Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins

Abstract

The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.

Figure 1.

GA biosynthesis in Arabidopsis. Solid arrows, Metabolic steps in GA biosynthesis. Dashed-line arrows, Inactivation steps. Bold text, Enzymes catalyzing these reactions. Italics, Arabidopsis gene names. T bar, Inhibition of the indicated enzyme. Boxed structures, Bioactive GAs. Brackets, Multiple steps catalyzed by one enzyme. The conversion of GA₁₂ to GA₅₃ is catalyzed by GA 13-oxidase, which has not been cloned in Arabidopsis. CDP, ent-Copalyl diphosphate. GA 20-oxidase catalyzes multiple steps, from GA₁₂ to GA₉ and GA₅₃ to GA₂₀. PAC, paclobutrazol.

Figure 2.

CPS and KS protein levels in overexpression lines. A, CPS levels in WT and CPS OE plants. B, CPS levels in CPS OE and CPS/KS OE plants. C, KS levels in WT, KS OE, and CPS/KS OE plants. Immunoblots show 30 (A and C) or 35 (B) μg of total protein extract per lane. Blots were probed with anti-CPS (A and B) or anti-KS (C) antisera. The 76-kD CPS protein is indicated by the arrow in A and B and is not detectable in WT. The upper band in C (indicated by the arrow) is the 88-kD KS protein, not visible in the ga2-1 mutant lane. ga2-1 protein is approximately 74 kD, similar in size to the lower band in C that represents a nonspecific background protein.

Figure 3.

Increased resistance of CPS/KS OE lines to PAC. A, Hypocotyl length of 6-d-old etiolated seedlings as an average of 10 seedlings ± se. WT and KS OE-a plants have reduced germination at PAC concentrations higher than 0.2 μm. B, Flowering time was determined by the appearance of the first flower bud. Values are indicated as ± se for the average of 20 plants per line. Plants were grown in long days (LDs) on Murashige and Skoog plates (–PAC, red bars) or Murashige and Skoog plus 1 μm PAC (+PAC, blue bars). Experiments in A and B have been repeated once each with similar results. C, Phenotypes of 26-d-old rosette plants representative of each line from the experiment described in B. Relative ent-kaurene levels in these lines are indicated below the photographs. Although the influorescence on PAC-treated CPS OE-a and CPS/KS OE-a is not clearly visible in this photograph, these plants have flower buds.

Figure 4.

Expression of downstream GA biosynthetic genes in CPS and/or KS overexpression lines. A, AtKAO1 mRNA levels in OE lines, relative to WT. Lines are as follows: 1, WT; 2, CPS OE-a; 3, CPS OE-b; 4, KS OE-a; 5, CPS/KS OE-a; and 6, CPS/KS OE-b. B, AtKAO2 mRNA levels in OE lines, relative to WT. Lines are designated as in A. For both A and B, numbers for WT, CPS OEa, and CPS OEb represent the average of duplicate qPCR reactions for each of three separate batches of tissue. Numbers for KS OE-a and CPS/KS OE lines represent the average of three qPCR reactions from a single batch of tissue. Bars = se. Total RNA was extracted from rosette tissue, and the amount of RNA in each reaction was determined by normalization to the housekeeping gene Act11. C, Autoradiogram of RNA blot containing 1 μg of poly(A⁺) RNA per lane. The blot was probed with a radiolabeled antisense AtGA20ox1 RNA probe (top) and then re probed with a random prime-labeled cyclophilin cDNA probe (bottom). Numbers below the top blot represent relative amounts of message based on loading control. WT was arbitrarily set to 1.0.