Architectural phenotypes in the transparent testa mutants of Arabidopsis thaliana

Abstract

Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants are compromised in the enzymatic steps or transcriptional regulators affecting flavonoid synthesis. tt mutant seedlings were grown on hard-slanted agar (a stress condition), under varying light conditions, and in soil to examine the resulting growth patterns. These tt mutants revealed a wide variety of architectural phenotypes in root and aerial tissues. Mutants with increased inflorescences, siliques, and lateral root density or reduced stature are traits that could affect plant yield or performance under certain environmental conditions. The regulatory genes affected in architectural traits may provide useful molecular targets for examination in other plants.

Fig. 1.

The flavonoid pathway in Arabidopsis. Shown are the locations of the transparent testa mutations included in these experiments (in brackets). Mutations tt1, tt2, tt8, and ttg are in regulatory genes involved in several points of the pathway. The gene products affected by these regulatory mutations are TT2 which is a R2R3 repeat MYB transcription factor; TT8 which is a bHLH transcription factor; and TTG which encodes a WD40 repeat gene. These proteins operate as a complex to induce BANYLUS expression to form anthocyanins as downstream products of the pathway. TT1 is a WIP family zinc finger transcription factor and TTG2 is a WRKY type transcription factor that acts downstream of TTG1. The mutated genes and the affected products are: TT3 (DFR: dihydroflavonol reductase); TT4 (CHS: chalcone synthase), TT5 (CHI: chalcone isomerase), TT6 (F3H: flavonol 3-hydroxylase), TT7 (F3′H: flavonol 3′-hydroxylase), and TT10 an enzyme for biflavonol conversion and oxidizing procyanidins to proanthocyanidins in the seed testa. The figure is adapted from Buer et al. (2007), (www.plantphysiol.org), Copyright American Society of Plant Biologists.

Fig. 2.

DPBA-flavonoid fluorescence in roots of transparent testa mutants varies according to the aglycone that accumulates in the organ. Most seedlings accumulate quercetin that fluoresces a bright golden yellow complexed with DPBA. The mutants tt4 and tt6 have very dim fluorescence from background sinapate esters and naringenin, respectively. Naringenin-DPBA fluoresces over 400 times less strongly than quercetin- or kaempferol-DPBA complexes (Buer et al., 2007). The tt7 mutant shows a dim fluorescence from dihydroquercetin-DPBA and tt5 shows fluorescence from the spontaneous reaction forming naringenin and subsequent downstream products. Several mutants have obvious DPBA fluorescence in the root hairs. Seedlings were analysed for fluorescence 5 d following germination. Seedlings were grown without sucrose in the medium. The bar=100 μm.

Fig. 3.

Root phenotypes of the various transparent testa mutants. Growing seedlings on 1.5% slanted agar induced several different phenotypes in the seedlings. All seedlings grew without sucrose in the medium under continuous light. The scans were performed at 9 d following germination. Interesting phenotypes were looped roots in tt4 and tt10 (arrow), increased (+) or decreased (–) lateral roots compared to wild type, greater root (>) or lesser (<) skewing, elongated hypocotyls in tt6 (‡), and elongated petioles in tt8 (§). The scale bar=8 mm.

Fig. 4.

The aerial phenotypes in some transparent testa mutants have multiple inflorescences. The multiple inflorescences of tt8, tt10, and ttg-1 are compared to the wild type and clearly have more inflorescences. The photographs were taken 9 weeks following transplanting to pots. The scale is provided by a ruler shown at the bottom of each panel with major gradations in cm.

Fig. 5.

Aberrant root outgrowths on tt4 roots at 40 d. (A–D) Aberrant growths that resemble root nodules are shown. Typically, each plant produced 100s of root outgrowths averaging 1 per mm root length, and often the structures were clustered in groups (B). A cleared root outgrowth is shown in (E). Cleared roots indicated that initiation is from the pericycle. Scale bars: (A)–(D) 50 μm; (E) 20 μm.

Fig. 6.

Feeding naringenin to tt4 restores flavonoid fluorescence to pollen. tt4 seedlings were watered three times weekly with 10 μM naringenin. Following pollen formation, flower tissue was analysed for flavonoid fluorescence using DPBA. The wild-type fluorescence is similar to that reported by Peer et al. (2001). Abbreviations: an, anther; st, stigma; po, pollen. Bar=100 μm.