Functional Characterization of MtrGSTF7, a Glutathione S-Transferase Essential for Anthocyanin Accumulation in Medicago truncatula

Abstract

Flavonoids are essential compounds widespread in plants and exert many functions such as defence, definition of organ colour and protection against stresses. In Medicago truncatula, flavonoid biosynthesis and accumulation is finely regulated in terms of tissue specificity and induction by external factors, such as cold and other stresses. Among flavonoids, anthocyanin precursors are synthesised in the cytoplasm, transported to the tonoplast, then imported into the vacuole for further modifications and storage. In the present work, we functionally characterised MtrGSTF7, a phi-class glutathione S-transferase involved in anthocyanin transport to the tonoplast. The mtrgstf7 mutant completely lost the ability to accumulate anthocyanins in leaves both under control and anthocyanin inductive conditions. On the contrary, this mutant showed an increase in the levels of soluble proanthocyanidins (Pas) in their seeds with respect to the wild type. By complementation and expression data analysis, we showed that, differently from A. thaliana and similarly to V. vinifera, transport of anthocyanin and proanthocyanidins is likely carried out by different GSTs belonging to the phi-class. Such functional diversification likely results from the plant need to finely tune the accumulation of diverse classes of flavonoids according to the target organs and developmental stages.

Keywords: Medicago truncatula; Tnt1 insertional mutants; anthocyanin transport; flavonoids; gluthatione S-transferase.

Figure 1

Pictures of wild -type plants (A–C,F) and R45 mutant line (D,E,G,H). Arrows in (F,H) point to the leaf blot (F) and to the corresponding position in R45 (H). Arrows in (C,G) point to the darkened region at the junction of pedicel and calyx (C) and to the corresponding position in R45 (G). Pictures (A,B,D) E were taken in a cold greenhouse where pigmentation in the aerial tissues was induced by low temperatures in the wild type (A,B), while no pigmentation was observable in R45 (D,E).

Figure 2

Intron-exon structure of MtrunR108HiC_012760 corresponding to MtrGSTF7 and position of Tnt1 insertions in exon 3 in R39 and R45 mutant lines (A). Segregation analysis on 10 plants in the R45 T₁ progeny. P1–P3 primed amplification fails in presence of the 5kbps Tnt1 insertion. Plants 3, 4 and 8 (circled in red) showed the mutant phenotype (B).

Figure 3

Anthocyanin content measured in leaves of plants cultivated in vitro on high sucrose (6%) containing medium. WT is wild-type R108. HET and MUT stands for heterozygous and homozygous referred to Tnt1 insertion in MtrGSTF7. Anthocyanins could not be detected in MUT. Data indicate mean values ±SD of four replicates (*** p < 0.001).

Figure 4

Contents of soluble and insoluble PAs measured in R45 and R108 (WT) seeds (* p < 0.05).

Figure 5

Phylogenetic tree with R108 MtrGSTFs and other GSTs involved in flavonoid transportation. The tree was constructed with the neighbor joining method (1000 replications of bootstrap test) and p-distance substitution model, pairwise deletion using the MEGA 6 program. PpRiant1 (Prunus persica ALE31199.1); MdGSTF6 (Malus domestica NP 001315851.1); FvRAP (Fragaria vesca XP 004288578.1); CsGST (Citrus sinensis ABA42223.1); VvGST4 (Vitis vinifera AAX81329.1); LcGST4 (Litchi chinensis ALY05893.1); AtTT19 (Arabidopsis thaliana OAO91277.1); CkmGST3 (Cyclamen persicum x Cyclamen purpurascens BAM14584.1); AcGST1 (Actinidia chinensis QCQ77644.1); PhAN9 (Petunia x hybrida CAA68993.1); PfGST1 (Perilla frutescens var. crispa BAG14300.1); DcGST (Dianthus caryophyllus BAM21533.1); PcGST (Pyrus communis ABI79308.1); VvGST3 (Vitis vinifera ABO64930.1); DcGSTa (Dracaena cambodiana ANH58194.1); DcGSTb (Dracaena cambodiana, ANH58192.1); ZmBz2 (Zea mays AAA50245.1); VvGST1 (Vitis vinifera AAN85826.1).

Figure 6

MtrGSTFs, expression in plant organs and in the seed coat.

Figure 7

Expression of MtrGSTFs in leaves of 35S::LAP1 (LAP1), showing hyperaccumulation of anthocyanins, and 35S::GUS used as control (GUS). Asterisk indicates log₂FC > 2.

Figure 8

MtrGSTFs expression in glandular (black bars) and non-glandular trichomes (white bars).

Figure 9

Expression levels of key genes of the phenylpropanoid pathway in M. truncatula leaves of wild-type R108 (WT) and mutant plants (R45) grown under controlled conditions. The relative expression of each gene is calculated using the [2^−(ΔCt)] algorithm using actin as housekeeping gene. Significance of differences between means was determined by t-test (*** p < 0.001).

Figure 10

Gene expression in leaves of M. truncatula R108 (WT) vs. mutant plants (R45) grown in cold conditions. Expression levels are calculated as in legend of Figure 9 (* p < 0.05).

Figure 11

Gene expression in pods of M. truncatula R108 (WT) and mutant (R45). The stage of the sampled pods is shown (bar = 1 mm). Expression levels are calculated as in legend of Figure 9 (* p < 0.05).

Figure 12

Functional complementation assay. From the top: seedlings; detail of anthocyanin colouration at the rosette base; seeds; relative content of soluble and insoluble PA in wt seeds vs. those from tt19-1 mutant and tt19-1 mutant transformed with the 35S::MtrGSTF7 construct. Significant differences (p < 0.05) as determined by Tukey’s multiple comparisons test are indicated with different letters.