The role of callose and beta(1,3)-glucanase in microsporogenesis.
2015-11-19T09:10:14Z (GMT) by
The significance of the tetrad callose wall and its hydrolysis at microspore release has been investigated in Brassica napus. Callose wall breakdown was analyzed by measuring activities of beta(1,3)-glucanase enzymes in relation to different stages of anther development. Assaying B. napus buds for endolytic and exolytic beta(1,3)-glucanases established that activity peaked at the time of callose dissolution. Initial experiments to clone genes encoding these enzymes involved the use of probes related to PR beta(1,3)-glucanases. Sequencing of an anther-specific cDNA, A6, which was isolated from a B. napus 'sporogenesis' library, revealed similarity to previously characterized endo-beta(1,3)- and beta(1,3;1,4)-glucanases. Polyclonal antibody raised to the A6 protein identified a temporally-regulated ~60 kD band in B. napus buds which attained maximum levels at microspore release stage. Further analysis revealed that this ~60 kD band represented several proteins of different charge but similar molecular weight. A6 thus appears to be part of a family of immunologically related proteins. The sequence data and the temporal expression pattern suggest that A6 may be part of the callase enzyme complex involved in microspore release. The role of the callose wall was investigated by engineering its premature removal in vivo. This was achieved by expressing a modified PR beta(1,3)-glucanase in the anther tapetum of transgenic Nicotiana tabacum. These transgenic plants exhibited reduced male fertility, ranging from partial to complete male sterility. The microspores from the reduced fertility transgenic plants had an abnormal exine and the tapetum demonstrated hypertrophy. Male sterility appeared to result from bursting of the aberrant microspores after microspore release. These results suggested that premature callose hydrolysis is sufficient to cause male sterility and that callose is involved in exine formation in N. tabacum.