TIR1-like auxin-receptors are involved in the regulation of plum fruit development

Abstract

Ethylene has long been considered the key regulator of ripening in climacteric fruit. Recent evidence showed that auxin also plays an important role during fruit ripening, but the nature of the interaction between the two hormones has remained unclear. To understand the differences in ethylene- and auxin-related behaviours that might reveal how the two hormones interact, we compared two plum (Prunus salicina L.) cultivars with widely varying fruit development and ripening ontogeny. The early-ripening cultivar, Early Golden (EG), exhibited high endogenous auxin levels and auxin hypersensitivity during fruit development, while the late-ripening cultivar, V98041 (V9), displayed reduced auxin content and sensitivity. We show that exogenous auxin is capable of dramatically accelerating fruit development and ripening in plum, indicating that this hormone is actively involved in the ripening process. Further, we demonstrate that the variations in auxin sensitivity between plum cultivars could be partially due to PslAFB5, which encodes a TIR1-like auxin receptor. Two different PslAFB5 alleles were identified, one (Pslafb5) inactive due to substitution of the conserved F-box amino acid residue Pro61 to Ser. The early-ripening cultivar, EG, exhibited homozygosity for the inactive allele; however, the late cultivar, V9, displayed a PslAFB5/afb5 heterozygous genotype. Our results highlight the impact of auxin in stimulating fruit development, especially the ripening process and the potential for differential auxin sensitivity to alter important fruit developmental processes.

Keywords: Auxin-receptors; Prunus salicina; auxin/ethylene crosstalk.; plum fruit development; protein–protein interaction; subcellular localization.

Fig. 1.

Variation in response to NAA application due to diversity in auxin sensitivity between early (EG) and late (V9) plum cultivars. In (A) and (B), the two plum cultivars were treated with two NAA concentrations (10 and 100 µM). Image (A) illustrates the effects of NAA concentration on flowering of EG and V9. Image (B) demonstrates the changes in flower developmental process due to auxin application. Floral stages were selected when ~70% of the flowers in the shoot were in the same developmental stage. I, II, III, and IV represent flower stages at 2, 3, 4, and 6 days after treatment (DAT). (C) Close-up views of plum fruits before and after auxin application, in which EG and V9 fruits were treated with 10 and 100 µM NAA, respectively. Images (D) and (E) show the alterations in EG and V9 fruit size and weight due to NAA treatment.

Fig. 2.

Representative fruit growth curves (A, B), changes in ethylene production (C, D), and IAA levels (E, F) during plum fruit development for the early- and late-ripening cultivars EG and V9, respectively. Values represent the mean ±SD, as derived from nine independent replicates (5 fruit per replicate). The x axis represents the developmental stages indicated by DAB. The developmental stages include flower (FL), and stages 1–4 (S1–S4) of fruit development. The y axis refers to the changes in fruit fresh mass (A, B), ethylene levels (C, D) and IAA content (E, F) during development, respectively. The grey arrow (C–F) represents the fruit at climacteric stage.

Fig. 3.

Differences in PslTIR1/AFBs–ASK1 and –Aux/IAAs interactions in yeast. Y2H assays of the interaction (indicated by formation of blue colour) were performed using PslTIR1/AFBs (as bait) in Y2HGold strain yeast and ASK1 or Aux/IAAs (as prey) in Y187 strain yeast. The mated yeast (DBD–PslTIR1/AFBs and AD–ASK1 or –Aux/IAAs) were grown in 96-well plates containing DDO/X/A selective medium in the presence or absence of 100 µM IAA. All experiments were repeated at least three times.

Fig. 4.

Functional characterization of PslTIR1, PslAFB2, and PslAFB5 domains. Constructs containing full-length ORFs are referred to as WT. Truncated derivatives, including independent deletion of the N-terminal, F-box, and C-terminal domains, are referred to as ∆N, ∆F, and ∆C, respectively. (A) Subcellular localization of WT and truncated derivatives fused to the GFP tag. All constructs were transiently transformed for the assay into Nicotiana tabacum protoplasts. NLS-mCherry was included in each transfection to indicate the location of the nucleus. GFP fluorescence is shown as green; the merged image is a digital merge of bright field and fluorescent images to illustrate the protein compartments. All experiments were repeated a minimum of three independent times. Bars, 10 µm. (B) Y2H assays were performed using previously indicated derivatives with ASK1 and AtIAA7 in the presence of IAA. Other details as described in Fig. 3.

Fig. 5.

Steady-state transcript levels of PslTIR1, PslAFB2, and PslAFB5 mRNAs assessed by qPCR during (A) EG flower and fruit development and (B) throughout ripening of early –EG and late –V9 fruit. The expression was determined from fruit pulp. Results represent data from three biological and three technical replicates. Standard curves were used to calculate the number of target gene molecules per sample. These were then normalized relative to PslAct expression. Error bars represent SD. The y axis refers to the mean molecules of the target gene per reaction/mean molecules of PslAct. The x axis in each figure represents the developmental stage as indicated by the number of days after bloom (DAB).

Fig. 6.

CAPS analysis of PslAFB5 alleles. The PCR products of PslAFB5 exon-1 were amplified using EG and V9 gDNA, digested with AccIII and then electrophoresed on 2.5% agarose gel. Lane L refers to 100-bp ladder; lanes indicated by AFB5 and afb5 are the digested products from subcloned exon-1 fragments. Lanes EG and V9 refer to digested PCR products using the gDNA of the plum cultivars indicated as a template.

Fig. 7.

Pslafb5 encodes an inactive auxin receptor. Constructs containing full-length ORFs of PslTIR1 and PslAFB5 were used as a positive control and referred to as WT. Mutations were created by a site-directed mutagenesis approach through substitution of Pro9 and Pro61 of PslTIR1 and PslAFB5, respectively, into serine to simulate Pslafb5. (A) Y2H interaction assays of PslTIR1 and PslAFB5 (WT) and mutated derivatives with ASK1 and AtIAA7 (other details as in Fig. 4). (B) BiFC interaction visualization of PslTIR1 and PslAFB5 (WT and mutated ORFs) with ASK1 and AtIAA7. PslTIR1- and PslAFB5-related proteins were fused with the N-terminus (NY) of YFP; ASK1 and AtIAA7 were fused with the C-terminus (CY) of YFP. Different combinations of NY and CY constructs were transiently co-expressed in tobacco protoplasts. NLS-mCherry was included in each transfection to highlight the location of the nucleus. YFP fluorescence is yellow; the merged image is a digital merge of bright field and fluorescent images to illustrate the interaction location. Bars, 10 µm.