Down-regulation of Kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly)phenols and tolerance to ultraviolet radiation

Abstract

Phenylpropanoid biosynthesis in plants engenders myriad phenolics with diverse biological functions. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway, directing primary metabolic flux into a phenylpropanoid branch. Previously, we demonstrated that the Arabidopsis (Arabidopsis thaliana) Kelch domain-containing F-box proteins, AtKFB01, AtKFB20, and AtKFB50, function as the negative regulators controlling phenylpropanoid biosynthesis via mediating PAL's ubiquitination and subsequent degradation. Here, we reveal that Arabidopsis KFB39, a close homolog of AtKFB50, also interacts physically with PAL isozymes and modulates PAL stability and activity. Disturbing the expression of KFB39 reciprocally affects the accumulation/deposition of a set of phenylpropanoid end products, suggesting that KFB39 is an additional posttranslational regulator responsible for the turnover of PAL and negatively controlling phenylpropanoid biosynthesis. Furthermore, we discover that exposure of Arabidopsis to ultraviolet (UV)-B radiation suppresses the expression of all four KFB genes while inducing the transcription of PAL isogenes; these data suggest that Arabidopsis consolidates both transcriptional and posttranslational regulation mechanisms to maximize its responses to UV light stress. Simultaneous down-regulation of all four identified KFBs significantly enhances the production of (poly)phenols and the plant's tolerance to UV irradiation. This study offers a biotechnological approach for engineering the production of useful phenolic chemicals and for increasing a plant's resistance to environmental stress.

Figure 1.

Phylogenetic analysis of Kelch domain-containing F-box proteins in Arabidopsis. AtKFB01 (At1g15670), AtKFB20 (At1g80440), AtKFB39 (At2g44130), and AtKFB50 (At3g59940) are in boldface.

Figure 2.

Interaction of PAL isozymes with the KFB39 protein. A, Y2H assay between BD-KFB39 and AD-GADT7-PAL (PAL1–PAL4). Yeasts were grown on two-amino acid dropout (−Leu/Trp; −LT) synthetic defined (SD) medium supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; top) or on three-amino acid dropout (−Leu/Trp/His; −LTH) selective medium (bottom). B, Domain-swapping validation of the interaction between KFB39 and PAL1 to PAL4 in Y2H assays. The assays were conducted between AD-KFB39 and BD-PAL (PAL1–PAL4). Yeasts were grown on SD (−Leu/Trp) medium in the absence (top) and presence (middle) of X-gal and on SD (−Leu/Trp/His) selective medium (bottom). C, BiFC assay for the interactions of PAL isozymes with KFB39 protein in transiently expressed tobacco leaves. PAL1 to PAL4 were fused with CFPc at their C termini, and the truncated KFB39(ΔF) was fused with nYFP at its N terminus. The truncated KFB39(ΔF) fusion construct was coinfiltrated with PAL-CFPc (or CFPc alone) in tobacco leaves. The pair of nYFP-KFB20(ΔF) with PAL1-CFPc served as a positive control. Bars = 50 μm.

Figure 3.

Attenuation of PAL stability and activity by AtKFB39. A, Relative activity of expressed PAL isozymes in crude extracts from tobacco leaves transiently expressing PAL alone (black bars) or coexpressing both PAL and KFB39 (white bars). B, Immunoblot detection of the stability of PAL-GFP proteins using the anti-GFP antibody in tobacco leaves coexpressing PAL-GFP and KFB39 genes. Ponceau S staining served as a control for the amount of protein loading.

Figure 4.

Overexpression of KFB39 and reduction of PAL protein level in Arabidopsis. A, Relative expression level of KFB39 in 5-DAG seedlings of the selected KFB39 overexpression lines. Its level in transgenic lines, after normalization to that of the UBIQUITIN10 control gene, was expressed relative to its level in an empty-vector control line. tt4, pal1, and c4h were the controls. WT, Wild type. B, Level of endogenous PAL protein in the KFB39 overexpression lines. The PAL proteins were detected with anti-PAL peptide antibody. The monoubiquitin immunoblot against the anti-ubiquitin antibody (Ub) served as a control for the amount of protein loading.

Figure 5.

Alteration of the accumulation of flavonoids, anthocyanins, and CTs in KFB39 overexpression plants. A, The 5-DAG seedlings of the KFB39 overexpression lines with control and tt4, pal1, and c4h mutants grown on one-half-strength MS medium containing 4% (w/v) Suc. Note the change in their pigmentation. B, Representative UV-HPLC profiles of methanolic soluble phenolics in the KFB39 overexpression lines. K1, Kaempferol 3-O-[6′′-O-(rhamnosyl)glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside 7-O-rhamnoside; K3, kaempferol 3-O-rhamnoside 7-O-rhamnoside; SM, sinapoyl malate. Metabolites were extracted from 0.1 g fresh weight of seedlings with 1 mL of 80% methanol, and 10-µL extracts were injected for HPLC profiling. C, Seed coat brown pigmentation of the control (left), KFB39 overexpression line 14-2 (middle), and KFB39 overexpression line 12-1 (right). D, Relative content of CTs in the mature seeds of KFB39-OE lines. Data represent means ± sd from three biological repeats. WT, Wild type.

Figure 6.

Reduction of total PAL activity and lignin content in KFB39 overexpression lines. PAL activity was measured using protein crude extracts from leaves of 4-week-old transgenic plants. Acetyl-bromide lignin was determined using the cell walls of 12-week-old Arabidopsis stems. Data represent means ± sd with three biological repeats. The enzyme activity and lignin content of the control plants were set at 1. WT, Wild type.

Figure 7.

Suppression of KFB39 and alteration of PAL protein level in KFB39 RNAi-mediated gene-silencing lines. A, qRT-PCR analysis of the expression level of KFB39 in RNAi silencing lines. Data represent means ± sd with three biological replicates. B, Immunoblot detection of the cellular concentration of the PAL protein using anti-PAL antibody. C, Relative PAL protein level in RNAi silencing lines calculated based on B. WT, Wild type.

Figure 8.

Alteration of the accumulation of anthocyanins, CTs, and lignin in KFB39-RNAi transgenic lines. A, Effect of KFB39 silencing on anthocyanin pigmentation. The top row shows the front view of 4-week-old control (left), kfb01/20/50 triple mutant (middle), and kfb01/20/50/KFB39-RNAi transgenic (right) rosettes; the bottom row shows the opposite view of control, mutant, and transgenic rosettes, illustrating the enhanced anthocyanin pigment in the petioles of the triple mutant and RNAi transgenic lines. Bar = 5 cm. B, Approximately 10-week-old kfb01/20/50/KFB39-RNAi transgenic plants showing pigmentation in the inflorescence stems. C, Change in the total lignin content in kfb01/20/50/KFB39-RNAi transgenic Arabidopsis. The acetyl-bromide lignin was measured in independent T2 lines. The data represent means ± sd with three biological replicates. CWR, Cell wall residue. D, Relative content of CTs in mature seeds of kfb01/20/50 triple mutant and kfb01/20/50/KFB39-RNAi transgenic plants. The data represent means ± sd from four biological repeats. WT, Wild type.

Figure 9.

Relative expression levels of PALs and KFBs in Arabidopsis exposed to UV light. Five-week-old Arabidopsis was illuminated with cool-white fluorescent light supplemented with UV-B radiation from an FS20-UVB lamp (0.2 J m⁻² s⁻¹) for 12 h. The error bars represent sd from three biological repeats. The expression level in untreated samples (black bars) was set at 1.

Figure 10.

Growth and injury of Arabidopsis seedlings under UV-B exposure. Seedlings at 5 DAG were grown on one-half-strength MS agar medium and illuminated with full-wavelength light supplemented with UV-B light from an FS20-UVB lamp (0.2 J m⁻² s⁻¹) for 8 h, then left to recover for 2 d. WT, Wild type.