The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening

Abstract

Fruit softening in apple (Malus x domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A cold-related gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the cold- and ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

Figure 1.

Postharvest regimes for ACO1-suppressed apples. Asterisks mark the point of 100 μL L⁻¹ ethylene treatment. NE, No ethylene treatment; E, ethylene treatment; H, harvest; w, weeks. Lowercase letters represent sampling points. A, For the 2005 harvest (E1), apples were harvested and stored at 4°C for 1 month before they were warmed to 20°C for 24 h, and half were treated with 100 μL L⁻¹ ethylene. Apple samplings are labeled a to e as follows: a, just before ethylene treatment; b, 4 h after ethylene treatment; c, 4 d (96 h) after ethylene treatment; d, 8 d (192 h) after ethylene treatment; e, 8-d no-ethylene control. Six apples were sampled at a time. B, For the 2007 harvest (E2), apples were sampled either immediately following ethylene treatment or after being store in the cold for 4 weeks. Sample times are labeled a to f as follows, with six apples in each group: a, sampled immediately; b, stored in an ethylene-free environment for 8 d before being sampled; c, sampled after being treated with 100 μL L⁻¹ ethylene for 8 d; d to f, stored at 4°C for 4 weeks and then transferred to 20°C for 1 d as follows: d, sampled immediately; e and f, sampled following an 8-d treatment either with or without 100 μL L⁻¹ ethylene. C, For the 2009 harvest (E3), apples were sampled following a room temperature treatment or a 0.5°C treatment. Sampling times are labeled a to i, with eight apples sampled at each time, as follows: a, sampled immediately; b, d, and e, stored in an ethylene-free environment at 20°C and sampled after 2 weeks of storage (b), after 4 weeks of storage (d), and after 6 weeks of storage (e); c, treated with 100 μL L⁻¹ ethylene for 2 weeks; f, treated with 100 μL L⁻¹ ethylene for 2 weeks after 4 weeks of storage at 20°C; g, h, and i, stored at 0.5°C in an ethylene-free environment for 4 weeks, then g was sampled immediately and h and i were transferred to 20°C and sampled after 2 weeks either with or without treatment with 100 μL L⁻¹ ethylene.

Figure 2.

Flesh firmness (n) of ACO1-suppressed apples following different treatments. A, Harvest E3. Letters represent sampling times, gray bars represent a no-ethylene treatment at 20°C, white bars represent a no-ethylene treatment after a 0.5°C treatment, and black bars represent apples treated with 100 μL L⁻¹ ethylene for 2 weeks (W). RT, Room temperature. B, Harvest E2. Apples were either treated at 20°C (gray bars) or stored at 4°C for 4 weeks before being transferred to 20°C (white bars). Firmness was assessed either immediately or following an 8-d treatment either with or without 100 μL L⁻¹ ethylene. Error bars represent se (n = 6).

Figure 3.

Patterns of gene expression measured by qPCR of either ACO1 or PG1. A and B, ACO1 expression (A) and PG1 expression (B) during fruit development of Royal Gala apples, from open flowers (0 DAFB) to eating ripe (146 DAFB). C, Expression of PG1 in Royal Gala ACO1-suppressed mutants from the E1 harvest at 0, 4, 96, and 192 h of 100 μL L⁻¹ ethylene treatment or 192 h of no-ethylene treatment. White bars represent expression in peel tissue, and gray bars represent expression in cortex tissue. D, Expression analysis of PG1 from the E2 harvest. The gray bar represents apples stored at 20°C, the white bars represent apples stored at 4°C for 4 weeks followed by a 20°C no-ethylene treatment, the black bar represents ethylene-treated apples stored at 20°C, and the hatched bar represents apples treated at 4°C followed by a 100 μL L⁻¹ ethylene treatment. Error bars represent se (n = 4).

Figure 4.

Phylogenetic clustering. A, Phylogenetic relationship among the six Arabidopsis EIL proteins and three apple EIL proteins. B, Phylogenetic relationship among the 147 Arabidopsis AP2 domain-containing proteins and 60 apple AP2 domain-containing proteins. The previously published Arabidopsis cluster groups are shown. For the full cluster and the number of apple and Arabidopsis genes per group, see Supplemental Figure S1 and Supplemental Table S1.

Figure 5.

Expression analysis of EILs and AP2D genes over fruit development in Royal Gala apples, from open flowers (0 DAFB) to eating ripe (146 DAFB). A, EIL1, EIL2, and EIL3. B, Relative expression patterns of 38 apple AP2 domain genes comparing 35 with 132 DAFB. A high ratio represents high expression late in fruit development. The letter a represents undetectable qPCR expression, y represents genes for which the error was too great to provide meaningful information, and n represents genes that were not assayed for this time point. The bar underneath represents the cluster groups for each of the AP2D genes; on this bar, a represents AP2 class genes, b represents RAV class genes, and c represents the VI-L class genes. Error bars represent se (n = 4).

Figure 6.

Relative expression analysis of apple AP2 genes in the E1 harvest of ACO1-suppressed apples that have been induced with ethylene. Values represent expression relative to the 0-h sample. A, Expression after a 4-h, 100 μL L⁻¹ ethylene treatment. B, Expression after a 192-h, 100 μL L⁻¹ ethylene treatment. C, Expression after 192 h in an ethylene-free environment. The genes are arranged in order of cluster groupings. Other features are as described in Figure 5.

Figure 7.

Tobacco transient assay of plants infiltrated with PG1-Luc. Transactivation of the promoter is measured as a ratio of the luciferase signal to the renillin signal. A, PG1-Luc-infiltrated plants with or without 100 μL L⁻¹ ethylene for 24 h prior to assay. PG1-Luc-infiltrated plants were kept at either 20°C or 4°C for 24 h prior to assay (cold a) or for 4°C for 24 h followed by 20°C for 2 d (cold b). B, Coinfiltration of PG1-Luc with EIL1 or EIL2 with or without a 24-h, 100 μL L⁻¹ ethylene treatment. C, Coinfiltration of PG1-Luc with 37 different apple AP2 domain-containing genes. The bar underneath represents the cluster groups as defined in Figure 5. Error bars represent se (n = 4).

Figure 8.

qPCR measuring gene expression in apple culture cells grown at 20°C (solid squares), 1°C (open squares), or 1°C for 2 d followed by 20°C (dashed lines). A, CBF-like gene (AP2D7- CBF2). B, PG1. Error bars represent se (n = 4).

Figure 9.

Tobacco transient assays of PG1-Luc coinfiltrated with a combination of empty vector control (pHex) and CBF2 with and without a 24-h, 100 μL L⁻¹ ethylene treatment. Error bars represent se (n = 4).