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Arabidopsis 3-Ketoacyl-CoA Synthase 4 is Essential for Root and Pollen Tube Growth

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Abstract

Very long-chain fatty acids (VLCFAs) are essential precursors of membrane lipids, such as phospholipids and sphingolipids, cuticular waxes, suberins, and Brassica seed oils. The first step of VLCFA synthesis is mediated by 3-ketoacyl-CoA synthase (KCS), which catalyzes the condensation of a C2 unit from malonyl-CoA to acyl-CoA. In the present study, we investigated the role of KCS4 in pollen tube and root growth. KCS4 was predominantly expressed in shoot and root apical meristems, leaf veins, mature and germinated pollen grains, and developing embryos. The fluorescent signals of KCS4 fused with enhanced yellow fluorescent protein (KCS4:eYFP) were detected in the endoplasmic reticulum of tobacco epidermis. KCS4 disruption inhibited pollen tube elongation and root growth, whereas KCS4 promoter-driven KCS4 expression rescued the growth-retarded phenotype to wild type (WT) in kcs4 complementation lines. Root growth assay of WT and kcs4 lines treated with metazachlor and mefluidide, which are specific KCS inhibitors, and fatty acid analysis of their roots and seeds revealed that KCS4 is involved in the elongation of longer than C24 VLCFAs, which are essential for root and pollen tube growth.

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Acknowledgements

This work was supported by grants from the National Research Foundation (NRF-2019R1A2B5B02070204) of South Korea and the New Breeding Technologies Development Program (Project No. PJ014781022020) of the Rural Development Administration, South Korea.

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Authors and Affiliations

  1. Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, 61186, Korea

    Juyoung Kim

  2. Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju, 54874, Korea

    Saet Buyl Lee

  3. Department of Life Science, Sogang University, Seoul, 04107, Korea

    Mi Chung Suh

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  1. Juyoung Kim
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Correspondence to Mi Chung Suh.

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Supplementary file1 (DOCX 18 KB)

Supplementary Fig. S1

Microarray analysis of mature (A) (Winter et al. 2007) and developing (B) pollen grains (Honys and Twell 2004) of 21 KCS isoforms. Dataset is derived from eFP-browser (https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi)(JPEG 64 KB)

Supplementary Fig. S2

Alexander staining in WT and kcs4 anthers. Whole flowers were submerged into Alexander staining solution for 20 min. The stained samples were washed with distilled water, and then anthers were photographed with under a light stereomicroscope (SteREO Lumar V12, Carl Zeiss) (JPG 55 KB)

Supplementary Fig. S3

Semi-thin section of WT and kcs4 anthers during pollen development. Open flowers of 6-week-old plants were tagged daily for 7 days. Flowers and buds were fixed and then embedded in Spurr resin. After obtaining semi-thin section, pollen developmental stages were divided as described previously (Smyth et al. 1990). Images were obtained with a AxioCam MRc5 camera coupled to a light stereomicroscope (SteREO Lumar V12, Carl Zeiss). Msp microspores, T tapetum layer, E epidermis, En endodermis, PG pollen grain, C connective, Fb fibrous bands, St stomium. Bars = 100 µm (JPG 212 KB)

Supplementary Fig. S4

Staining of mature WT and kcs4 pollens with Fluorol yellow 088. Mature pollens were incubated with 0.01 % (w/v) Fluorol yellow in 100% ethanol for 5–10 min, washed with distilled water, and then visualized under a microscope with fluorescent (A) and UV (B) light as described in Brundrett et al. (1990) and Regan and Moffatt (1990). A Bars = 10 µm. B Arrows indicate the pollen surface (JPG 69 KB)

Supplementary Fig. S5

Composition (A) and levels (B) of suberin monomers in WT and kcs4 roots. Roots of 2-week-old seedlings were harvested and delipidated. Completely dried root residues were depolymerized and acetylated according to a previously reported method (Li-Beisson et al. 2013). Composition and levels of suberin monomers were analyzed and quantified by GC-MS and GC-FID. Student’s t-test was used for statistical analysis between WT and kcs4 (n = 4) (JPG 82 KB)

Supplementary Fig. S6

Cuticular wax composition (A) and levels (B) in WT and kcs4 stems. Cuticular waxes were extracted from the stems of 5-week-old plants using chloroform. The levels and composition of cuticular waxes were analyzed by GC-MS and GC-FID. Student’s t test was used for statistical analysis between WT and kcs4 (n = 3) (JPG 60 KB)

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Kim, J., Lee, S.B. & Suh, M.C. Arabidopsis 3-Ketoacyl-CoA Synthase 4 is Essential for Root and Pollen Tube Growth. J. Plant Biol. 64, 155–165 (2021). https://doi.org/10.1007/s12374-020-09288-w

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