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Research ArticleGROWTH AND DEVELOPMENT
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Brassinosteroid/Sterol Synthesis and Plant Growth as Affected bylka and lkb Mutations of Pea

Takahito Nomura, Yukiko Kitasaka, Suguru Takatsuto, James B. Reid, Motohiro Fukami, Takao Yokota
Takahito Nomura
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Yukiko Kitasaka
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Suguru Takatsuto
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James B. Reid
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Motohiro Fukami
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Takao Yokota
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Published April 1999. DOI: https://doi.org/10.1104/pp.119.4.1517

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    Fig. 1.

    Proposed biosynthesis pathways for brassinolide from campesterol. Conversions marked with asterisks are hypothetical. Lesions in the BR biosynthesis of Arabidopsis mutantsdet2, dwf4, and cpd are shown.

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    Fig. 2.

    Effects of brassinolide and its biosynthetic precursors on the internode length of lkb plants of pea. The lengths of the fourth and fifth internodes were measured 3 d after the fourth internodes were treated. Results are means ±se (n = 5). Campesterol and campestanol were applied in lanolin paste.

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    Fig. 3.

    The sterol biosynthesis pathways. Lesions in the BR biosynthesis of the lkb mutant of pea are shown.

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    Fig. 4.

    Proposed synthetic pathway from [25,26,27-2H7]24-methylenecholesterol to [26,27-2H6] campesterol.

Tables

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    Table I.

    Growth data for 49-d-old pea shoots

    GenotypeNo. of PlantsPlant Height-aNo. of Expanded Leaves per Shoot-aTotal WtAveraged Wt per Shoot
    cm   g
    WT19028.9  ± 0.97.80  ± 0.14352.29
    lka 19220.0  ± 0.37.04  ± 0.13952.06
    lkb 21819.8  ± 0.37.48  ± 0.14812.21

    These plant materials were used for analyses of endogenous BRs (TableII) and sterols (Table III).

      • ↵F0-a Data are means ± se of 25 plants randomly selected.

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      Table II.

      Endogenous levels of BRs in 49-d-old shoots of WT, lka, and lkb of pea

      BRBR Level
      WTlkalkb
      ng kg−1 fresh wt
      BrassinolideND1-a NDND
      Castasterone491  ± 0.33005  ± 2.5112  ± 0.2
      Typhasterol26  ± 0.139  ± 1.54  ± 0.0
      3-Dehydroteasterone20  ± 1.421  ± 1.915  ± 0.4
      Teasterone5  ± 0.15  ± 0.54  ± 0.2
      6-Deoxocastasterone2937  ± 224053  ± 73208  ± 7.8
      6-Deoxotyphasterol835  ± 2.1983  ± 17245  ± 13
      3-Dehydro-6-deoxoteasterone182  ± 1.9339  ± 4.2101  ± 4.1
      6-Deoxoteasterone180  ± 2.8197  ± 1.1176  ± 2.9

      The levels of BRs were determined by GC-SIM using 2H internal standards. Data are means ± se of duplicate determinations.

        • ↵F1-a ND, Not detected.

        • View popup
        Table III.

        Endogenous levels of sterols in 49-d-old shoots and mature seeds of WT, lka, and lkb of pea

        SterolSterol Level
        WTlkalkb
        ShootSeedShootSeedShootSeed
        μg g−1 fresh wt
        24-Methylenecholesterol 0.7  ± 0.0 ND2-a 0.5  ± 0.0 ND 17.5  ± 0.2 14.5  ± 0.4
        Campesterol/24-epicampesterol 21.1  ± 0.1 85.8  ± 1.0 25.0  ± 0.4 88.2  ± 2.0 2.3  ± 0.0 6.0  ± 0.0
        Campestanol/24-epicampestanol 1.3  ± 0.0 5.3  ± 0.1 1.4  ± 0.0 5.7  ± 0.0 0.1  ± 0.0 0.5  ± 0.0
        Isofucosterol 20.3  ± 0.2 6.8  ± 0.1 16.5  ± 0.3 6.1  ± 0.1 252.5  ± 2.0 1514.5  ± 20
        Sitosterol 122.4  ± 0.8 872.0  ± 17 143.5  ± 0.8 943.2  ± 39 11.7  ± 0.1 21.7  ± 0.2
        Stigmasterol 46.9  ± 0.2 42.0  ± 1.1 57.5  ± 0.4 60.4  ± 2.1 0.6  ± 0.0 4.4  ± 0.0
        Sitostanol 5.8  ± 0.0 15.9  ± 0.3 6.3  ± 0.0 15.7  ± 0.1 ND ND
        Cholesterol 1.4  ± 0.0 1.4  ± 0.0 1.2  ± 0.0 2.2  ± 0.0 2.0  ± 0.0 1.9  ± 0.0
        Total sterols 219.9 1029.2 251.9 1121.5 286.7 1563.5
        End-pathway sterols2-b 191.8 1001.2 227.2 1094.0 16.6 34.0

        The levels of sterols were determined by GC-SIM using [2H6]campestanol as an internal standard. Data are means ± se of duplicate determinations.

          • ↵F2-a ND, Not detected.

          • ↵F2-b Campesterol plus 24-epicampesterol plus sitosterol plus stigmasterol plus cholesterol.

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          Table IV.

          Effects of brassinolide on the growth and sterol levels of WT, lka, and lkb shoots of pea

          Growth and LevelWTlkalkb
          ControlBrassinolide (100 ng)ControlBrassinolide (100 ng)ControlBrassinolide (100 ng)
          Profile
           Fourth internode length (mm ±se)11.2  ± 0.417.2  ± 0.67.7  ± 0.211.5  ± 0.48.7  ± 0.221.4  ± 0.7
           Averaged weight per apical segment (g)  0.18  0.22  0.19  0.22  0.19  0.27
          Sterol (μg g−1 fresh wt)
           24-Methylenecholesterol1.2  ± 0.01.4  ± 0.01.5  ± 0.01.9  ± 0.143.7  ± 0.432.1  ± 0.3
           Campesterol/24-epicampesterol38.3  ± 0.239.0  ± 0.149.9  ± 0.146.0  ± 0.32.0  ± 0.11.3  ± 0.0
           Campestanol/24-epicampestanol0.9  ± 0.00.8  ± 0.01.3  ± 0.01.4  ± 0.00.1  ± 0.00.1  ± 0.0
           Isofucosterol32.2  ± 0.331.1  ± 0.121.4  ± 0.026.8  ± 0.3311.8  ± 8.0161.1  ± 2.5
           Sitosterol100.5  ± 0.4100.2  ± 2.2131.7  ± 1.4121.1  ± 0.39.5  ± 0.06.5  ± 0.1
           Stigmasterol132.0  ± 0.0132.5  ± 1.3144.9  ± 0.3137.8  ± 1.10.8  ± 0.00.5  ± 0.0
           Sitostanol3.3  ± 0.03.2  ± 0.04.7  ± 0.24.4  ± 0.0ND3-a ND
           Cholesterol7.2  ± 0.18.4  ± 0.116.9  ± 0.213.9  ± 0.17.7  ± 0.03.4  ± 0.0
          Total sterols (μg g−1 fresh wt)315.6316.6372.3353.3375.6205.0
          Total sterols (μg per segment) 56.9 69.6 70.8 77.7 71.4 55.5

          Third leaves of 8-d-old seedlings (n = 20) were treated with brassinolide. After 2 d growth data and sterol levels were determined. The levels of sterols were determined by GC-SIM using [2H6]campestanol as an internal standard. Sterol levels are expressed as means ± se of duplicate determinations.

            • ↵F3-a ND, Not detected.

            • View popup
            Table V.

            Subcellular distribution of sterols in 23-d-old WT, lka, and lkb seedlings of pea

            SterolSterol Composition
            Microsomal fractionSoluble fractionWhole shoot
            WTlkalkbWTlkalkbWTlkalkb
            % wt of total sterols
            24-MethylenecholesterolND4-a ND3NDNDNDNDND9
            Campesterol/24-epicampesterol910411111111121
            Campestanol/24-epicampestanol110.51520.50.50.1
            Isofucosterol8764NDND2141088
            Sitosterol52522573747338381
            Stigmasterol24250.511234370.4
            Sitostanol33111122ND
            Cholesterol32213890.50.50.5

            Sterols were quantified by GC-SIM using [2H6]campestanol as an internal standard. Data determined in duplicates were averaged and used to calculate sterol compositions.

              • ↵F4-a ND, Not detected.

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            Brassinosteroid/Sterol Synthesis and Plant Growth as Affected bylka and lkb Mutations of Pea
            Takahito Nomura, Yukiko Kitasaka, Suguru Takatsuto, James B. Reid, Motohiro Fukami, Takao Yokota
            Plant Physiology Apr 1999, 119 (4) 1517-1526; DOI: 10.1104/pp.119.4.1517

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            Brassinosteroid/Sterol Synthesis and Plant Growth as Affected bylka and lkb Mutations of Pea
            Takahito Nomura, Yukiko Kitasaka, Suguru Takatsuto, James B. Reid, Motohiro Fukami, Takao Yokota
            Plant Physiology Apr 1999, 119 (4) 1517-1526; DOI: 10.1104/pp.119.4.1517
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            Apr 1999
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