Transcription factor StABI5-like 1 binding to the FLOWERING LOCUS T homologs promotes early maturity in potato

Abstract

Potato (Solanum tuberosum L.) maturity involves several important traits, including the onset of tuberization, flowering, leaf senescence, and the length of the plant life cycle. The timing of flowering and tuberization in potato is mediated by seasonal fluctuations in photoperiod and is thought to be separately controlled by the FLOWERING LOCUS T-like (FT-like) genes SELF-PRUNING 3D (StSP3D) and SELF-PRUNING 6A (StSP6A). However, the biological relationship between these morphological transitions that occur almost synchronously remains unknown. Here, we show that StABI5-like 1 (StABL1), a transcription factor central to abscisic acid (ABA) signaling, is a binding partner of StSP3D and StSP6A, forming an alternative florigen activation complex and alternative tuberigen activation complex in a 14-3-3-dependent manner. Overexpression of StABL1 results in the early initiation of flowering and tuberization as well as a short life cycle. Using genome-wide chromatin immunoprecipitation sequencing and RNA-sequencing, we demonstrate that AGAMOUS-like and GA 2-oxidase 1 genes are regulated by StABL1. Phytohormone profiling indicates an altered gibberellic acid (GA) metabolism and that StABL1-overexpressing plants are insensitive to the inhibitory effect of GA with respect to tuberization. Collectively, our results suggest that StABL1 functions with FT-like genes to promote flowering and tuberization and consequently life cycle length in potato, providing insight into the pleiotropic functioning of the FT gene.

Figure 1

Interaction between StABL1 and FT-like paralogs. A, Interactions between FT and StABL1 with St14a in Y2H assays. StSP6A mutant F99A and StSP5G mutant F98A were tested; Alanine substitution (S344A) and phosphomimic mutation (S344E) on S/TAP motifs of StABL1 were also tested. −WL, medium without tryptophan and leucine; −WLHA, medium without tryptophan, leucine, histidine, and adenine. BD (pGBKT7) and AD (pGADT7) are the bait and prey vectors, respectively. 3-aminotriazole. B, Self-activation test of domain of StABL1 and its truncated fragments. StABL1-N (N-terminal of StABL1), StABL1-bZIP (bZIP fragment of StABL1), StABL1-C (C-terminal of StABL1). C, Y2H interaction analysis of StABL1-C and St14-3-3s. −WLH (medium without tryptophan, leucine, and histidine). D, Y2H interaction analysis of StSP3D with and St14-3-3s. E, Interactions between StSP6A and its mutant (2mStSP6A: R60K/P92L mutation), and StSP3D with StABL1-C in Y2H assays. F, BiFC analysis of the interaction among StSP6A, 2mStSP6A, StSP3D, StABL1, and St14a in E3 protoplasts. Chl, Chloroplast auto-fluorescence; YN, nYFP; YC; cYFP. Scale bar: 10 µm. G, Interaction between StABL1 and St14-3-3s in the Co-IP assays. The proteins were extracted from young coinjected leaves of N. benthamiana and immunoprecipitated by anti-GFP agarose beads. Gel blots were probed with anti-HA or anti-GFP antibody. The asterisk indicates the specific eGFP-StABL1 band. These nonspecific bands represent breakdown products resulting from protein turnover during IP.

Figure 2

Tuberization, maturity, and flowering of StABL1 transformants. A, The tuberization phenotype of representative E3, RNAi-StABL1, and StABL1-overexpressing (StABL1ox) transgenic plants in vitro 4 and 9 WAP, respectively. Scale bar: 1.5 cm. B, Percentage of in vitro cultured transgenic plants with micro-tubers. Data are presented as mean ± se. Data were obtained from 72 plants for each genotype. C, The tuberization phenotype of representative soil-grown WT E3 and StABL1ox transgenic plants at 5 DAT to SDs. Scale bar: 5 cm. D, Tuber number per plant of soil-grown WT E3 and StABL1 transgenic plants. Data collected at 5 and 10 DAT to SDs. n = 6. The error bar indicates the standard deviation. E, Percentage of soil-grown WT E3 and StABL1 transgenic plants with tubers. Data were obtained at 5 and 10 DAT to SD after 4 weeks grown under LD conditions., n = 6. Three independent experiments were performed. F, Representative photos of StABL1-overexpressing plants and WT E3 plants grown in pots under LDs for 50 and 90 d in net house. Scale bar: 25 cm. G, Percentage of plant survival with the plant aging (DAP) in WT E3 and StABL1ox transgenic plants, n = 24. H, Representative photos of StABL1ox plants and WT control E109 plants grown in pots for 40 d under LDs. Scale bar: 10 cm. I, Percentage of plant with flower buds in StABL1ox plants and its WT control E109, n = 8. J, Percentage of plant with open flower in StABL1ox plants and its WT control E109, n = 8. The asterisks in (B and D) indicate a statistically significant difference (Student’s t test, *P < 0.05, **P < 0.01).

Figure 3

Tuber phenotype of WT and StABL1 transformants. A, Representative microtuber photographs of WT E3 and StABL1 transgenic plants, grown in 8% sucrose MS medium for 70 d under SD conditions. Scale bar: 1 cm. B, Representative tuber photographs of six soil-grown WT E3 and StABL1-overexpressing transgenic plants. Picture was taken at 60 DAT to SDs. C, Tuber number per plant and (D) yield for soil-grown WT E3 and StABL1-overexpressing transgenic plants. Data were obtained at 60 DAT to SDs with six plants for each genotype. Data are presented as mean ± standard deviation. E, Yield for net house-grown WT E3 and StABL1-overexpressing transgenic plants. Data were obtained at 120 DAP, n ≥ 14. Data are presented as mean ± standard deviation. The asterisks indicate a statistically significant difference (Student’s t test, *P < 0.05, **P < 0.01).

Figure 4

Genome-wide identification of StABL1 binding sites. A, Distribution of peak in functional regions of genome. Promoter-TSS: ±2,000 bp of TSS; TES: ±1,000 bp of TES; Intergenic: gene-free region from 1,000-bp downstream of TES to 2,000-bp upstream of the TSS of the closest gene. Exon and intron are corresponding to the gene models in genome annotation. B, Heat maps showing the ChIP-Seq distribution of reads across TSS. C, HOMER de novo motif enrichment analyses of StABL1 binding peaks. The top five significantly enriched binding motifs and their matched TF family were presented.

Figure 5

Identification of StABL1 target genes. A, Venn diagram showing the number and overlapping genes between the StABL1-binding targets revealed by ChIP-Seq and the DEGs, identified by RNA-Seq in StABL1-overexpressing lines. B, Heatmap showing the P-values of the functional categories significantly enriched (P < 0.05) in either upregulated or downregulated DEGs in StABL1-overexpressing lines. P-values were calculated by one-side Fisher’s exact test. C, StABL1 binding profiles to AGAMOUS-like genes visualized with the IGV. Scale bar: 1 kb. D, Heatmap showing the normalized expression levels of AGAMOUS-like genes. Genes are represented with their gene ID, annotation, and Arabidopsis homolog. Data represent the mean of three CPM values in RNA-Seq.

Figure 6

Gibberellin metabolism and activity is affected in StABL1-overexpressing plants. A, StABL1 binding profile and control (input) peaks at StGA2ox1 visualized with the IGV. The position of G-box motif was indicated from the TSS. Scale bar: 1 kb. B and C, Time course of StGA2ox1 relative expression in WT E3 and StABL1-overexpressing plant leaves (B) and stolon tips (C). L0, L2, L4, and L6 represent leaves sampled at 0, 2, 4, and 6 DAT to SD, respectively. S0, S2, S4, and S6 represent stolon tips sampled at 0, 2, 4, and 6 DAT to SD, respectively. Data are presented as mean ± standard deviation, n = 3. D, Major reactions in GA biosynthetic pathway (Yamaguchi, 2008). E–L, The endogenous levels of gibberellins in WT E3 and StABL1-overexpressing stolon tips which were sampled at 7 DAT to SD. Data are presented as mean ± standard deviation, n = 3. M and N, Time course of StGA20ox1 relative expression in WT E3 and StABL1-overexpressing plant leaves (M) and stolon tips (N) sample mentioned in (B and C). O, Microtuber initiation time of WT E3 and StABL1-overexpressing plants on tuber-inducing medium (Control) or tuber-inducing medium supplemented with 0.2 mg L⁻¹ GA₃, or 5-μM ABA. Data are presented as mean ± standard deviation, n ≥ 30. nt represents no tuber. All the asterisks in this figure indicate a statistically significant difference (Student’s t test, *P < 0.05, **P < 0.01). ns means no significant difference. P, Western blot detection of GFP-StABL1 protein in lowest node of OE-StABL1–13 line under control and GA₃ treatment within 24 h. Rubisco large subunit (Rbc L) was used as a loading control.

Figure 7

Model for aTAC and aFAC in promoting potato maturation. Under SD conditions, the accumulated StSP6A interact with StABL1 bridged by St14-3-3s, to form aTAC, which block GA activity in stolon tips by altering GA metabolism, to promote tuberization transition; under both LD and SD conditions, the expressed StSP3D associate with StABL1 to trigger floral transition. Collectively, StABL1 functions with florigen (StSP3D) and tuberigen (StSP6A) to promote flowering and tuberization and thereby synchronizes potato maturity syndrome in potato.