CYP704B1 Is a Long-Chain Fatty Acidω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis    

Abstract

Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.

The Gibberellin perception system evolved to regulate a pre-existing GAMYB-mediated system during land plant evolution

“…The genomic fragment containing the 3′-flanking region of each PpGAMYB gene was inserted into the 3′-region of the NPTII expression cassette of this plasmid. To transform these plasmids into P. patens, polyethylene glycol-mediated transformation was performed essentially as described 30 , using 10 µg of linearized plasmid. Stable transformants were screened by Southern hybridization using a probe that detected the homologous region of the transgene.…”

“…The collapsed spores observed in PpGAMYB-KOs suggest that the structural and physical protection against the vacuum condition of SEM analysis might be reduced in PpGAMYB-KOs spores, compared with WT spores. We next treated spores with a solution of acetic anhydride and sulphuric acid, which generally disrupts all spore/pollen content except for the exospore/exine by hydrolysis and esterification (acetolysis) 30,31 . The spores in PpGAMYB-KOs, especially in the PpGA-MYB2-KO, were easily damaged by acetolysis treatment (percentage of acetolysis-sensitive spores in PpGAMYB1-KO, 23.32 ± 2.68%; PpGAMYB2-KO, 60.33 ± 8.97%; and WT, 14.63 ± 3.21%; Fig.…”

Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis

“…Pollen grains from the other genotypes were examined by fluorescence microscopy after staining with auramine O, a fluorescent dye that reveals exine patterns (Dobritsa et al, 2009a(Dobritsa et al, , 2009b. Fluorescence of the few tkpr1-2 mutant pollen grains that were produced ( Figure 6B) was strongly attenuated compared with the wild-type pattern ( Figure 6A), whereas only subtle alterations were seen in the exine patterns of tkpr2-1 pollen whose overall fluorescence intensity was slightly reduced ( Figure 6C).…”

“…Because the role of CYP450 hydroxylases in modification of fatty acid sporopollenin precursors has been demonstrated previously (Morant et al, 2007;Dobritsa et al, 2009b), we tested the ability of TKPR to reduce hydroxylated tetraketide a-pyrone compounds. These were generated by first incubating 16-OH palmitic or 12-OH stearic acids with ACOS5, ATP, and CoA.…”

“…ACOS5, originally annotated as a 4-coumarate:CoA ligase-like protein, displays fatty acyl-CoA synthetase activity with medium-to long-chain fatty acids (C8-C18 fatty acids) (de Azevedo Souza et al, 2009), and DEX2 encodes CYP703A2, which has been shown to in-chain hydroxylate lauric acid (C12:0 fatty acid) (Morant et al, 2007). Recently, CYP704B1 has been demonstrated also to be implicated in exine formation and to catalyze the v-hydroxylation of long-chain fatty acids (Dobritsa et al, 2009b). These studies unequivocally demonstrate that fatty acids are essential precursors of sporopollenin.…”