Department of Botany, University of British Columbia, Vancouver, BC, Canada
The patterned pollen cell wall contains sporopollenin, a resistant biopolymer that protects the microspores. Sporopollenin precursors are made in the tapetum of the anther during a narrow developmental window of pollen production, but its composition remains elusive. In order to examine intact live anthers during pollen development, we used two-photon microscopy to visualize intrinsic fluorescent properties of metabolites contributing to sporopollenin production. This approach was used in conjunction with Arabidopsis molecular genetics to test hypotheses of transport and the metabolite origins of sporopollenin. Two-photon microscopy of intact anthers revealed intrinsic autofluorescent compounds that are found on the surface of wild-type pollen, but in the tapetum of an ABC transporter (AtABCG26) mutant. A series of double mutant anthers from plants defective in both ABCG26 and enzymes of the polyketide and hydroxycinnamoyl spermidine biosynthetic pathways suggest that polyketide precursors of sporopollenin are transported by ABCG26, while hydroxycinnamoyl spermidines are transported independent of ABCG26 but prior to programmed cell death of the tapetum. The phenotyping of mutants by precise metabolite localization in tapetum and microspore cells indicates that multiple pathways contribute to pollen wall production.