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. 2008 Jan;20(1):201-12.
doi: 10.1105/tpc.107.056531. Epub 2008 Jan 18.

Sad3 and sad4 are required for saponin biosynthesis and root development in oat

Panagiota Mylona  1 , Amorn Owatworakit, Kalliopi Papadopoulou, Helen Jenner, Bo Qin, Kim Findlay, Lionel Hill, Xiaoquan Qi, Saleha Bakht, Rachel Melton, Anne Osbourn
Affiliations

Sad3 and sad4 are required for saponin biosynthesis and root development in oat

Panagiota Mylona et al. Plant Cell. 2008 Jan.

Abstract

Avenacins are antimicrobial triterpene glycosides that are produced by oat (Avena) roots. These compounds confer broad-spectrum resistance to soil pathogens. Avenacin A-1, the major avenacin produced by oats, is strongly UV fluorescent and accumulates in root epidermal cells. We previously defined nine loci required for avenacin synthesis, eight of which are clustered. Mutants affected at seven of these (including Saponin-deficient1 [Sad1], the gene for the first committed enzyme in the pathway) have normal root morphology but reduced root fluorescence. In this study, we focus on mutations at the other two loci, Sad3 (also within the gene cluster) and Sad4 (unlinked), which result in stunted root growth, membrane trafficking defects in the root epidermis, and root hair deficiency. While sad3 and sad4 mutants both accumulate the same intermediate, monodeglucosyl avenacin A-1, the effect on avenacin A-1 glucosylation in sad4 mutants is only partial. sad1/sad1 sad3/sad3 and sad1/sad1 sad4/sad4 double mutants have normal root morphology, implying that the accumulation of incompletely glucosylated avenacin A-1 disrupts membrane trafficking and causes degeneration of the epidermis, with consequential effects on root hair formation. Various lines of evidence indicate that these effects are dosage-dependent. The significance of these data for the evolution and maintenance of the avenacin gene cluster is discussed.

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Figures

Figure 1.
Figure 1.
Synthesis of Sterols and Triterpenoid Saponins in Oat. The first committed step in the avenacin pathway is catalyzed by the oxidosqualene cyclase β-amyrin synthase (encoded by Sad1). sad3 and sad4 mutants accumulate avenacin A-1 lacking the β-1,4–linked d-glucose.
Figure 2.
Figure 2.
Root Phenotypes of sad3 and sad4 Mutants. Roots of 3-d-old oat seedlings showing the reduced fluorescence (A) and root hair–deficient (B) phenotypes of sad3 mutant #1139 and sad4 mutant #9. Bar = 2 mm.
Figure 3.
Figure 3.
Characterization of New Mutant Alleles of sad3 and sad4. (A) and (B) Thin-layer chromatography analysis of root extracts from wild-type oat and from sad3 (A) and sad4 (B) mutant lines. Avenacin A-1 and monodeglucosyl avenacin A-1 (MDG A-1) were visualized under UV illumination. (C) Mean root lengths (±se) of 3-d-old wild-type and mutant seedlings (sample size, 25).
Figure 4.
Figure 4.
Degeneration of the Epidermal Layer in sad3 and sad4 Mutants. (A) Longitudinal optical sections of roots of 2-d-old wild-type and mutant propidium iodide–stained seedlings, showing the epidermal cell layer in the meristematic zone. Bar = 25 μm. (B) Longitudinal sections through the epidermal layer of roots of 3-d-old wild-type and mutant propidium iodide–stained seedlings, showing the cellular organization at the beginning of the differentiation zone. Bar = 25 μm. (C) Optical cross sections of roots shown in (B). Gaps in the epidermis of sad3 and sad4 are indicated by arrows, and exposed cortical cells are indicated by asterisks. Bar = 25 μm.
Figure 5.
Figure 5.
Localization of UV Fluorescent Material in Wild-Type and Mutant Oat Root Epidermal Cells. UV confocal microscopy of oat root epidermal cells showing uniform distribution of avenacin A-1 in vacuoles of wild-type roots (A) and patchy distribution of monodeglucosyl avenacin A-1 in sad3 roots (B) (arrow). v, vacuole. Bars = 100 μm.
Figure 6.
Figure 6.
Epidermal Cells of Roots of sad3 and sad4 Mutants Contain Aggregates That Stain with Calcofluor. Cross sections of roots of 2-d-old wild-type ([A] and [B]) and mutant ([C] to [F]) seedlings (meristematic zone), showing Calcofluor-staining aggregates (arrows). C, cortex; E, epidermis; V, vasculature. Bars = 50 μm ([A], [C], and [E]), 16 μm (B), 12 μm (D), and 17 μm (F).
Figure 7.
Figure 7.
sad3 and sad4 Mutants Have Membrane Trafficking Defects. Transmission electron micrographs of cross sections of the epidermal cells of roots of 2-d-old wild-type (A) and sad3 mutant ([B] and [C]) seedlings in the meristematic zone. C, cortex; E, epidermis; N, nucleus. Arrows indicate wavy/thickened appearance of cell margins (B) and sac-like structures (C) in sad3 mutants. Bars = 2 μm (A), 6 μm (B), and 1.8 μm (C).
Figure 8.
Figure 8.
Root Phenotypes of sad1/sad1 sad3/sad3 and sad1/sad1 sad4/sad4 Double Mutants. (A) sad1/sad1 sad3/sad3 double mutants have normal root morphology. (B) The sad4 root morphology phenotype is dependent on the dosage of Sad1. Sad1/sad1 sad4/sad4 and sad1/sad1 sad4/sad4 mutants both have normal root morphology. Four-day-old seedlings are shown.
Figure 9.
Figure 9.
Effects of Sad1 Gene Dosage on Avenacin A-1 Content. (A) Liquid chromatography–mass spectrometry (LC-MS) analysis of the avenacin A-1 and monodeglucosyl avenacin A-1 content of root extracts of F3 progeny derived from a cross between sad1 and sad4 mutants. Values are means ± se of 25 seedlings. (B) Growth rates (measured over a 4-d period) of roots of seedlings with the genotypes shown in (A). Values are means ± se for 25 seedlings. (C) LC-MS analysis of root extracts of F2 seedlings derived from a cross between wild-type oat and a homozygous sad1 mutant (in the absence of sad3 or sad4 mutations). Values are means ± se of 25 seedlings.
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