Post-pollination development


The post-pollination or progamic phase of pollen development encompasses events from Growing Pollen Tubespollination to fertilisation. To identify essential male progamic phase genes we developed a genetic screen in the model species Arabidopsis thaliana based on segregation ratio distortion of an insertion-linked resistance marker. Our rationale was that if DNA insertions inactivate essential male gametophytic genes, then the ratio of resistant to sensitive seedlings among self progeny would deviate significantly below the expected 3:1 ratio [Howden et al., 1998]. Progamic phase mutants that block male-transmission but produce morphologically normal pollen can then be selected by cytological analysis and reciprocal crossing to determine gametophytic transmission.
We have screened both T-DNA and transposon insertion populations and found that the proportion of lines showing segregation ratio distortion is similar at about 1 % [Lalanne et al ., 2004]. In collaboration with Prof. Ueli Grossniklaus we screened ~3,500 Ds transposon insertion lines and isolated 20 independent gametophytic mutants. Male-specific mutants were named seth and those with reduced transmission through both sexes were named ungud . We identified 10 seth and 5 ungud mutants that act during male progamic development. Gene sequences disrupted by Ds insertions encode proteins with diverse functions including protein anchoring (SETH1, SETH2), cell wall biosynthesis (SETH3), calcium sensing, signaling (SETH6, SETH7 and UNG6) and metabolism (SETH9, UNG10) [Lalanne et al., 2004].

seth.jpgSeth (left): brother and murderer of Osiris, god of fertility in Egyptian mythology.ungud.jpg

 

Ungud (right): hermaphrodite snake god in the mythology of the Aboriginals in north-western Australia .

 

 

Some mutants affecting pollen germination and tube growth are being characterised in more detail (see below) . These and other mutants, which could act later, during pollen tube guidance or fertilisation will facilitate new avenues of research concerning cellular functions during this key phase of pollen development.
Mutant seth1 pollen fails to germinate or produces pollen tubes with abnormal callose deposition.

SETH1 and SETH2 encode conserved proteins involved in the phosphotidylinositol-glycan (GPI) synthase complex, the first step of the GPI biosynthetic pathway. Reduced pollen seth1.jpggermination and tube growth in seth1 and seth2 is associated with abnormal callose deposition demonstrating an essential role for GPI-anchor biosynthesis in pollen tube wall deposition or metabolism. Our results suggest an essential role for GPI-anchoring of proteins in the establishment and maintenance of polarized pollen tube growth. Using transcriptomic and proteomic approaches in collaboration with Dr David Honys and Dr Paul Dupree we identified 47 genes encoding potential GPI-anchored proteins expressed in pollen and demonstrated that at least 11 of these are GPI-anchored. Many of these show homology to proteins involved in cell wall synthesis and remodelling or intercellular signalling and adhesion, and are likely to play important roles in the establishment and maintenance of polarised pollen tube growth [see Lalanne et al., 2004 for further details] .

SETH4 defines a small protein family in Arabidopsis that contains Armadillo (ARM) repeats as predicted protein-protein interactions domains. seth4 mutations completely block WTseth4.jpgmale transmission and pollen grains appear to be normally polarised but are unable to initiate pollen tube growth. Two proteins named SETH-FOUR-LIKE (SFL1 and SFL2) exhibit strong homology with SETH4. In contrast to SETH4 that is strongly expressed only in some floral organs and mature pollen, SFL1 and SFL2 genes show complementary patterns of expression and are only expressed in sporophytic tissues. We are currently investigating the cellular role(s) and protein interactions of SETH4 and SFL proteins in Arabidopsis.

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