Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants

Abstract

Arabinogalactan proteins (AGPs) are structurally complex plasma membrane and cell wall proteoglycans that are implicated in diverse developmental processes, including plant sexual reproduction. Male gametogenesis (pollen grain development) is fundamental to plant sexual reproduction. The role of two abundant, pollen-specific AGPs, AGP6, and AGP11, have been investigated here. The pollen specificity of these proteoglycans suggested that they are integral to pollen biogenesis and their strong sequence homology indicated a potential for overlapping function. Indeed, single gene transposon insertion knockouts for both AGPs showed no discernible phenotype. However, in plants homozygous for one of the insertions and heterozygous for the other, in homozygous double mutants, and in RNAi and amiRNA transgenic plants that were down-regulated for both genes, many pollen grains failed to develop normally, leading to their collapse. The microscopic observations of these aborted pollen grains showed a condensed cytoplasm, membrane blebbing and the presence of small lytic vacuoles. Later in development, the generative cells that arise from mitotic divisions were not seen to go into the second mitosis. Anther wall development, the establishment of the endothecium thickenings, the opening of the stomium, and the deposition of the pollen coat were all normal in the knockout and knockdown lines. Our data provide strong evidence that these two proteoglycans have overlapping and important functions in gametophytic pollen grain development.

Arabinogalactan Proteins in Arabidopsis thaliana Pollen Development

“…Ds transposon insertion mutant lines for these two genes were available from RIKEN BioResource Center. However, as with so many other plant single gene null mutants, these did not produce recognizable phenotypes (Coimbra et al, 2009). At least in the case of AGP6 and AGP11, a double agp6 agp11 null mutant did produce identifiable phenotypic traits which could be ascribed to the simultaneous lack of both gene products.…”

“…This approach has been used to confirm and characterize the developmental expression of two pollen-specific Arabidopsis classical AGPs (AGP6 and AGP11), and one pollen-specific FLA (FLA3), using either GFP, GUS, or the red fluorescent www.intechopen.com protein RFP as reporter genes (Coimbra et al, 2008(Coimbra et al, , 2009Levitin et al, 2008;Li et al, 2010). The temporal expression of AGP6 was determined with great accuracy in Arabidopsis plants that were transformed with a ProAGP6::GFP gene construct.…”

“…6). The collapsed pollen phenotype of agp6 agp11 was characterized both by scanning electron microscopy and by transmission electron microscopy of pollen grains that clearly showed the degeneration of pollen contents (Coimbra et al, 2009). …”

“…In this case, it is possible that the genes sharing the same function are not those with higher identity. Therefore, OsAGP7 and OsAGP10 may play a conserved role in pollen development, like AtAGP6 and AtAGP11 which redundantly control pollen development and fertility (Coimbra et al, 2009(Coimbra et al, , 2010Levitin et al, 2008).…”

Pipeline to Identify Hydroxyproline-Rich Glycoproteins

“…For the 16 Arabidopsis GPI-AGPs, there are some robust groupings (greater than 70% bootstrap values), but only between pairs of sequences. For example, the pollen-specific AtAGP6 and AtAGP11 (Pereira et al, 2006;Coimbra et al, 2009Coimbra et al, , 2010 A, Protein disorder (PONDR) plots (see "Materials and Methods") for AtAGP6 (At5g14380; left), AtEXT3 (At1g21310; middle), and prolyl-4-hydroxylase (AtP4H1; At2g43080; right) using VL-XT (red), VL3 (green), and VSL2 (blue). PONDR prediction scores above the threshold line (0.5) predict disorder; below the line, they predict order.…”