Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots

Abstract

Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT. Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [(3)H]-indole-3-acetic acid tracer. The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport. These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.

Keywords: Arabidopsis thaliana; flavonol biosynthesis; flavonol glycoside; flavonol glycosyltransferases; plant growth; polar auxin transport.

Figure 1

Flavonoid biosynthesis pathway in Arabidopsis thaliana. (a) Scheme of flavonoid biosynthesis. CHS (TT4), chalcone synthase; F3′H (TT7), flavonoid 3′-hydroxylase; DFR (TT3), dihydroflavonol 4-reductase; FLS, flavonol synthase; ANS (TT18), anthocyanidin synthase; UGT78D1, flavonol 3-O-rhamnosyltransferase; UGT78D2, flavonoid 3-O-glucosyltransferase; UGT89C1, flavonol 7-O-rhamnosyltransferase. As shown by Yin et al. (2012), the combined loss of UGT78D1 and UGT78D2 does not imply an accumulation of flavonol aglycones because of a feedback inhibition of flavonol biosynthesis. (b) Glycosylation reactions catalyzed by UGT78D1, UGT78D2 and UGT89C1 (Jones et al., ; Tohge et al., ; Yonekura-Sakakibara et al., 2007). Abbreviations: Kaempferol (k); rhamnoside (rha); glucoside (glu). k1, k-3-O-rha-7-O-rha; k2, k-3-O-glu-7-O-rha; k3, k-3-O-[rha (1->2 glu)]-7-O-rha; q1, q2 and q3 are quercetins structurally equivalent to k1, k2 and k3, respectively.

Figure 2

Phenotypes of flavonoid biosynthesis-related mutants. Shoots of Arabidopsis thaliana wild-type and the indicated mutants were imaged after 28 d of growth. Bar, 5 cm.

Figure 3

Flavonol glycoside pattern in Arabidopsis thaliana mutants affecting flavonoid biosynthesis and conjugation. (a) Flavonols extracted from stems (lower region of 2.5 cm) of the wild-type and flavonoid metabolism-related mutants. The scale of all representative high-performance liquid chromatograms is identical. Peaks u1–u8 represent unknown flavonoids. SM, sinapoyl malate. (b) Quantification of k1 (Fig.1b) in the wild-type and flavonoid metabolism-related mutants. The means of k1 ± SD from four biological samples (a pool of three to five individual stem segments for one sample) are shown. k1 was quantified using kaempferol aglycone as a standard. Letters above the error bars represent a significant difference between genotypes by a paired t-test (P < 0.01).

Figure 4

Basipetal auxin transport in inflorescence stems. (a) Basipetal auxin transport in Arabidopsis thaliana wild-type, ugt78d2, tt7 and tt7 ugt78d2 basal stem segments. Data represent radioactivity accumulated in basal segments (means ± SE obtained from 10 individual plants without N-1-naphthylphthalamic acid (NPA) treatment and from two individual plants for NPA treatment)). Closed bars, +NPA; open bars, –NPA. Letters above the error bars represent a significant difference between genotypes by a paired t-test (P < 0.05). (b) Basipetal auxin transport in relation to k1 levels. Mean value and standard error are plotted. A linear relationship was observed (R² = 0.9911).

Figure 5

Gravitropic response of wild-type and ugt78d2 inflorescence stems. Four-week-old Arabidopsis thaliana plants were laid down from an upright to a horizontal position in darkness. The curvature of the primary inflorescence stems was measured at different time points to assess the gravitropic response. The angles refer to a horizontal positioning of the tip of the inflorescence (0°) and a fully upright position of the tangential approximation of the bent tip (90°). Wild-type, blue line; ugt78d2, red line. Means and SE are shown (n = 15).