Ph.D. - Botanical Sciences (Plant Physiology)

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    Ecology of the silversword, Argyroxiphium sandwicense DC. (Compositae), Haleakala Crater, Hawaii
    ( 1973) Kobayashi, Herbert K.
    Argyroxiphium sandwicense DC. is an endemic composite of the alpine zone in Hawaii. Western man and his herbivores have restricted its present distribution mostly to the cinder cones and lava flows within Haleakala Crater, Maui. Here the silversword is able to sustain a high regeneration rate under dynamic substrate conditions that eliminate all but a few exotic and endemic species. Long-term field measurements of environmental parameters, and growth cabinet experiments on germination and seedling survival established the following optimal conditions. Germination and seedling survival depend on a temperature not exceeding about 30°C, and the relatively high moisture content of a stable sandy substratum completely covered by cinder fragments no thicker than about 5 cm. The maintenance of the thin complete cover over an area larger than a hectare for a period of a few decades, is best met by a shelf of agglutinates supplying tabular fragments which slide over a sloped sandy germination layer. The slope angle is about 35 degrees; low enough to stabilize the sandy layer, yet steep enough to be slightly unstable for tabular fragments. A thin cover is maintained by removal of fragments at the foot of the slope by wind and water during winter storms. Vandalism and browsing are probably not important under the protection of the Park Service, but root breakage by trampling may become a problem with further increase in visitors. Seed damage by insects may not be important as previously reported, but confirmation awaits further investigation during a good flowering season.
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    The growth and phenology of Metrosideros in Hawaii
    ([Honolulu], 1972) Porter, John Richard
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    Molecular cloning and characterization of a tobacco calmodulin binding protein
    ( 1996) Dash, Sagarika
    Calmodulin-binding proteins (CaMBP) play important roles in Ca2+ICaMmediated cellular activities. While many CaMBPs have been isolated and characterized in animals, very little is known about plant calmodulin-binding proteins (CaMBPs). A cDNA clone, pTCB 60, encoding a recombinant tobacco CaMBP was isolated previously by screening a tobacco (Nicotiana tabacum L. cv Wisconsin 38) cDNA library using S35_ labeled CaM as a ligand probe. The cDNA clone contained a 1572 bp cDNA insert with an 1184 bp open reading frame encoding 393 amino acid residues. Northern blot analysis of tobacco total RNA with radiolabeled cDNA insert as a probe detected a 2.4 kb mRNA, indicating that pTCB60 is not a full length cDNA clone. The presence of a poly (A) tail at 3' end and the lack of an initiation methionine codon at the beginning of the open reading frame (ORF) of the cDNA sequence, suggest that approximately 830 bases from the 5'end of the message are missing in pTCB60 cDNA clone. The complete sequence of the messenger was determined by two approaches. First, the 800 bp unknown 5'downstream region of the messenger was synthesized and amplified by a slightly modified 5'RACE (rapid amplification of cDNA ends) protocol, cloned and sequenced. Second, a tobacco cDNA library was constructed in Agt11 and rescreened with pTCB60 cDNA probe to isolate a full-length clone. The combined nucleotide sequence of the 5'RACE clone and pTCB60 partial cDNA clone indicated the full length cDNA sequence is 2409 bp containing a 1656 bp ORF encoding 551 amino acid residues. Database searches revealed no similarity with known gene or protein sequences. The 48.5 kD recombinant tobacco CaMBP encoded by pTCB60 cDNA clone was purified by a two step procedure using CaM-Sepharose chromatography and Protosorb immunoaffinity chromatography. Polyclonal antisera raised against the purified recombinant protein recognized a 60 kD polypeptide in western blots of tobacco cell extracts. Northern blot and immunoblot analyses showed differential expression of TCB60 mRNA and the corresponding protein during tobacco cell culture growth and heat shock response. The western blot of tobacco leaf, stem and root proteins indicated the expression of the protein only in leaf tissues. Secondary structure analysis of the deduced amino acid sequence of the recombinant CaMBP suggested the presence of a basic amphiphilic a-helix (BAA) motif at the C-terminus of the protein. A synthetic peptide was made corresponding to the putative BAA motif spanning amino acids 520-538 and its interaction with CaM was analyzed by a variety of methods. Calmodulin exhibited a Ca2+-dependent mobility shift upon binding the synthetic peptide in 4 M urea and native PAGE. The synthetic peptide competitively inhibits CaM-stimulated POE activity (Ki = 15 nM). Upon binding CaM, the fluorescence emission spectra of the peptide containing two tryptophanyl residues shifted toward blue and increased in intensity. The circular dichroism (CD) spectra show the helicity of CaM and peptide increase upon complex formation. IH NMR studies indicate that the peptide interacts with the aromatic residues in the leading helices of domain I and III of CaM. Taken together, these data provide direct evidence that a structurally conserved BAA CaM-binding domain similar to most CaM-binding proteins characterized in animal systems is present in a plant protein.
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    Molecular cloning and characterization of a heat-shock induced calmodulin binding protein gene and cDNAs encoding glutamate decarboxylase from tobacco
    ( 1995) Dharmasiri, M.A. Nihal
    Two individual heat shock induced/enhanced tobacco (Nicotiana tabacum L. cv Wisconsin-38) CaMBP cDNAs, pTCB48 and pTCB15, were characterized. Both of these clones were partial cDNAs. The 5' end of pTCB48 was obtained using 5' RACE technique. The ORF of this cDNA codes for a protein with a molecular weight of 50 kD. Using several deletions coupled with CaM gel overlay, CaM binding domain of pTCB48 was localized to the very end of the C-terminus of the protein. Structural prediction of this domain revealed a possibility of forming a BAA structure but an alternative β-strand and β-turn structure could not be excluded. Polyclonal antibody was raised against purified TCB48 fusion protein and used in Western analysis. The gene (TG48) corresponding to the pTCB48 cDNA was isolated and characterized. TG48 contains a total of 6149 bp including 1064 bp of 5' flanking region and 364 bp of 3' flanking sequence. The TG48 gene is consists of 6 exons and 5 introns. The 5' flanking sequence contains common promoter elements such as TATA and CAAT boxes, and 5 putative HSEs, characteristic of heat shock induced genes. These HSEs may be involved in heat shock induction of this gene. The structure of both TG48 gene and full length cDNA indicate that of two heat shock induced mRNAs, the 1.8 kb transcript is encoded by this gene. Northern analysis indicates this gene is subjected to tissue specific, developmental and environmental regulation. Sequence analysis of pTCB15 indicated that it encodes a CaM binding glutamate decarboxylase (GAD). Two full length GAD clones were isolated by screening a N. tabacum L. cv Xanthi cDNA library. Both clones had similar nucleotide sequences except that GAD9 had a shorter 3' UTR. CaM binding domain of the tobacco GAD may be located at C-terminus and possibly a BAA structure. Results presented here indicate GAD transcript level is subjected to developmental, environmental and tissue specific regulation.
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    Purification and characterization of violaxanthin de-epoxidase, an enzyme of the xanthophyll cycle
    ( 1995) Rockholm, David C.
    Violaxanthin de-epoxidase (VDE) catalyzes the conversion of violaxanthin to zeaxanthin in chloroplast thylakoids in the presence of ascorbate and an acidic lumen. In higher plants zeaxanthin plays a role in photoprotection by mediating non-radiative (heat) dissipation of light energy in the light-harvesting complexes of photosystem II (PSII) whenever the light intensity exceeds a plant's capacity for carbon-fixation. Because of VDE's central role in this process, its purification and characterization are of interest. VDE had been partially purified from Lactuca sativa var. Romaine by differentially extracting sonicated thylakoids at both neutral and acid pH's, followed by size exclusion chromatography. I have now purified VDE by anion-exchange chromatography (Pharmacia Mono Q column) and a unique lipid-affinty precipitation step to one major polypeptide detectable by two-dimensional 50S-PAGE. VDE at pH 5.20 associates with monogalactosyl diacylglycerol (MGOG), the principal thylakoid lipid, forming a complex that can be precipitated by ultracentrifugation. In contrast to MGDG, very little VDE precipitated in the presence of eight other lipids, indicating a specific association of VDE for MGDG. The uniqueness of VDE's affinity for MGDG is further exemplified through a negative result: established" protein purification procedures using various surfactants to form reverse micelles failed to isolate VDE. The purified enzyme has an apparant molecular mass of 43 kD and a pI of 5.4. The KM values of VDE for its substrates were 0.352 µM for violaxanthin and 8.54 mM for ascorbate. Neutral red, 9-aminoacridine, and dibucaine (substances used to determine pH intracellularly and/or selectively uncouple the lumen proton gradient) directly inhibited VDE. VDE's amino acid composition and a number of partial amino add sequences from VDE were determined (its N-terrninus and four tryptic fragments). Various diverse characteristics of the purified enzyme are also reported, including how TO and BSA affect VDE activity and stability as well as the absence of mobility shifts on polyacrylamide gels. Polyclonal antibodies were generated to purified VDE (in conjunction with Katrin Hinderhofer)which inhibited the de-epoxidase reaction and reacted principally to the 43 kD polypeptide on a Western blot.
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    Molecular cloning and characterization of nucleoside diphosphate kinase in cultured sugarcane cells
    ( 1995) Dharmasiri, Sunethra
    A low molecular weight autophosphorylating protein (pp18) in cultured sugarcane cells was identified and characterized as nucleoside diphosphate (NDP) kinase. The purified NDP kinase separated into five isoforms of 16.5kD (NDP kinase I) and two isoforms of 18kD (NDP kinase IT) on two dimensional IEF/SDS-PAGE. The apparent K, values of the purified enzyme containing both size classes were 2.3mM and 0.2mM for ATP and GDP, respectively. Twenty one positive clones were isolated by screening a λgt11 cDNA library derived from cultured sugarcane cells with spinach NDP kinase I cDNA probe, and four were sequenced. The cDNA pSCNDK8 contained a 447 bp coding region (149 amino acids), and 58 bp and 238 bp 5' and 3' flanking sequences respectively. This cDNA hybridized to a 0.91kb mRNA. The deduced amino acid sequence of sugarcane NDP kinase cDNA clone showed over 60% sequence identity to many eukaryotic NDP kinases. The cDNA pSCNDK8 showed a frame shift resulting in 55 amino acid residues at the carboxyl terminus with no homology to NDP kinases. The sugarcane NDP kinase showed approximately a 3 to 4-fold enhancement in in situ autophosphorylation and 1.5 to 2 fold increase in NDP kinase activity in response to HS at 40-42°C for 2h. However, NDP kinase protein or mRNA levels did not show an increase during HS. The synthesis and phosphorylation of NDP kinase appears to be developmentally and heat shock regulated. NDP kinase levels were highest and showed a greater enhancement in autophosphorylation in response to HS in cells in fresh culture. In contrast to cultured sugarcane cells, mRNAs for NDP kinase I enhanced at least 2-fold in response to a 2 h HS at 40°C in young sugarcane shoots.
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    Structure and expression of ribulose-1, 5-biphosphate carboxylase/oxygenase small subunit genes in sugarcane
    ( 1994) Tang, Wendong
    Sugarcane (Saccharum spp. hybrid cultivar H32-8560), a C4 polyploid, is estimated to contain about 16 copies of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcs) gene. Seven rbcs genes were isolated from a sugarcane genomic library, and were partially or completely sequenced. Unlike rbcs gene members of other plant species that are heterogeneous in their 3' UTRs, these genes are highly conserved in their coding sequences as well as their 3' and 5' untranslated regions (UTRs). The nested Rapid Amplification of cDNA 3' Ends (nested-RACE) technique was used to clone the 3' UTR of rbcs mRNA from sugarcane leaves of different developmental stages. There is no significant difference among the RACE-products from different organs in terms of their overall electrophoretic patterns. The sequences of the 3 '-RACE products have more than 95% homology with the 3' UTR of the cloned rbcs genes. However, their lengths (from translation stop codon to polyadenylation site) are quite different and can be classified into five major size groups, i.e. Group I, 501bP; II, 351bP; III, 254bP; VI, 184bP; and V, 103bp. Northern analysis of total RNA from mature leaf, 51 mapping, and sequence determination of the rbcs cDNA clones isolated from a mature leaf cDNA library confirm the existence of different lengths of rbcs mRNA. The AAUAAA-like motifs for Groups I and II can be identified; however, we fail to detect other sequence motifs that have been suggested to be important in directing correct cleavage and polyadenylation. Differential amplification of sugarcane genomic DNA suggests that the genes encoding Groups I and II mRNAs are similar in length. Groups I and II mRNAs continue to be expressed during development of the sugarcane leaf and the ratio of these two classes of mRNAs in various sugarcane samples is similar. Sugarcane leaf bombardment experiments showed that both the scrbcs-1 and scrbcs-3 promoters are able to direct GUS gene expression in mesophyll cells and bundle sheath cells during C3-C4 transition, although at a ratio of 1 to 3 in favor of the bundle sheath cells. These finding supports the notion that the rbcs genes in C4 plants have a default C3-type expression pattern (Schaffner and Sheen, 1991).
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    Sulfur-rich 2S proteins in Lecythidaceae and their methionine-enriched forms in transgenic plants
    ( 1993) Zuo, Wei-Neng
    The 28 seed proteins from three members of the Brazil nut family (Lecythidaceae), monkeypot, cannonball, and Brazil nut, were isolated, characterized, and compared in regard to physical and chemical properties including subunit structure, amino acid composition, immunoreactivity, N-terminal sequence, and processing. These 28 proteins contain about 24% sulfur amino acids, thus they are sulfur-rich proteins (8RPs). The 28 8RPs are major seed proteins in the three plant species and all consist of two subunits, 9 kD and 3 kD, linked by disulfide bonds. In addition, these 28 8RPs share a high degree of similarity in subunit structure, amino acid composition, immunoreactivity, and N-terminal sequence. However, the precursor processing patterns of these 28 proteins are distinctly different. In monkeypot and Brazil nut, the precursor is cleaved into mature subunit polypeptides in three steps, i.e. 18 kD → 15 kD → 12 kD → 9 + 3 kD, while in cannonball, only two steps are detected, i.e. 18 kD → 15 kD → 9 + 3 kD. A total of 9 cDNA clones encoding the 28 8RPs in monkeypot and cannonball have been isolated and characterized. DNA sequence analysis reveals that the 28 8RPs isolated from different members of the Brazil nut family share a high degree of homology in amino acid (>80%) and nucleotide (>90%) sequences. The methionine (Met) residues are clustered in two areas of the variable region (between the 6th and 7th Cys residues) of the larger subunit. The 2S SRPs are encoded by multigene families and can be classified into two subfamilies. Native monkeypot 2S SRP (MP2S) contains 16 Mol% Met. The variable region of the MP2S gene was modified through nucleotide sequence alternations to increase the protein's Met content. Eight Met-enriched MP2S genes were engineered to increase the Met varying amounts ranging from 18 to 24 Mol%. To test the effect of these modifications on the stability of the MP2S, chimeric genes containing coding sequences of three modified (19, 21, and 23% Met) and the wild type MP2S cDNA were transferred into tobacco plants via the Agrobacterium transformation system. Northern and Western blot analyses demonstrated that the genes for the modified MP2S were expressed in the transgenic tobacco seeds at levels comparable to that of the wild type MP2S gene. Both the wild type and modified MP2S proteins were correctly processed into mature subunits. These results suggest that the wild type as well as the modified MP2S genes are suitable candidates for use in protein quality improvement.