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. 2020 Sep 25;37(3):311-318.
doi: 10.5511/plantbiotechnology.20.0427a.

Isolation of dominant Arabidopsis seiv mutants defective in VND7-induced xylem vessel cell differentiation

Phookaew Pawittra  1 , Takaomi Suzuki  1 , Harunori Kawabe  1 , Arika Takebayashi  2 , Taku Demura  1   2 , Misato Ohtani  1   2   3
Affiliations

Isolation of dominant Arabidopsis seiv mutants defective in VND7-induced xylem vessel cell differentiation

Phookaew Pawittra et al. Plant Biotechnol (Tokyo). .

Abstract

The plant-specific NAC transcription factor VASCULAR-RELATED NAC-DOMAIN 7 (VND7) functions in xylem vessel cell differentiation in Arabidopsis thaliana. To identify novel factors regulating xylem vessel cell differentiation, we previously performed ethyl methanesulfonate mutagenesis of a transgenic 35S::VND7-VP16-GR line in which VND7 activity can be induced post-translationally by glucocorticoid treatment. We successfully isolated mutants that fail to form ectopic xylem vessel cells named seiv (suppressor of ectopic vessel cell differentiation induced by VND7) mutants. Here, we isolated eight novel dominant seiv mutants: seiv2 to seiv9. In these seiv mutants, ectopic xylem vessel cell differentiation was inhibited in aboveground but not underground tissues. Specifically, the shoot apices of the mutants, containing shoot apical meristems and leaf primordia, completely lacked ectopic xylem vessel cells. In these mutants, the VND7-induced upregulation of downstream genes was reduced, especially in shoots compared to roots. However, endogenous xylem vessel cell formation was not affected in the seiv mutants. Therefore, the seiv mutations identified in this study have repressive effects on cell differentiation in shoot meristematic regions, resulting in inhibited ectopic xylem vessel cell differentiation.

Keywords: Arabidopsis; VND7; seiv mutants; xylem vessel cell.

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Figures

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Figure 1. The seiv mutants show inhibited xylem vessel cell differentiation caused by effects on DEX-induced VND7 activity. Photographs of the dominant suppressor mutants seiv2–9, the parental line VND7-VP16-GR, and the vector control. Seven-day-old seedlings were treated without (−DEX, in A) or with (+DEX, in B) DEX for 4 day. Bar=1 cm.
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Figure 2. Effects of the seiv mutations on ectopic xylem vessel cell differentiation in cotyledons. Seven-day-old seedlings were treated with 10 µM DEX for 4 day and subjected to microscopic observation. Cotyledons of the vector control (A), VND7-VP16-GR (B), seiv2 (C), seiv3 (D), seiv4 (E), seiv5 (F), seiv6 (G), seiv7 (H), seiv8 (I), and seiv9 (J) are shown. Bar=200 µm.
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Figure 3. Effect of the seiv mutations on ectopic xylem vessel cell differentiation in the shoot apical region. Seven-day-old seedlings were treated with 10 µM DEX for 4 day and subjected to microscopic observation. The shoot apical regions of the vector control (A), VND7-VP16-GR (B), seiv2 (C), seiv3 (D), seiv4 (E), seiv5 (F), seiv6 (G), seiv7 (H), seiv8 (I), and seiv9 (J) are shown. Arrows and asterisks indicate shoot apical meristems and leaf primordia, respectively. Bar=50 µm.
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Figure 4. qRT-PCR analysis of the expression of genes downstream of VND7 in shoot regions of the seiv mutants. Seven-day-old vector control, VND7-VP16-GR, and seiv seedlings were treated with 10 µM DEX for 1 day, separated into shoot and root regions, and subjected to qRT-PCR analysis. The UBQ10 gene was used as an internal control. The expression levels of each gene are shown relative to the vector controls (set to 1). The results are means±SE (n=3). Statistically significant differences between VND7-VP16-GR and each seiv mutant are indicated by asterisks (Student’s t test, * p<0.05).
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Figure 5. qRT-PCR analysis of the expression of genes downstream of VND7 in the root regions of the seiv mutants. Seven-day-old vector control, VND7-VP16-GR, and seiv seedlings were treated with 10 µM DEX for 1 day, separated into shoot and root regions, and subjected to qRT-PCR analysis. The UBQ10 gene was used as an internal control. The expression levels of each gene are shown as relative values compared to the vector controls (set at 1). The results are means±SE (n=3). Statistically significant differences between VND7-VP16-GR and each seiv mutant are indicated by asterisks (Student’s t test, * p<0.05).
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