Fig.2.Expression ofthe four members of the FaPIN (A,B) and FaAUX/LAX (C,D) gene families in theachene (A,C) and the receptacle (B,D) at four developmental stages (green;white; turning; red), by RNAseq. FPKM, fragments per kilobase of exon permillion fragments mapped.

Auxin distributionby polar auxin transport is mediated by the PIN and AUX/LAX proteins. Only fourFaPIN genes (FaPIN1, FaPIN4, FaPIN5, and FaPIN10) were expressed in the fruitsof the cultivated strawberry (Fragaria ananassa), showing adevelopmental specific pattern (Figures 2A,B). While FaPIN10 had higher expressionin the achene than in the receptacle, the other three FaPIN genes displayedhigher expression in the receptacle. In all cases, the pattern corresponded todecreased expression in the transition from the green to red stage, both in theachene and the receptacle. Regarding theF.ananassaAUX/LAX genes (FaAUX/LAX1, FaAUX/LAX2, FaAUX/LAX3, andFaAUX/LAX4), all were expressed in the receptacle and achenes (Figures 2C,D).In achenes, the highest expression corresponded to FaAUX/LAX4, while the otherthree members showed higher expression in the receptacle. In all cases, as occurredfor FaPIN genes, the pattern was a continuous decrease from green to red stagesin both the achene and receptacle. It is remarkable that most of the transportgenes showed higher expression in the receptacle compared to the achene.

Fig.3.Expression ofthe three members of the tryptophan aminotransferase gene family (FaTAA1,FaTAR1, and FaTAR2) and four members of the flavin-dependent monooxygenases(FaYUC2, FaYUC4, FaYUC7, FaYUC10, and FaYUC11) in achene (A,C) and receptacle(B,D) at four developmental stages (green; white; turning; red), by RNAseq.FPKM, fragments per kilobase of exon per million fragments mapped.

The expression ofthe correspondingF. ananassagenes,analyzed by RNA-seq, shows that only three (FaTAA1, FaTAR1, and FaTAR2) areexpressed in fruits (Figures 3A,B). In theachene, the three genes show the same pattern,i.e., a steep decrease in the transition from the green to the white stage (Figure 3A) and then decreasingto the red stage. The highest absolute valuefor expression is shown by FaTAR1. In thereceptacle, the only gene showing expression over 1 FPKM is FaTAR2 (Figure 3B). Interestingly, theexpression of this gene continuously increasesfrom the green to the red stage. In relationto the YUCCA genes, only five of the nine genes mappedinF. vescaare expressed inF. ananassa(Figures 3C,D). In the achene, the common pattern isa decrease from green to red, with the highestexpression level being that of the FaYUC11gene (Figure 3C). Even with low expression, some YUCCA genes were identified in the red receptacle (Figure3D). In the transition from the green to the red receptacle, thereis a significant increase in the expression of both FaTAA1 and FaTAR2. Theincrease was higher for FaTAR2, which also showed expression levels that werehigher than those of FaTAA1 in the receptacle (Figure 3B).

Fig.4. Auxindistribution in strawberry. DR5-GUS expression pattern in strawberry roots (A)and green fruits (B–D). For the NAA treatment, in (A), plants were grown invitro and treated with a solution of 50 mM NAA in water for 24 h (right), orjust with water for control plants (left). Scale bars(B) = 500 mm, (C,D) = 250 mm,. P, pericarp; E, endosperm; Em, embryo;Aq,achene;R, receptacle. (E) Relative expression by qRT-PCR of the GUS gene in receptacleat three developmental stages. Different letters indicate a significantdifference between samples according to the corresponding ANOVA (P < 0.05).

To investigate the role played by auxin in strawberry fruit development, thesynthetic auxin response promoter DR5 fused to the b-glucuronidase (GUS) gene wasused to generate transgenic strawberry plants. The roots of the transgenicstrawberry plants showed a GUS expression pattern that was similar to that observed in the corresponding transgenic Arabidopsis (Figure 4A),indicating that GUS expression in DR5-GUS transgenic strawberry plants is abona fide indicator of auxin output signaling. Moreover, treatment with NAAincreased the GUS staining in the root (Figure 4A), indicative of afunctional response of the DR5 promoter to auxin in this organ. In the greenfruit, DR5-driven GUS staining was observed in the receptacle around the acheneattachment (Figure 4B). A closer view of a sectioned achene showedstrong GUS staining in the embryo (Figure 4C). In the green receptaclesurrounding the achenes, we observed a gradient of GUS staining that initiatedat the achene (Figure 4D). Then, GUS transcription was evaluated inthe receptacle at three stages. The result showed that in the red receptacle, theexpression was maintained, and slightlyhigher, compared to the green and white stages(Figure 4E). Overall, the pattern of GUS activity and GUS expression in strawberry fruits confirms thepreviously reported auxin activity in theachene and green receptacle and the auxin responsivenessin red receptacle.

The pathway startswith the auxin receptor genes TIR1 and AFB. In the F. vesca genome, four auxin receptorsare present, while three are expressed in F.ananassa. Their study in the receptacle shows that FaTIR1 slightlyincreases its expression from the green to red stage, while the expression of FaAFB2decreases, and FaAFB5 remains constant (Figure 5A). Therefore, the expression in the developing receptacle of thecorresponding genes of F. ananassa (FaAux/IAA and FaARF) was analyzed in the RNA-seq expression data. A total of 19FaAux/IAA genes were expressed in ripeningreceptacle of strawberry fruit with differentpatterns (Figure 5B). Most of them showed the highest expression at thegreen stage and then continuously decreased up to the red stage, in agreement withthe occurrence of auxin in the green receptacle (Figure 5B).However,three genes showed a different pattern. The FaAux/IAA8a gene showed the highestexpression at all stages, with a small increase from the green to increase from77 FPKM at the green to 236 FPKM at the red stage (Figure 5C).Clustering by co-expression analysis of the FaAux/IAA and FaARF genes in thereceptacle during ripening was performed to identify the possible positiveinteractions between members of the two gene families (Figure 5D). Twomain clusters are formed. One of them (upper) includes those genes with the highestdecrease in the transition from the green to red stage. The other clusterincludes genes that showed variable and not very drastic changes in expressionduring the growth and ripening of the receptacle. In the lower part of thiscluster are grouped, in a sub-cluster, those genes whose expression increased with theprogression of ripening (FaAux/IAA11, FaARF6a, FaAux/IAA14b, FaAux/IAA33, andFaARF16c). In this group, the two genes showing the highest expression leveland highest change in the transition from the green to red stage are FaAux/IAA11and FaARF6a.