS3(AAB48945), SlACS4(AAA03164), SlACS6(BAA34923), SlACS7(AAC32317)], Solanum tuberosum [StACS1a (Z27233), StACS1b (Z27234), StACS2 (Z27235)] and Vigna radiata [VrASC1 (CAA77688), VrACS6 (U34986), VrACS7 (U34987)]. ACC oxidase sequences used for phylogenetic tree construction are: Musa acuminata [MaACO1(AY804252), MaACO2 (X95599), MAO1B(AF030410)], Arabidopsis thaliana [AtACO1 (AEC06898), AtACO2 (AEE33960), AtACS3 (Q8H1S4), AtACS5 (Q43383), AtACS6 (AAG48754), AtACS7 (AAG50090), AtACS9 (AAG48755), AtACO10 (Q9LSW6)], Cucumus sativum [CsACO1 (AB006806), CsACO2 (AB006807)], Diospyros kaki [DkACO1 (AB073008), DkACO2 (AB073009)], Doritaenopsis sp. [DsACO1 (L37103), DsACO2(L07912)], Solanum lycopersicon [SlACO1 (X58273), SlACO4 (AB013101), SlACO5 (AJ715790)], Solanum tuberosum [StACO1 (AF384820), StACO2 (AF384821)] and Vigna radiata [VrASO1 (U06046), VrACO2(AM180697)].