Isolation and regulation of genes expressed during Douglas-fir germination and post-germination




Tranbarger, Timothy John

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To identify genes expressed during Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) germination and early seedling development, a cDNA library was constructed with mRNA pooled from 4-6-day-old seedlings. The library was then screened differentially with cDNA probes synthesized using mRNA isolated from mature seeds and 6-day-old seedlings. Partial DNA sequence analysis and predicted amino acid sequences revealed cDNA clones that encoded polypeptides with similarity to several plant proteins including: a chaperonin 606 (cpn60β), a low molecular weight heat shock protein (LMW HSP), a luminal binding protein (BiP), a type II chlorophyll a/b-binding protein (CAB), and a cysteine protease (CysP). In northern blots, each cDNA clone detected transcripts that increased during seed germination. A clone detected RNA at similar levels in both mature seeds and in 6-day-old seedlings was isolated and found to share similarity to a NADPH-cytochrome P450 reductase (CPR) (EC The cDNA clones encoding the CysP and the CPR were selected for further sequence and gene expression analysis. The CysP cDNA consists of a 5’ untranslated region (UTR) of 153 bp followed by an open reading frame (ORF) of 1362 bp encoding a putative mature CysP flanked by N- and C-terminal propeptides. A 364 bp 3’ UTR contains multiple putative AU-rich elements that may be involved in the destabilization of transcripts. The CysP from Douglas-fir (pseudotzain) contains the same invariant amino acid residues that are involved in the catalytic reaction and make up the catalytic center of CysP from other plants and animals. Pseudotzain transcripts were most abundant in the megagametophyte (MG) after germination and were not detected in the MG or embryo during embryogenesis. Various osmotic stresses slightly enhanced pseudotzain transcript quantities during early seedling development, whereas abscisic acid, gibberellic acid and other plant growth regulators and changes in environmental conditions had little or no effect. Pseudotzain transcripts were present in different amounts in the cotyledons, root and seed coat of 10-day-old seedlings, but were most abundant in the MG, suggesting a role for this protease in storage protein mobilization. Phylogenetic analysis of mature pseudotzain groups it with other angiosperm CysP having both N- and C-terminal propeptides, suggesting a conserved function and/or targeting of this subgroup of enzymes. The CPR cDNA encodes a polypeptide of approximately 79.6 kDa. A cDNA probe detected a single transcript of 3 kb that was expressed differentially in cotyledons, radicle and MG. CPR transcript quantities were low during seed maturation, higher in mature seeds, and remained constant throughout germination and early seedling development before they declined in 14-day-old seedlings. An antiserum against a synthetic CPR-peptide was produced and western blot analysis detected a single 80 kDa polypeptide in the membrane fraction of microsomal extracts from seeds and seedlings. CPR accumulation during germination and early seedling development indicated regulation is at the transcriptional or post-transcriptional level. However, CPR activity (measured by NADPH-cytochrome-c reduction) present in the microsomes increases during stratification, germination and post-germination and decreases in 7-14-day-old seedlings. These results indicate CPR may be post-translationally activated during Douglas-fir stratification and germination. This study describes the isolation of the first cDNAs that share identity with a CysP, cpn60β, a LMW HSP, BiP and CPR (EC from a gymnosperm. The developmental expression of these cDNAs suggests that their gene products play critical roles during the process of germination and post-germination and provides the necessary framework for future studies.



Douglas fir