Characterization and expression of the Douglas-fir luminal binding protein
Date
2018-06-20
Authors
Forward, Benjamin Spencer
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Abstract
The endoplasmic reticulum (ER) molecular chaperone, BiP, plays a role in the
translocation and subsequent folding and assembly of newly synthesized
proteins targeted to the ER and secretory pathway. The sequence encoding a
Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) BiP homologue (PmBiP) was
identified by differential screening of a seedling cDNA library. Southern
blotting indicated that PmBiP is most likely present as a single copy although
other BiP alleles likely exist within a given seedlot. The deduced amino acid
sequence of PmBiP contains a HEEL tetrapeptide sequence which functions to
retain PmBiP in the ER and is different from HDEL commonly found in
angiosperm plant BiPs. Amino acid sequence alignment and phylogenetic
analysis show that PmBiP is highly similar to other plant BiPs yet forms a
distinct phylogenetic subgroup separate from angiosperm BiPs. Northern and
western blotting revealed that PmBiP is subject to developmental regulation
during seed development, germination, and early seedling growth and is
seasonally regulated in needles of young seedlings. The expression of PmBiP is
developmentally regulated during seed development with higher amounts
present in seeds prior to embryo development and the deposition of storage
proteins. Increased PmBiP expression correlates with seedling growth and
developm ent and the mobilization of seed storage proteins. Increased
synthesis during germination is likely due to increased synthesis of cell wall
proteins and other secretory traffic. This idea is supported by
immunolocalization of PmBiP in root tip cells showing staining around the new
cell wall in telophase cells and at the periphery of cells in the elongation zone. PmBiP may also play a role in mediating homotypic ER and nuclear envelope membrane fusion during mitosis in actively dividing tissues. PmBiP is
seasonally regulated in the needles of young seedlings and increased
expression was observed in tissues treated with low temperature suggesting
that PmBiP plays an important role in the adaptation of seedlings to low
temperatures. This is most likely accomplished through the maintenance of
secretory traffic through the ER necessary for the synthesis of proteins with a
more direct role in cold acclimation. Proteins were associated with PmBiP in an
ATP dependent manner in mature seeds and 2-day-old seedlings but were only
detectable in minute amounts. ATP associated proteins were more readily
detectable in embryonal suspensor mass (ESM) cultures but only in small
amounts unsuitable for N-terminal sequencing and identification.
The Douglas-fir BiP promoter (PmBiPProl) contains a variety of cis-acting
regulatory elements commonly found in the promoters of storage protein
genes, light regulated genes, and phenlypropanoid and cell wall protein genes.
The presence of different cis-element groups suggests the transcriptional
regulation of PmBiP is controlled by a variety of signal transduction pathways
depending upon the developmental and/or physiological state of a given
tissue.
Transient expression analysis showed that PmBiPProl is functional in germinating Douglas-fir embryos. The expression of PmBiPProl in transgenic
Arabidopsis is associated with actively dividing and secretory tissues. Deletion
analysis showed that minimal promoter elements lie within a 263 bp region
directly upstream of the PmBiP cDNA sequence although upstream flanking
sequences are necessary for higher level expression. G-box motifs residing within the 263 bp fragment together with a quantitative activator region (QAR)
and a negative regulatory region (NRR) present in upstream areas are likely
involved in transcriptional control in young seedlings. PmBiPProl was also
wound inducible in transgenic Arabidopsis cotyledons that correlated with
similar experiments conducted in Douglas-fir seedlings. Elements involved in
conferring wound inducibility are located in PmBiPProl-5 but upstream
elements are necessary for higher level expression. G-box motifs may also play
a role in the wound inducibility of the Douglas-fir BiP promoter.
Description
Keywords
Douglas fir, Seedlings