Comparative transcriptome analysis reveals distinct ethylene-independent regulation of ripening in response to low temperature in kiwifruit

Abstract

Background: Kiwifruit are classified as climacteric since exogenous ethylene (or its analogue propylene) induces rapid ripening accompanied by ethylene production under positive feedback regulation. However, most of the ripening-associated changes (Phase 1 ripening) in kiwifruit during storage and on-vine occur largely in the absence of any detectable ethylene. This ripening behavior is often attributed to basal levels of system I ethylene, although it is suggested to be modulated by low temperature.

Results: To elucidate the mechanisms regulating Phase 1 ripening in kiwifruit, a comparative transcriptome analysis using fruit continuously exposed to propylene (at 20 °C), and during storage at 5 °C and 20 °C was conducted. Propylene exposure induced kiwifruit softening, reduction of titratable acidity (TA), increase in soluble solids content (SSC) and ethylene production within 5 days. During storage, softening and reduction of TA occurred faster in fruit at 5 °C compared to 20 °C although no endogenous ethylene production was detected. Transcriptome analysis revealed 3761 ripening-related differentially expressed genes (DEGs), of which 2742 were up-regulated by propylene while 1058 were up-regulated by low temperature. Propylene exclusively up-regulated 2112 DEGs including those associated with ethylene biosynthesis and ripening such as AcACS1, AcACO2, AcPL1, AcXET1, Acβ-GAL, AcAAT, AcERF6 and AcNAC7. Similarly, low temperature exclusively up-regulated 467 DEGS including AcACO3, AcPL2, AcPMEi, AcADH, Acβ-AMY2, AcGA2ox2, AcNAC5 and AcbZIP2 among others. A considerable number of DEGs such as AcPG, AcEXP1, AcXET2, Acβ-AMY1, AcGA2ox1, AcNAC6, AcMADS1 and AcbZIP1 were up-regulated by either propylene or low temperature. Frequent 1-MCP treatments failed to inhibit the accelerated ripening and up-regulation of associated DEGs by low temperature indicating that the changes were independent of ethylene. On-vine kiwifruit ripening proceeded in the absence of any detectable endogenous ethylene production, and coincided with increased expression of low temperature-responsive DEGs as well as the decrease in environmental temperature.

Conclusions: These results indicate that kiwifruit possess both ethylene-dependent and low temperature-modulated ripening mechanisms that are distinct and independent of each other. The current work provides a foundation for elaborating the control of these two ripening mechanisms in kiwifruit.

Keywords: Ethylene; Fruit ripening; Low temperature–modulated ripening; On–vine ripening; Transcription factor.

Fig. 1

Ethylene production patterns of ‘Sanuki Gold’ kiwifruit as affected by storage technique. In grouped storage technique, kiwifruit at commercial maturity were divided into groups of ten, placed into containers and then covered lightly to reduce water loss (a). Four groups were stored at 20 °C without any treatment (b). Four other groups were pre–treated with a mixture of fungicides (0.015 g/L oxytetracycline, 0.15 g/L streptomycin, 0.5 g/L iprodione, 1 × 10^− 10 cfu/L Bacillus subtillis HAI–0404 spores and 0.5 g/L benomyl) prior to storage and fortnightly during storage at 20 °C (c). Ethylene production pattern of individual fruit in each group was monitored periodically. Each line represents the ethylene production pattern of a single fruit. Lines of the same color represent fruit stored within the same container. In individual storage technique, kiwifruit at commercial maturity were pretreated with a mixture of fungicides and individually wrapped in perforated polythene bags before being placed in containers, about 10 cm apart (d). One group contained non–treated fruit (e), while another group of fruit were treated twice a week with 1–MCP at 5 μLL^− 1 for 12 h (f). Fruit in all groups were stored at 20 °C in ethylene–free chambers. Ethylene production pattern of each fruit in the respective groups was monitored periodically. The proportion of ethylene–producing fruit was determined as a percentage of the total number of fruit in the respective groups (g)

Fig. 2

Ethylene–induced ripening in ‘Sanuki Gold’ kiwifruit. Kiwifruit at commercial maturity were divided into two groups. The first group was continuously exposed to propylene (5000 μLL^− 1) at 20 °C to induce the ethylene effect. The second group was stored in air at 20 °C as a non–treated control. Ethylene production (a), flesh firmness (b), soluble solids content (c), and titratable acidity (d) were determined periodically using five independent biological replicates. Different letters indicate significant differences at p < 0.05

Fig. 3

Changes in fruit ripening characteristics of ‘Sanuki Gold’ kiwifruit during storage at 20 °C and 5 °C. Kiwifruit at commercial maturity were divided into four groups. Each storage temperature had one group that was treated with 1–MCP, and another non–treated group. 1–MCP was applied twice a week at 5 μLL^− 1 for 12 h. Fruit in all groups were stored in ethylene–free chambers using the individual storage technique (10 cm apart), and any fruit that produced detectable ethylene (> 0.01 nLg^− 1 h^− 1) were eliminated from storage chambers. Flesh firmness (a), titratable acidity (b) and soluble solids content (c) were determined using fruit that did not produce any detectable ethylene (five independent biological replicates). Different letters indicate significant differences at p < 0.05

Fig. 4

Comparison of transcriptome datasets between kiwifruit samples exposed to propylene and during storage. a Venn diagram for the differentially expressed genes (DEGs) in response to propylene and during storage at 5 °C. DEGs were selected based on a discriminatory criterion of > 3–fold change between propylene–treated/harvest, and 5 °C/20 °C samples. b A heat map showing the expression pattern of DEGs in kiwifruit at harvest (Day 0), after exposure to propylene for five days, and after storage at 20 °C and 5 °C with 1–MCP treatment for four weeks

Fig. 5

Reverse Transcriptase–Quantitative PCR analysis of selected kiwifruit genes that were up–regulated by both propylene and low temperature. Kiwifruit were continuously treated with 5000 μLL^− 1 propylene (PROP) at 20 °C, alongside a non–treated group (NT). For storage, kiwifruit were kept at either 5 °C or 20 °C with (1–MCP) or without regular 1–MCP treatment (NT). 1–MCP was applied twice a week at 5 μLL^− 1 for 12 h. Gene–specific primers were designed for (a) AcPG: POLYGALACTURONASE (Achn051381/AF152756); (b) AcEXP1: EXPANSIN 1 (Achn336951/AY390358); (c) AcXET2: XYLOGLUCAN ENDOTRANSGLUCOSYLASE 2 (Achn38797); (d) Acβ–AMY1: β–AMYLASE 1 (Achn141771/FG525163); (e) AcGA2ox1: GIBBERELLIC ACID OXIDASE 1 (Achn209941); (f) AcNAC6 (Achn289291); (g) AcMADS1 (Achn061601) and (h) AcbZIP1 (Achn135561). AdACTIN (EF063572) was used as the housekeeping gene and the expression of fruit at harvest (D0) was calibrated as 1. Values are means of three independent biological replicates. Error bars represent SE. Different letters indicate significant differences at p < 0.05. Symbols are D = Day, W = Week, NT = non–treated and PROP = propylene treatment

Fig. 6

Reverse Transcriptase–Quantitative PCR analysis of selected kiwifruit genes that were exclusively up–regulated by propylene. Kiwifruit were continuously treated with 5000 μLL^− 1 propylene (PROP) at 20 °C, alongside a non–treated group (NT). For storage, kiwifruit were kept at either 5 °C or 20 °C with (1–MCP) or without regular 1–MCP treatment (NT). 1–MCP was applied twice a week at 5 μLL^− 1 for 12 h. Gene–specific primers were designed for (a) AcACS1: ACC SYNTHASE 1 (Achn364251); (b) AcACO2: ACC OXIDASE 2 (Achn326461); (c) AcPL1: PECTATE LYASE 1 (Achn070291); (d) AcXET1: XYLOGLUCAN ENDOTRANSGLUCOSYLASE 1 (Achn349851); (e) Acβ–GAL: β–GALACTOSIDASE (Achn123061); (f) AcAAT: ALCOHOL ACYLTRANSFERASE (Contig15634/ KJ626345); (g) AcERF6 (GQ869857) and (h) AcNAC7 (Achn104221). AdACTIN (EF063572) was used as the housekeeping gene and the expression of fruit at harvest (D0) was calibrated as 1. Values are means of three independent biological replicates. Error bars represent SE. Different letters indicate significant differences at p < 0.05. Symbols are D = Day, W = Week, NT = non–treated and PROP = propylene treatment

Fig. 7

Reverse Transcriptase–Quantitative PCR analysis of selected kiwifruit genes that were exclusively up–regulated by low temperature. Kiwifruit were continuously treated with 5000 μLL^− 1 propylene (PROP) at 20 °C, alongside a non–treated group (NT). For storage, kiwifruit were kept at either 5 °C or 20 °C with (1–MCP) or without regular 1–MCP treatment (NT). 1–MCP was applied twice a week at 5 μLL^− 1 for ggmxf. Gene–specific primers were designed for (a) AcACO3: ACC OXIDASE 3 (Achn150611); (b) AcPL2: PECTATE LYASE 2 (Achn315151/HQ108112); (c) AcPMEi: PECTIN METHYLESTERASE INHIBITOR (Achn319051/FG458520); (d) AcADH: ALCOHOL DEHYDROGENASE (Achn262421); (e) Acβ–AMY2: β–AMYLASE 2 (Achn212571); (f) AcGA2ox2: GIBBERELLIC ACID OXIDASE 2 (Achn218871); (g) AcNAC5 (Achn169421) and (h) AcbZIP2 (Achn227711). AdACTIN (EF063572) was used as the housekeeping gene and the expression of fruit at harvest (D0) was calibrated as 1. Values are means of three independent biological replicates. Error bars represent SE. Different letters indicate significant differences at p < 0.05. Symbols are D = Day, W = Week, NT = non–treated and PROP = propylene treatment

Fig. 8

On–vine fruit ripening characteristics in kiwifruit. ‘Sanuki Gold’ kiwifruit were left attached to the vines after commercial harvesting date (indicated by the black arrow). Flesh firmness and SSC (a), ethylene production and titratable acidity (b) were determined using fruit that did not produce any detectable ethylene (five independent biological replicates). Error bars represent SE

Fig. 9

Reverse Transcriptase–Quantitative PCR analysis of selected genes during on–vine ripening in ‘Sanuki Gold’ kiwifruit. Genes for analysis were selected based on (i) DEGs that were up–regulated by both propylene and low temperature (a, b, c, d); (ii) DEGs that were exclusively up–regulated by low temperature (e, f, g, h, i) and (iii) DEGs that were exclusively up–regulated by propylene (j, h, l, m, n, o). AdACTIN (EF063572) was used as the housekeeping gene and the expression of fruit at harvest (D0) was calibrated as 1. Values are means of three independent biological replicates. Error bars represent SE. Values are means of three independent biological replicates. Error bars represent SE. Different letters indicate significant differences at p < 0.05

Fig. 10

A simplified model showing ethylene–dependent and low temperature–modulated ripening pathways in kiwifruit. In ethylene–dependent ripening pathway, ethylene exclusively regulates the expression of specific genes (orange) as well as common genes (green). The expression of these ethylene–induced genes can be suppressed by 1–MCP application. Equally, low temperature regulates the expression of specific genes (blue) as well as common genes (green). Induction of these genes by low temperature is not suppressed by 1–MCP application, implying that they are independent of ethylene. Ripening–related gene classes are represented as rectangle (ethylene biosynthesis–related genes), oval (cell wall modification–related genes), wavy (carbohydrate metabolism–related genes), diamond (flavor–related genes), and pentagon (Gibberellin–2–oxidase genes)