DEX1, a novel plant protein, is required for exine pattern formation during pollen development in Arabidopsis

Abstract

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.

Figure 1

General pollen wall structure. Schematic representation of the main features of a mature pollen grain wall. The innermost layer adjacent to the plasma membrane is the intine. The exine is comprised of the sexine and nexine, a continuous layer covering the entire pollen grain. The bacula and tectum make up the sculpted sexine in this representation.

Figure 2

Map of the DEX1 locus and exon patterns. A, Map of a 10-kb region of chromosome 3. The extent of the DEX1 coding region (shaded box) is shown relative to the region used in the complementation experiments (medium black line). Heavy black lines represent regions that hybridized to LB and RB probes. Restriction sites shown are: H, HindIII; E, EcoRI; X, XhoI; and Xb, XbaI. X* is an XhoI site that is derived from λ DNA and was used for cloning the complementation clone. B, Partial restriction map and exon pattern of DEX1. The positions of exons are shown as solid boxes. The position and direction of primers used in this study are shown as horizontal arrows below the map. The position of the T-DNA insertion site is shown as a vertical arrow. Restriction sites are as in A.

Figure 3

DEX1 expression analysis. A, Northern blot of poly(A⁺) RNA, isolated from buds of wild-type and dex1 mutant plants probed with a mixture of the 2.7-kb HindIII/EcoRI fragment 1 and the 3.0-kb SacI fragment. B, Northern blot of total RNA isolated from wild-type buds, leaves, roots, and seedlings, was probed with the partial DEX1 cDNA clone. Equal loadings were determined by subsequent hybridization with an rRNA probe.

Figure 4

DEX1 cDNA and deduced amino acid sequence. The deduced DEX1 amino acid sequence is shown below the cDNA sequence. The 5′ end of the cDNA sequence shown is the longest clone obtained by IPCR. A potential polyadenylation signal is double underlined. The upstream AUG is marked with asterisks.

Figure 5

Transmission electron micrographs of the edges of tetrad stage, high-pressure frozen, and freeze-substituted microspores of Arabidopsis. All micrographs are shown at equal magnifications. Size bar = 0.25 μm. A through D, Developmental series of WT microspores. A, Within the callose wall (C), primexine is first evident as discrete electron-dense deposits (arrowheads) directly outside the microspore (M) membrane. B, Later in development, portions of the microspore plasma membrane display regular undulations (arrowheads), although other portions of the plasma membrane are straight (arrows). The primexine varies in both thickness and electron density at this stage, with the thickest and most electron-dense areas located within the membrane undulations. C, Later in development the primexine matrix has increased in thickness and is evenly distributed along the surface of the straight microspore membrane. Electron-dense deposits of material have formed within the primexine matrix (arrowheads). These deposits appear to be in contact with the wall on the callose-facing surface of the primexine matrix, but are not in contact with the membrane on the microspore-facing surface of the primexine matrix. D, At a later stage, electron-dense material within the primexine matrix is clearly recognizable as the developing exine. Fine fibrillar material (arrows) is evident in the primexine matrix between the probaculae (P), which are not in direct contact with the microspore membrane. The pattern of the future tectum has been established (asterisks) within the callose-facing surface of the primexine. E through H, Developmental series of dex1 microspores. E, As in WT, primexine is first evident as discrete electron-dense deposits (arrowheads) directly outside the microspore (M) membrane within the callose wall (C). F, The primexine has increased slightly in thickness outside a straight microspore membrane. Areas of two different electron densities can be distinguished within the primexine. Regularly spaced along the microspore membrane, the more electron-dense areas (arrowheads) are lens shaped and thicker than the more electron-lucent regions between them. G, Later in development the primexine matrix has increased in thickness on some areas of the microspore, but in other areas is extremely thin (arrow). Electron-dense deposits of varying sizes (arrowheads) have formed within the primexine matrix. H, At a later stage the electron-dense deposits (arrowheads) and primexine that enclose them have become thicker. Linear electron-lucent regions (arrows) are present within some of the deposits.