A Novel Fatty Acyl-CoA Synthetase Is Required for Pollen Development and Sporopollenin Biosynthesis inArabidopsis   

Abstract

Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by selffertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein-ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes.

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

“…botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi) (Winter et al, 2007), to determine the tissue expression patterns of At4g35420 (DRL1) and At1g68540 (CCRL6). The results of this search indicated that both were preferentially expressed in young flower buds, in accordance with their high coregulation scores, similar to the expression patterns reported for PKSA, PKSB, and ACOS5 (de Azevedo Souza et al, 2009;Kim et al, 2010). To facilitate their designation in the following paragraphs, genes corresponding to At4g35420 (DRL1) and At1g68540 (CCRL6) were named TET-RAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2, respectively, in anticipation of the enzymatic activities described below for the two corresponding proteins.…”

“…Therefore, considering the fact that these two putative oxidoreductase genes are tightly coexpressed with ACOS5 and the polyketide synthases PKSA and PKSB in tapetal cells during pollen development (Figures 1 and 2) (de Azevedo Souza et al, 2009;Kim et al, 2010), we examined the possibility that these enzyme activities could function in a common biochemical pathway. Particularly, the tri-and tetraketide compounds produced No amplicon was detectable in tkpr1-1, tkpr1-2, and tkpr2-1 samples, whereas a weak signal was recorded in tkpr2-2 buds, using primers shown in Supplemental Table 1 online.…”

“…Significant progress in our understanding of exine formation processes has been made recently, thanks to genetic and molecular studies of Arabidopsis mutants exhibiting defects in exine structure and deposition (Aarts et al, 1997;PaxsonSowders et al, 2001;Ariizumi et al, 2003Ariizumi et al, , 2004Ito et al, 2007;Morant et al, 2007;Yang et al, 2007;Guan et al, 2008;Suzuki et al, 2008;de Azevedo Souza et al, 2009;Dobritsa et al, 2009a;Tang et al, 2009). Unfortunately, in most cases, annotations of the mutated Arabidopsis genes found to be responsible for pollen phenotypes are primarily based on sequence similarity, and the exact functions of the corresponding proteins in pollen development remain unknown.…”

“…Recently acos5, a male-sterile mutant, and dex2, a malesemisterile mutant, have both been shown to be impaired in exine formation (Morant et al, 2007;de Azevedo Souza et al, 2009). For both ACYL-COA SYNTHETASE5 (ACOS5) and DEX2, the catalytic activities of the encoded proteins were characterized biochemically, providing evidence for specific roles in sporopollenin biosynthesis.…”

“…For both ACYL-COA SYNTHETASE5 (ACOS5) and DEX2, the catalytic activities of the encoded proteins were characterized biochemically, providing evidence for specific roles in sporopollenin biosynthesis. 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).…”