Developmental Staging of Maize Microspores Reveals a Transition in Developing Microspore Proteins

Abstract

A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady-state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period.acious remnants of the tapetal cells are deposited on the surface of the pollen grain, which desiccates and is shed from the anther upon dehiscence.The study of pollen development provides an opportunity to examine the gametophyte generation of higher plants. While recent studies have suggested that multiple molecular events are involved in the progression of microspores through the pollen developmental pathway (4,(23)(24)(25), the scope of these events was unknown. The work presented here provides a method for the preparation of developmentally staged microspores and young pollen. These preparations are of sufficient purity and quantity to allow the characterization of protein and RNA populations associated with each stage. A major transition in steady-state protein populations has been observed between the large vacuole and starch-filling stages of microsporogenesis. MATERIALS AND METHODS MaterialsMicrosporogenesis in higher plants has been well described at the genetic and cytological levels (1,2,9,11,20). The maize microspore developmental pathway can be briefly summarized as follows (1,3,6,21)

Developmentally regulated loss of ubiquitin and ubiquitinated proteins during pollen maturation in maize.

“…In keeping with this idea is the finding that, although there is a heat shock response in higher plant somatic and embryonic tissues, pollen is distinctive in the lack of induction of heat shock protein mRNA levels and synthesis of heat shock proteins upon heat treatment (39). The timing of the loss of both ubiquitin and the heat shock response correlates with the event of microspore mitosis, which is thought to mark commitment to the gametophytic developmental program (16,40). Pollen, which survives only for a short burst of pollen tube growth after release from the anthers, may not require tight regulation of protein turnover or of protein stabilization and therefore has eliminated both systems from its physiological repertoire.…”

“…As pollen matures, it accumulates storage substances and proteins necessary for the rapid growth of a pollen tube that delivers the gametes to the embryo sac for fertilization. Some of the proteins accumulating to high levels at this time are cytoskeletal proteins such as actin (16) and tubulin (39). These normally filamentous proteins are stored in a nonfilamentous form, ready to be rapidly polymerized upon tube germination.…”

“…This division is thought to signal commitment of microspores to gametophytic development (14). Previous studies have shown that the RNA and protein populations of the pollen undergo a substantial alteration at this time (15,16 (16). Mature pollen grains were collected from shedding tassels.…”

The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography

“…We have used the DNase I affinity chromatography procedure of Zechel (30) (4). Maize pollen that we have collected is relatively free of ATPases and phenolics and does not contain large acidic vacuoles.…”

The making of gametes in higher plants

“…Quantitative estimates of the amount of gene expression in the gametophyte generation and of the extent of gametophyte-sporophytic overlap were described (Stinson et al, 1987;Hanson et al, 1989;Brown and Crouch, 1990;Theerakulpisut et al, 1991; Weterings et al, 1992a). In maize, a major switch of gene expression after microspore mitosis was demonstrated by differences on the mRNA populations isolated from different developmental stages (Bedinger and Edgerton, 1990). Mandaron et al (1990) used in vivo labelling and twodimensional gel electrophoresis to show that protein synthesis was extremely active from tetrad stage to the vacuolated stage of pollen development, stopped for a short period during starch accumulation and rapidly increased just before anther dehiscence, indicating that presumably these proteins were required for pollen germination and tube growth.…”