Hormonal diterpenoids derived from ent-kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens

Abstract

Gibberellins (GAs) are diterpenoid hormones that regulate growth and development in flowering plants. The moss Physcomitrella patens has part of the GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid via ent-kaurene, but it does not produce GA. Disruption of the ent-kaurene synthase gene in P. patens suppressed caulonemal differentiation. Application of ent-kaurene or ent-kaurenoic acid restored differentiation, suggesting that derivative(s) of ent-kaurenoic acid, but not GAs, are endogenous regulator(s) of caulonemal cell differentiation. The protonemal growth of P. patens shows an avoidance response under unilateral blue light. Physiological studies using gene mutants involved in ent-kaurene biosynthesis confirmed that diterpenoid(s) regulate the blue-light response. Here, we discuss the implications of these findings, and provide data for the ent-kaurene oxidase gene-disrupted mutant.

Keywords: CPS, ent-copalyl diphosphate synthase; CYP701B1; GA, gibberellin; KAO, ent-kaurenoic acid oxidase; KO, ent-kaurene oxidase; KS, ent-kaurene synthase; P450, cytochrome P450 monooxygenase; Physcomitrella patens; blue light; cytochrome P450 monooxygenase; ent-kaurene; ent-kaurenoic acid; gene disruption; gibberellin; nptII, neomycin phosphotransferase II; protonema.

Figure 1.

The biosynthetic pathway of diterpenoid regulator(s) and physiological responses under light sources in P. patens. (A), In P. patens, novel diterpenoid (GA-like) regulator(s) synthesized from ent-kaurenoic acid. PpCPS/KS and PpKO (CYP701B1) are involved in ent-kaurene and ent-kaurenoic acid biosynthesis, respectively. Black arrow shows the GA biosynthetic pathway in flowering plant. White arrow shows the GA-like biosynthetic pathway in Physcomitrella patens. (B), GA-like regulator(s) from ent-kaurenoic acid regulates the differentiation to caulonemal cells under red light and the avoidance response of protonemal cells to blue light. Mutants show the suppression of caulonemal differentiation under red light and that of avoidance response under unilateral blue light. These phenotypes are restored by application of ent-kaurenoic acid.

Figure 2.

Generation of CYP701B1 mutant. (A), Structure of genomic locus of CYP701B1 and disruption constructs. White boxes represent exons; the line between the boxes represents an intron. Arrows show the primers used for PCR in (B). (B), Insertion of nptII in the correct region of CYP701B1. (C), The open reading frame of CYP701B1 was amplified from cDNA by PCR.

Figure 3.

Photomorphogenesis of protonema in the wild type and mutant of P. patens under red-light irradiation. (A), Ppcyp701b1 mutant. (B), Ppcps/ks mutant. (C), Wild type. (D), Ppcyp701b1 mutant with application of 1 μM ent-kaurene. (E), Ppcps/ks mutant with application of 1 μM ent-kaurene. (F), Ppcyp701b1 mutant with application of 1 μM ent-kaurenoic acid. (G), Ppcps/ks mutant with application of 1 μM ent-kaurenoic acid. Bars = 1 mm.

Figure 4.

Photomorphogenesis of protonema in the wild type and mutant of P. patens under unilateral blue-light irradiation. (A), Wild type. (B), Ppcps/ks mutant. (C), Ppcyp701b1 mutant. (D), Ppcyp701b1 mutant with application of 1 μM ent-kaurene. (E), Ppcyp701b1 mutant with application of 1 μM ent-kaurenoic acid. Bars = 5 mm.