Seasonal Variation of Western White Pine (Pinus monticola D. Don) Foliage Proteins

Recently, a western white pine protein, Pin m III, was shownto be associated with overwintering and frost hardiness of westernwhite pine foliage. To examine whether Pin m III is directlyinvolved in frost hardiness by functioning as an antifreezeprotein, work is underway to clone the gene encoding this proteinand to assess the function of this gene in freezing toleranceby incorporating the gene in a test plant, such as tobacco.Here, we examined in more detail, by SDS-PAGE and also by twodimensional gel electrophoresis, the seasonal variation of additionalproteins in western pine foliage. SDS-PAGE analysis of threeseedlots showed that different proteins reached a maximum levelin different months, although most proteins (5 to 11) reacheda maximum level in winter months (December, January and February).The 2-D gel analysis of foliage sampled on three harvest dates(October, January and April) of one seedlot revealed a seasonalvariation of a large number proteins (76 to 184). Of the seasonallyvaried proteins, the amino terminal sequence of several proteinsincluding Pin m III was determined. One of the sequences wasidentified by homology to that of the small subunit of ribulosebiphosphate carboxylase, whose level increased substantiallyfrom fall to spring. The amino terminal sequence of Pin m IIIhad 89% homology to a sugar pine protein, Pin 11. The anti-photosystemII antibody was used to monitor the annual variation of theextrinsic 23-kDa photosystem II protein. The level of the extrinsic23-kDa photosystem II protein decreased slowly as fall progressedand reached its lowest level in December and then increasedin early spring indicating that this variation is due to photosyntheticactivity of the foliage during the season. (Received July 29, 1995; Accepted March 5, 1996)     

Cloning and characterization of a cDNA of cro rI from the white pine blister rust fungus Cronartium ribicola

White pine blister rust (WPBR) is caused by the fungus Cronartium ribicola which has five spore stages on two unrelated hosts, the five-needle pines and Ribes spp. Recently, during the molecular analysis of the proteins and genes involved in host-pathogen interaction, the WPBR fungal protein Cro rI was identified in infected white pine tissues. To further characterize Cro rI, an expression cDNA library from poly(A)(+) mRNA of C. ribicola axenic mycelial culture was constructed and immunoscreened and the cDNA was cloned. Sequence analysis indicated an open reading frame of 462 bases, which encodes a protein of 153 amino acid residues with a molecular mass of 16.7 kDa and a predicted isoelectric point (pI) of 8.93. Based on the N-terminal amino acid sequences of Cro rI, the secreted portion of Cro rI protein should be 136 amino acids long with several putative posttranslational modification sites and a molecular mass of 14.8 kDa. The predicted pI for the secreted portion was 9.34. The predicted N-terminal signal peptide was 17 amino acids long. The N-terminal 42-amino acid sequence of the predicted mature protein (secreted portion) was identical to the amino terminal sequence of Cro rI that was previously determined. Southern blot hybridizations indicated that the C. ribicola genome contained at least two copies of the cro rI gene. Isolation of the genomic PCR fragment, which was approximately 400 bp longer than the cDNA, and subsequent cloning and sequencing analyses confirmed that there were three introns within the coding regions. Western immunoblot analyses revealed that Cro rI protein accumulated in large amounts only in the infected white pine tissues while no trace was detectable in the alternate Ribes stage or the five different spores, suggesting a critical role of Cro rI in the haploid stage of the fungus (in pine). The translocation of Cro rI was only found to occur in cankered trees, and not in the young infected seedlings. The implications of Cro rI in pathogenesis are discussed.     

Site preparation alters soil distribution of roots and ectomycorrhizae on outplanted western white pine and Douglas-fir

This report documents root and ectomycorrhizal development on container-produced (1-0), outplanted, western white pine and Douglas-fir seedlings growing in site-prepared forest soils typical of the Inland Northwestern US. The following site preparations were used: 1) mounding organic and surface mineral horizons; 2) mounding with subsequent physical removal or chemical control of competing vegetation; 3) scalping to reduce competing vegetation; and, 4) a control or no post-harvest disturbance. Treatments were applied on relatively harsh and moderate sites in northern Idaho. Most ectomycorrhizae on the seedling population were found in the mineral substrates that dominated planting sites. However, compared to mineral substrates, highest seedling ectomycorrhizal tip counts were recorded in organic matter, particularly decayed wood or mixtures containing decayed wood. Strong ectomycorrhizal development was characteristic of western white pine. It supported highest ectomycorrhizal activity in organic substrates on the harshest treatments (scalps). Douglas-fir showed even stronger relative increases of ectomycorrhizae in organic substrates on harsh treatments. Three of the four common ectomycorrhizal morphological types were concentrated in mineral substrates with all treatments. A treatment-induced change of behavior was shown by the principal pine type. It occurred at highest numbers in organic substrates of the mound with competing vegetation treatment and in mineral substrates with the control. If relative availability to seedling roots was considered, organics (especially decomposed wood) were generally equal or superior to mineral substrates for supporting ectomycorrhizal activity on planted seedlings.     

Characterization and expression of the Douglas-fir luminal binding protein (PmBiP)

The endoplasmic reticulum (ER) molecular chaperone, BiP, plays a role in the cotranslational 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. The deduced amino acid sequence of PmBiP contains an 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 which is separate from the 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. Received: 21 February 2000 / Accepted: 13 April 2000     

NADPH : protochlorophyllide oxidoreductases in white pine (Pines strobes) and loblolly pine (P. taeda)

Summary NADPH : protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a gt11 library constructed from poly(A)⁺ RNA prepared from the cotyledons of dark-grown white pine (Pines strobes) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.     

Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11

Complementary DNAs encoding alpha-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting alpha-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the raw-starch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.     

质粒pRSET-A前导肽串联多聚体的构建及其多克隆抗体制备*

质粒pRSET-A是一个常用的高效原核表达载体,编码一N端含组氨酸标签（6×His）的34aa前导肽序列,以方便利用抗组氨酸标签抗体鉴定或纯化所表达的重组蛋白。本实验设计一对两侧含编码疏水性氨基酸密码子的引物,经过扩增前导序列10~34aa基因序列,并重新克隆入质粒pRSET-A构建串联二聚体后,再利用质粒pRSET-A的BamH I / Bgl II同尾酶克隆位点,经一系列简单的酶切和连接,快速构建这一前导肽中不含组氨酸标签序列的串联多聚体基因,并成功表达其六聚体重组蛋白。将此重组蛋白主动免疫山羊,获得了能够特异地识别pRSET-A编码的N端前导肽序列的抗体。结果显示,所制备的羊抗10~34aa前导肽抗体能够识别pRSET-A指导表达的含有完整前导肽的重组蛋白,但不能识别不含10~34aa序列的重组蛋白;同时,利用同位酶技术可以快速高效构建短肽的串联多聚体以制备具有高免疫原性的亚单位疫苗或免疫调控物质。     

A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.     

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). I. The amino acid sequence of cyanogen bromide fragment II.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is one of the major protein constituents of human plasma high density lipoproteins. The protein has 245 amino acid residues, including 3 residues of methionine, and is lacking isoleucine, cystine, and cysteine. Cleavage of apoLP-Gln-I with cyanogen bromide yields four fragments, designated in their order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the present study, we report the complete amino acid sequence of the NH2-terminal fragment, CNBr II, a peptide that contains 90 amino acid residues.     

Effects of NaCl on responses of ectomycorrhizal black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) to fluoride

Black spruce ( Picea mariana ), white spruce ( Picea glauca ) and jack pine ( Pinus banksiana ) were inoculated with Suillus tomentosus and subjected to potassium fluoride (1 m M KF and 5 m M KF) in the presence and absence of 60 m M NaCl. The NaCl and KF treatments reduced total dry weights in jack pine and black spruce seedlings, but they did not affect total dry weights in white spruce seedlings. The addition of 60 m M NaCl to KF treatment solutions alleviated fluoride-induced needle injury in ectomycorrhizal (ECM) black spruce and white spruce, but had little effect in jack pine seedlings. Both KF and 60 m M NaCl treatments reduced E values compared with non-treated control seedlings. However, with the exception of small reductions of K_r by NaCl treatments in black spruce, the applied KF and NaCl treatments had little effect on K_r in ECM plants. Chloride tissue concentrations in NaCl-treated plants were not affected by the presence of KF in treatment solutions. However, shoot F concentrations in ECM black spruce and white spruce treated with 5 m M KF + 60 m M NaCl were significantly reduced compared with the 5 m M KF treatment. The results point to a possible competitive inhibition of F transport by Cl. We also suggest that the possibility that aquaporins may be involved in the transmembrane transport of F should be further investigated.     

Antifungal activity of a Pinus monticola antimicrobial peptide 1 (Pm-AMP1) and its accumulation in western white pine infected with Cronartium ribicola

Pinus monticola antimicrobial peptide 1 (Pm-AMP1) was expressed and purified from bacterial cell lysate and its identity and purity confirmed by Western blot analysis using the Pm-AMP1 antibody. Application of Pm-AMP1 resulted in visible hyphal growth inhibition of Cronartium ribicola , Phellinus sulphurascens , Ophiostoma montium , and Ophiostoma clavigerum 3-12 days post-treatment. Pm-AMP1 also inhibited spore germination of several other phytopathogenic fungi by 32%-84% 5?days post-treatment. Microscopic examination of C. ribicola hyphae in contact with Pm-AMP1 showed distinct morphological changes. Seven western white pine ( Pinus monticola Douglas ex D. Don) families (Nos. 1, 2, 5, 6, 7, 8, 10) showing partial resistance to C.?ribicola in the form of bark reaction (BR) were assessed by Western immunoblot for associations between Pm-AMP1 accumulation and family, phenotype, canker number, and virulence of C. ribicola. There was a significant difference (p?< 0.001) in mean Pm-AMP1 protein accumulation between families, with higher levels detected in the full-sib BR families (Nos. 1, 2, 5) than the half-sib BR families (Nos. 6, 7). Family 8, previously described as a Mechanism 'X' BR family, had the highest number of BR seedlings and displayed high Pm-AMP1 levels, whereas the susceptible family (No. 10) showed the lowest levels (p?< 0.05). Family 1 showed a significant association between Pm-AMP1 accumulation and overall seedling health (p?< 0.01, R?= 0.533), with higher protein levels observed in healthy versus severely infected seedlings. In general, low Pm-AMP1 levels were observed with an increase in the number of cankers per seedling (p?< 0.05), and seedlings inoculated with the avirulent source of C. ribicola showed significantly higher Pm-AMP1 levels (p?< 0.05) in the majority of BR families. Cis-acting regulatory elements, such as CCAAT binding factors, and an AG-motif binding protein were identified in the Pm-AMP1 promoter region. Multiple polymorphic sites were identified within the 5' untranslated region and promoter regions. Our results suggest that Pm-AMP1 is involved in the western white pine defense response to fungal infection, as observed by its antifungal activity on C. ribicola and a range of phytopathogens as well as through its association with different indicators of resistance to C.?ribicola.     

Sperm-egg binding mediated by sperm alpha-L-fucosidase in the ascidian,Halocynthia roretzi

Spermatozoa bind to the vitelline coat in the ascidians and many other animals. The binding of sperm in Halocynthia roretzi is mediated by a sperm alpha-L-fucosidase and complementary-L-fucosyl residues of glycoproteins in the vitelline coat. cDNA clones for alpha-L-fucosidase were isolated from growing testis mRNA. It contained a 1398 bp full-length cDNA insert (HrFuc'ase) that encoded the 466 amino acid residues of H. roretzi sperm alpha-L-fucosidase. A putative signal peptide of 21 amino acid residues proceeded the sequence for the mature protein (M.W. 52.4 kDa). The coding sequence for HrFuc'ase showed 47.7% sequence identity to the human liver fucosidase sequence. The polyclonal antibody was prepared against a lacZ-HrFuc'ase fusion protein expressed in E. coli. The antibody crossed to a 54 kDa protein in sperm on western blotting and inhibited fertilization in a dose dependent manner. These data suggest that sperm-egg binding is mediated by the sperm alpha-L-fucosidase, HrFuc'ase in the ascidian, H. roretzi.     

Isolation and characterization of a cDNA clone for the amino-terminal portion of the pro-alpha 1(II) chain of cartilage collagen

We have isolated a cDNA clone (pRcol 2) which is complementary to the 5'-terminal portion of the rat pro-alpha 1(II) chain mRNA. A synthetic oligonucleotide was used both as a primer for cDNA synthesis and as a probe for screening a cDNA library. The probe was a mixture of sixteen 14-mers deduced from an amino acid sequence present in the amino-terminal telopeptide of the rat cartilage alpha 1(II) chain. This primer was chosen so that the resulting cDNA would contain the sequence of the 5' end of the mRNA. The nucleotide sequences of the cDNA were determined and compared with that of three other interstitial procollagen chain mRNAs (pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) chain mRNA). pRcol 2 contains a 521-base pair (bp) insert, including 153 bp of the 5' untranslated region plus 368 bp coding for the signal peptide, the amino-terminal propeptide, and a part of the telopeptide. The signal peptide of the type II collagen chain is composed of about 20 amino acids. There is little homology between the amino acid sequence of the signal peptide in the pro-alpha 1(II) chain and that of three other interstitial procollagen chains. The NH2-terminal propeptide is deduced to contain short nonhelical sequences at its amino and carboxyl ends and an internal helical collagenous domain comprising 25 repeats of Gly-X-Y with one interruption. There is a strong conservation of the amino acid sequence of the carboxyl-terminal part of the NH2-terminal propeptide in the pro-alpha 1(II), pro-alpha 1(I), and pro-alpha 2(I) chains. Type II collagen mRNA does not contain a sequence corresponding to a uniquely conserved nucleotide sequence around the translation initiation site which occurs in mRNA for other procollagen chains.     

Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana)

Opening of apical and axillary buds of mature Douglas-fir and sugar pine trees was obtained on a newly formulated basal medium (DCR) without growth regulators. Elongation of buds was observed on 1/2 strength DCR with 0.3% activated charcoal (DCR-1). In sugar pine, multiple shoots were obtained when explants on DCR with 0.5 mg/1 BAP for 5–6 weeks were transferred to DCR-1 medium. On subculture, axillary buds again developed when shoots were cultured on DCR with 0.2 mg/1 BAP for Douglas-fir and 0.5 mg/1 BAP for sugar pine. These buds were again elongated on DCR-1 medium. By subculturing 7–10 shoots of Douglas-fir and 2–3 shoots of sugar pine, over 100 shoots can be obtained in a year.Abbreviations BAP N⁶-benzylaminopurine - KN kinetin - NAA -naphthalene acetic acid - IAA Indole-3-acetic acid - MS Murashige-Skoog medium - WPM woody-plant medium