The expression pattern of PgPINs in different rootedness pomegranate self-rooted seedling adventitious root
Two different rootedness pomegranate self-rooted seedling are screened in this study, compared with each other, the two self-rooted seedling show different rootedness adventitious roots branch angle (Table 1). The two self-rooted seedling provide appropriate material development for the study of GSA. We had screened the shallow-rooted pomegranate breed ‘Taishanhong’(TSH) and the deep-rooted pomegranate breed: ‘Lanbaoshi’(LBS) ( Fig. 1a). The PINs have been found to conducive to nearly every step of auxin fluxes [3, 18, 35, 20] and the auxin distribution patterns have been predicted and explained through auxin transport [2, 6, 1]. In this study, the expression of PgPIN1 and PgPIN10 showed higher in LBS than TSH (Fig. 1b). We also investigated the expression of other PgPINs, PgPIN3 and PgPIN7 showed lower in LBS than TSH (Additional file 1: Figure S1). These results indicated that PgPINs may participate in the regulation of GSA in pomegranate adventitious root.
Molecular cloning of PgFLP
To further characterize the function of PgPINs in pomegranate, we performed the Y1H screening via pomegranate cDNA library. Eventually, we used the promoter sequences of PgPIN10 as baits to obtained a cDNA fragment of PgFLP. To identify the pomegranate PgFLP that is involved in adventitious root gravitropism, a genome-wide analysis was performed. Comparison of amino acid sequences of PgFLP with their close homologs, the PgFLP protein is highly conserved identity to R2R3-MYB protein (Fig. 2a). To understand the evolutionary relationship of PgFLP, using the neighbor-joining (NJ) method [24], we constructed a phylogenetic tree. As a result, We found that PgFLP had been clustered within the R2R3-MYB clade including AtFLP, MdFLP and others for which root gravitropism have been identified. Within the R2R3-MYB clade, PgFLP is closely associated with AtFLP, which regulate lateral root gravitropism, indicating that PgFLP may participate in the regulation of GSA in pomegranate adventitious root (Fig. 2b).
Subcellular localization analysis of PgFLP
To further characterize the function of PgFLP in pomegranate, we used the coding sequence of PgFLP and GFP reporter to constructed a 35S::PgFLP-GFP translational fusion. We found that in contrast to 35S::GFP where a fluorescent signal was observed throughout the whole epidermal cell, the 35S::PgFLP-GFP showed a signal in nucleus (Fig. 3a-b), suggesting that as a transcription factor, PgFLP may be involved in pomegranate adventitious root gravitropism.
PgFLP expression pattern in pomegranate
To further determine the function of PgFLP, we performed the qRT-PCR to determine the expression patterns of PgFLP in pomegranate. We found that the expressing PgFLP were detected in various organs of pomegranate, especially higher expressing in the leaves and the fruit peels (Fig. 4a). The auxin participate in triggering lateral root development [4], we investigated whether PgFLP expression is induced by auxin. The expression of PgFLP was rapidly up-regulated in auxin-treated roots (Fig. 4b). We also investigated the expression of PgMYB88 was not significantly induced by auxin (Additional file 1: Figure S2b). Subsequently, we found that the transcripts of PgFLP in LBS was increased (~2-fold) upon the TSH (Fig. 4c). We also investigated the IAA content in different rootedness pomegranate self-rooted seedling and found that the level of IAA was slightly increased in LBS compared to that in TSH on shoot cutting at 30 d and 40 d (Fig. 4d). These results indicated that PgFLP plays an key role in pomegranate adventitious root gravitropism.
PgFLP directly regulate PgPIN10 transcription
To further characterize the mechanism of PgFLP to regulate GSA of adventitious root in pomegranate, we performed the Y1H assays. Y1H assays showed that PgFLP could interact with the promoter of PgPIN10 to activate its expression (Fig. 5a). To further investigate whether PgFLP could activate the transcriptional expression of PgPIN10 in planta, we conducted a transient expression assay. As a result, the proPgPIN10::GUS plus PgFLP showed significantly higher GUS activity in tobacco leaves than the control (Fig. 5b-d). These results showed that PgFLP could activate the expression of PgPIN10 in pomegranate.
It is well established that FLP can bind directly to the promoters of PINs genes that harbour an [A/T/G][A/T/G]C[C/G][C/G] motif [31]. We found that there was two potential binding sites including AGCGG and TACCC in the promoter region of PgPIN10 (Fig. 5e and Additional file 1: Figure S3). To further determine if PgFLP could directly bind to these binding sites in plants, we performed Y1H assays. We found that PgFLP could interact with FBS (AGCGG). On the other hand, PgFLP could not interact with the mutated FBS motif (mFBS) (Fig. 5f). These data demonstrate that PgFLP directly regulate PgPIN10 transcription by binding to the AGCGG motif.
Ectopic overexpression of PgFLP in Arabidopsis seedlings results in a smaller GSA of lateral root
As it is difficult to obtain transgenic pomegranate plants overexpressing PgFLP, PgFLP was ectopically expressed in Arabidopsis to further characterize if PgFLP functions in the regulation GSA of pomegranate self-rooted seedling adventitious root in planta. Three independent transgenic lines, OE3, OE4 and OE7, were chosen for further analysis (Fig. 6a). To investigate phenotypes of the WT and PgFLP overexpression lines grown for 21 days on MS media, we found that PgFLP (OX-3, OX-4, OX-7) (Fig. 6b-e), exhibited a smaller GSA and grew downwards faster than WT. For example, over 35% of PgFLP-OX lateral root increased within a 30-50° range, meanwhile, within a 50-70° range, over 30% of lateral root was decreased (Fig. 6f). These results indicated that PgFLP functions in the regulation GSA of pomegranate self-rooted seedling adventitious root in planta.