LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase essential for pollen exine development in Arabidopsis

Abstract

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.

Figure 1.

lap5 and lap6 mutants have defects in anther and pollen exine morphology. A to C, Compared with the wild-type (WT) anthers (A), anthers of lap5-1 (B) and lap6-1 (C) mutants appear glossy and do not easily shed pollen (arrowhead in A). D to I, SEM of pollen grains and exine. Wild-type grains (D and G) have a regular reticulate exine pattern, while lap5-1 (E and H) and lap6-1 (F and I) mutations cause pollen to collapse more easily and disrupt exine, changing the pattern or resulting in a more extensively covered surface. J to L, Similar to the wild type (J), lap5-1 (K) and lap6-1 (L) pollen does not demonstrate sensitivity to acetolysis. The lap6-1 grains, however, exhibit decreased reactivity to acetolysis-dependent staining. Bars = 100 μm (A–C), 10 μm (D–F and J–L), and 5 μm (G–I).

Figure 2.

The lap5 and lap6 defects map to At4g34850 and At1g02050, respectively. LAP5, LAP6, and At4g00040 have gene structures similar to CHS/STS family members, including the conserved position of an intron separating the first and second nucleotides in a Cys codon (TGC). Exons are shown as black rectangles. Positions of the lap5-1 and lap6-1 point mutations and the lap6-2, lap6-3, and At1g00040 insertions are indicated with black triangles.

Figure 3.

LAP5, LAP6, and At1g00040 belong to a male-specific clade of the CHS/STS family. A, Amino acid alignment of protein sequences from LAP5, LAP6, At1g00040, CHS, STS, and CHS/STS-like proteins from several plant species (two-thirds of the protein length is shown; the remainder shows a similar trend). Residues identical to LAP5 are shaded blue; those identical to the Arabidopsis CHS TT4 are shaded yellow; and those that match the majority consensus from 17 sequences are shaded orange. Amino acid numbers correspond to CHS2 from M. sativa. Proteins with demonstrated male-specific expression patterns are marked with asterisks; the lap5-1 and lap6-1 lesions are indicated, and two of the four amino acids critical for active center formation (C164 and F215) are boxed. M137, important for dimer formation, is conserved in the CHS-like enzymes (boxed) but replaced with L/F in enzymes more similar to LAP5. B, A phylogenetic tree demonstrates a clear segregation of the LAP5/LAP6-like proteins in a clade separate from the other members of the CHS superfamily. Red bar, CHS; blue bar, sequences similar to LAP5; green bar, four CHS-like proteins with demonstrated non-CHS activity (2PS, aloesone synthase [ALS], acridone synthase [ACS], and STS). Numerical values indicate bootstrap support for each node. PKS18 from M. tuberculosis was used as an outgroup.

Figure 4.

LAP5 and LAP6 are expressed specifically in developing anthers. LAP5pr::GUS (A–D) and LAP6pr::GUS (E–H) promoter fusion constructs are expressed in anthers of stage 9 and 10 buds (B and F) but not at earlier stages (A and E) or later stages (stage 11 [C and G] and mature anthers [D and H]). All images are to the same magnification. Bar = 100 μm.

Figure 5.

A lap5-1 lap6-1 double mutant is sterile and lacks pollen. A, Left to right: siliques from the wild type, lap5-1, lap6-1, and a lap5-1 lap6-1 double mutant. B, Mature anther from lap5-1 lap6-1 demonstrates complete absence of pollen grains (compare with Fig. 1A for a wild-type anther).

Figure 6.

Pollen produced by a lap5-1 lap6-1 double mutant lacks exine and degenerates. Anther development is shown in the wild-type (WT) Arabidopsis (A–D) and a lap5-1 lap6-1 double mutant (E–H). Anther locules at stages 6 (A and E), 7 (B and F), 9 (C and G), and 11 (D and H) are shown. Exine is deposited around developing pollen grains at stage 9 in the wild type (green halo around the pollen grains; C) but not in lap5 lap6 (G). Insets in C and G are higher magnification views of boxed areas. Little pollen is visible in lap5 lap6 by stage 11 (H). Bars = 20 μm in A to H and 10 μm in the insets.

Figure 7.

Levels of chalcone and naringenin are affected in the lap5 and lap6 mutants. Examples of UPLC-QTOF-MS results obtained from lap5-1, lap6-1, the lap5-1 lap6-1 double mutant, and wild-type (WT) anther extracts are shown.

Figure 8.

HPLC-UV-MS results of the enzymatic assays with 4-coumaroyl-CoA and malonyl-CoA as substrates. A, Empty-vector control. B, TT4/CHS. Insets are UV and MS spectra of the enzymatic reaction product (naringenin). C, Naringenin standard. The insets are the UV and MS spectra. D, LAP5. The insets are closeup views of peaks 1 and 2 and their UV spectra. E, LAP6. The insets are closeup views of peaks 1 and 2 and their UV spectra.

Figure 9.

HPLC-ITMS UV analyses of the products of the enzymatic reactions with octanoyl-CoA and malonyl-CoA as substrates. A to C, UV chromatograms (287 nm) of the products of enzymatic reactions in the presence of empty-vector control (A), LAP5 (B), or TT4/CHS (C). D, Mass spectrum of the major enzymatic product, 4-hydroxy-6-heptyl-2-pyrone, of the reaction catalyzed by LAP5. E, Mass spectrum of the major enzymatic product, 4-hydroxy-6-heptyl-2-pyrone, of the reaction catalyzed by TT4.

Figure 10.

Exine from the lap5-1 and cyp703a2 (lap4-1) double mutant has defects stronger than either single mutant. Confocal images of the exine surface from wild-type (A and E), lap5-1 (B and F), lap4-1 (C and G), and lap4-1 lap5-1 double mutant (D and H) pollen grains stained with auramine O. The arrow in A indicates an aperture; the arrow in D indicates a longitudinal area of increased fluorescence. E and F show optical sections through the middle of pollen grains, allowing visualization of exine thickness. All images are to the same magnification. Bar = 10 μm.