No abstract

In angiosperms, the formation of the flower meristem is controlled by partially redundant flower meristem identity genes of which FLORICAULA (FLO)/LEAFY (LFY) plays a central role. It is not known whether formation of reproductive organs of pre-angiosperm species is similarly regulated. Recently, a FLO/LFY-like cDNA, NEEDLY (NLY), has been cloned in a conifer species Pinus radiata (D. Don). Here we report cloning of a different Pinus radiata FLO/LFY-like cDNA, PRFLL. PRFLL had two large regions of high similarity to angiosperm FLO/LFY orthologues: amino acids 61-126 and 247-406 (50% and 81% identity, and 75% and 88% similarity, respectively, to LFY) and shorter regions of local similarity. Overall identity was 53% to LFY and 61% to NLY. Phylogenetic analysis of deduced protein sequences including partial LFY-like sequences from Pseudotsuga menziesii indicated that conifer proteins constituted a separate clade that could be divided into two groups represented by NLY and PRFLL. In contrast to angiosperms, both conifers had two paralogous proteins resembling LFY. Northern hybridisation analysis revealed expression of PRFLL in vegetative buds of juvenile, adolescent and mature trees. The transcript was not detected in vascular cambium, roots or secondary needles. To follow PRFLL expression during the early stages of cone development we analysed a temporal series of buds containing cone primordia, and developing cones, using Northern hybridisation and confocal microscopy in parallel. PRFLL mRNA was detected in buds from dominant and subordinate branches, in which cone and shoot primordia develop, and in developing male cones but not in developing female cones. Expression was particularly high in buds containing axillary primordia prior to their differentiation as male cone primordia. This is consistent with PRFLL being involved in determination of the male cone primordium identity.

We describe the isolation and characterization of 13 cDNA clones that are differentially expressed in male cones of Pinus radiata (D. Don). The transcripts of the 13 genes are expressed at different times between meiosis and microspore mitosis, timing that corresponds to a burst in tapetal activity in the developing anthers. In situ hybridization showed that four of the genes are expressed in the tapetum, while a fifth is expressed in tetrads during a brief developmental window. Six of the seven cDNAs identified in database searches have striking similarity to genes expressed in angiosperm anthers. Seven cDNAs are homologs of defense and pathogen response genes. The cDNAs identified are predicted to encode a chalcone-synthase-like protein, a thaumatin-like protein, a serine hydrolase thought to be a putative regulator of programmed cell death, two lipid-transfer proteins, and two homologs of the antherspecific A9 genes from Brassica napus and Arabidopsis. Overall, our results support the hypothesis that many of the reproductive processes in the angiosperms and gymnosperms were inherited from a common ancestor.

Recombinant exo‐β‐(1‐3)‐glucanase from Candida albicans was expressed in Saccharomyces cerevisiae and purified. The enzyme contains a number of short blocks of sequence homology with several genes for cellulases of the family A glycanases including the conserved sequence motif NEP which has previously been shown to be important in the catalytic function of several cellulases. Site directed mutagenesis of this glutamic acid residue in the 1,3 glucanase (E230D, E230Q) decreased the enzymatic activity 15,000‐ and 400‐fold, respectively. This suggests that the E of the NEP participates in catalysis of the exoglucanase and other related glycanases.