Proteins In Higher Plants Research Articles

Occurrence of five different chromosomal HMG1 proteins in various maize tissues.

The nuclear HMG1 proteins of higher plants are small non-histone proteins that have DNA-bending activity and are considered architectural factors in chromatin. The occurrence of the chromosomal HMG1 proteins, HMGa, HMGc1/2 and HMGd, in various maize tissues was analyzed, and in the course of these studies a novel HMG1 protein, now termed HMGe, was identified. Purification and characterization of HMGe (M(r) 13,655) and cloning of the corresponding cDNA revealed that it displays only moderate similarity to other members of the plant HMG1 protein family. The five maize HMG1 proteins could be detected in kernels, leaves, roots and suspension culture cells, indicating that these proteins can be expressed simultaneously and occur relatively ubiquitously. However, the various HMG1 proteins are present in significantly different quantities with HMGa and HMGc1/2 being the most abundant HMG1 proteins in all tissues tested. Furthermore, the relative amounts of the various HMG1 proteins differ among the tissues examined. The HMG1 proteins were found to be relatively stable proteins in vivo, with HMGc1/2, HMGd and HMGe having a half-life of ca. 50 h in cultured cells, while the half-life of the HMGa protein is ca. 65 h. Collectively, these findings are compatible with the concept that the different plant HMG1 proteins might act as general architectural proteins in concert with site-specific factors in the assembly of certain nucleoprotein structures involved in various biological processes.

The First Caldesmon-like Protein in Higher Plants

Using anti-caldesmon polyclonal and monoclonal (raised against the N-terminal fragment of chicken gizzard caldesmon) antibodies, a plant caldesmon-like protein, 107 kDa as determined by SDS-gel electrophoresis, has been identified based on Western blotting of total extracts of Ornithogalum virenspollen tubes. Biochemical investigations showed common properties of this protein with animal caldesmon—it binds to actin and, in a Ca 2+-dependent manner, to calmodulin. In contrast to animal caldesmon, this plant cell counterpart is relatively resistant to proteolysis by endogenous proteases and sensitive to heat treatment. Our results show the presence of a caldesmon-like protein in higher plants for the first time.

Pigment-binding properties of the recombinant photosystem II subunit CP26 reconstituted in vitro.

CP26 is the most recently described antenna protein in higher plants which has been reported to be involved in xanthophyll-dependent regulation of the light-harvesting function but is largely unknown due to the difficulties of purification. In this study we have overexpressed in Escherichia coli the Lhcb5 gene product and reconstituted CP26 in vitro by refolding the recombinant protein in the presence of chlorophyll a, chlorophyll b and xanthophylls. The resulting pigment-protein complex is stable enough to be isolated by partially denaturing gel electrophoresis. Reconstitution and isolation conditions for CP26 are similar to those used for other chlorophyll a/b complexes like the major light-harvesting complex of photosystem II (LHCII) and CP29; however, CP26 differs with regard to its lower specificity in carotenoid binding. Most significantly, rather stable recombinant CP26 can be reconstituted containing violaxanthin as the only carotenoid. This enhanced plasticity with respect to carotenoid binding is consistent with CP26 being the major binding protein of violaxanthin involved in the xanthophyll cycle. The availability of recombinant CP26 opens the way to a better characterisation of this pigment-protein complex with regard to its biochemistry and its physiological functions.

Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes.

The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.

Identification of mRNAs and proteins in higher plants using probes from the Band 3 anion transporter of mammals

Band 3 is a well characterized ion transporter in mammalian cells. To determine if Zea mays expressed Band 3-related polypeptides, polyclonal antibodies to a 13-amino acid sequence at the carboxyl terminal end of the membrane domain of the mouse Band 3 anion transporter was used in immunoblot studies. This antiserum detected three classes of polypeptides from maize roots. The polypeptides had apparent molecular masses of 43, 46 and 59 kDa and appeared to be integral membrane components associated with various cellular membranes including the plasma membrane. Maize genomic DNA did not contain sequences that cross-hybridized with the mouse cDNA encoding the C-terminal membrane domain of Band 3. However, an oligonucleotide corresponding to a portion of the 13-amino acid sequence used for antibody preparation detected mRNAs from both monocotyledonous and dicotyledonous plants. The maize root and leaf mRNAs that were detected were 1.75, 1.45, and 0.88kb in size. The levels of the 1.75- and 1.45 kb mRNAs in maize roots did not change dramatically in response to nitrate; however, the 0.88kb transcript levels increased in response to nitrate. Using the 32 P-labelled Band 3 oligonucleotide, a partial cDNA clone (λB3-2758) encoding a 0.88 kb transcript was isolated. The abundance of the B3-2758 and nitrate reductase transcripts increased markedly in response to nitrate in an amino acid-grown maize cell line and was well correlated with the induction of nitrate transport.

Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein.

The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots.

Protein phosphorylation and magnesium status regulate the degradation of the D1 reaction centre protein of Photosystem II

Illumination of higher plant leaves (pumpkin, wheat and pea) at a photon flux density of 1200 μmol m −2 s −1 induced strong phosphorylation of the D1 reaction centre protein of photosystem II. Phosphorylation of D1 protein was not observed in a moss Ceratodon purpureus. To study the effect of protein phosphorylation on D1 degradation, thylakoids isolated from pumpkin leaves and Ceratodon were phosphorylated in vitro in the presence of ATP before being subjected to photoinhibitory illumination. The rate of photoinhibition of Photosystem II oxygen evolution was not influenced by phosphorylation conditions in pumpkin or Ceratodon thylakoids. D1 protein in its phosphorylated form in pumpkin thylakoids was degraded significantly more slowly whereas in Ceratodon the degradation of D1 protein occurred rapidly with or without the presence of ATP in the photoinhibitory medium. Depletion of Mg 2+ ions also blocked the degradation of unphosphorylated D1 protein in pumpkin thylakoid membranes. We conclude that degradation of the D1 protein in higher plants is regulated by phosphorylation on the substrate level and its optimal rate requires the presence of Mg 2+.

Ca2+homeostasis in plant cells: intracellular Ca2+stores and Ca2+binding proteins

Abstract Plants share with animals the cellular machinery for regulating cytosolic free calcium concentrations. Nevertheless, the components involved in Ca2+ homeostasis have not yet been fully characterized in plants. In the last years there has been growing interest in identifying the plant counterparts of animal Ca2+ buffering proteins within intracellular Ca2+ storage pools. Evidence is accumulating for the expression of calreticulin as the main ER resident Ca2+ binding protein in higher plants. The fundamental role of this protein, involved in many cellular processes, is presented here in light of the latest findings obtained in animal cells.

A Chlamydomonas homologue to the 14-3-3 proteins: cDNA and deduced amino acid sequence

We have isolated and sequenced a 1464 bp cDNA from the unicellular green alga Chlamydomonas reinhardtii encoding an acidic polypeptide (259 aa) with considerable homologies to the 14-3-3 proteins of animals, yeasts and higher plants. Like the other members of this highly conserved protein kinase regulatory protein family, the deduced amino acid sequence of the Chlamydomonas 14-3-3 protein includes two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca 2+-binding sites is located within the C-terminal part of this polypeptide (positions 208–219). EF hand motifs are also present in the 14-3-3 proteins of some higher plants but not in those of animals and yeasts.

Nucleotide Sequence of a cDNA Encoding the ADP/ATP Carrier from Wheat (Triticum turgidum)

The adenine nucleotide carrier is the most abundant mitochondrial membrane protein. It catalyzes the exchange of adenine nucleotides between mitochondrial matrix and cytosol and plays a central role in the integration of mitochondrial metabolism. We have characterized an ADP/ ATP carrier cDNA from wheat (Triticum turgidum) with a view toward gaining an understanding of the expression and biogenesis of this protein in higher plants. The cDNA sequence has been obtained from a wheat single-stranded cDNA by PCR utilizing sequence information from the Zea mays homolog (Bathgate et al., 1989). Overlapping cDNA fragments were obtained. To extend the sequence to the 5’ end a modified cDNA tailed with a poly(A) was used. The cDNA of the ADP/ATP carrier is 1035 bp in length. The open reading frame encodes the mature protein consisting of 331 amino acids (Table I). The amino acid sequence of the wheat ADP/ATP carrier is 90.3 and 79.6% identical to that of the ADP/ATP carrier from maize (Bathgate et al., 1989) and rice (Hashimoto et al., 1993). Most of the differences are located at the N-terminal region. Since the function of this region is not known, the significance of these differences is not understood.

A cytoskeletal 50 kDa protein in higher plants that forms intermediate-sized filaments and stabilizes microtubules

Filamentous structures of 7–10 nm in diameter were regenerated in vitro from a soluble 50 kDa protein (p50) that had been isolated from mung bean seedlings and from cultured tobacco cells. The polymerization of p50 in vitro was dependent on the presence of guanosine nucleotides, in particular, guanosine monophosphate (GMP). Unlike tubulin, p50 is a stable basic protein with the ability to polymerize even after heat treatment for 1 min at 70 °C. Furthermore, the freeze-dried powder of p50 retained the ability to regenerate filamentous structures after it had been dissolved in polymerization buffer to which GMP was then added. Two monoclonal antibodies against p50 were obtained. These antibodies stained the filamentous structures that extended from the surface of the nucleus to the cell periphery in interphase tobacco cells. They stained spindles and phragmoplasts as did tubulin-specific antibodies. They also stained fibrillar structures that were present around the spindle poles and the telophase daughter nuclei in which no microtubules were present. These results suggest that a part of the cell's complement of p50 may be associated with microtubules in dividing cells while the rest may itself form unique fibrillar structures. The antibodies against p50 did not stain cortical microtubules or the pre-prophase band of microtubules. The antibody against p50 also stained intermediate filament-like structures in cultured animal cells. The formation of microtubules in vitro was markedly stimulated and the assembled microtubules were greatly stabilized by p50. Further investigation of p50 is indispensable for the understanding of properties and function of intermediate-sized filaments in higher plant cells.

Nucleotide sequence of two cDNAs encoding fucoxanthin chlorophyll a/c proteins in the diatom Odontella sinensis

Two cDNA clones encoding fucoxanthin chlorophyll a/c-binding proteins (FCP) in the diatom Odontella sinensis have been cloned and sequenced. The derived amino acid sequences of both clones are identical, comparison of the corresponding nucleic acids reveals differences only in the third codon position, suggesting a recent gene duplication. The derived proteins are similar to the chlorophyll a/b-binding proteins of higher plants. The presequences for plastid import resemble signal sequences for cotranslational import rather than transit peptides of higher plants. They are very similar to the presequences of FCP proteins in the diatom Phaeodactylum, but different from the presequences of the gamma-subunit of CF0CF1 of Odontella and the peridinin chlorophyll a binding proteins (PCP) of the dinoflagellate Symbiodinium.

Structural cell wall proteins in higher plants

Abstract The architecture and chemical composition of plant cell wall change dynamically according to developmental stage of different cell type and in response to environmental conditions. In this review, attention has been given to the tissue- and organ-specific expression of the cell-wall structural proteins. The best characterized cell-wall structural proteins are: hydroxyproline-rich glycoproteins (HRGPs) known as extensins, proline-rich proteins (PRPs) and glycine-rich proteins (GRPs). Two novel classes of cell-wall structural proteins have recently been discovered; they have been named tyrosine- and lysine-rich protein (TLRP) and asparagine- glycine- and tyrosine-rich protein (AGTRP). The meaning of these proteins in the cell-wall has been discussed.

Purification and properties of the soluble acid invertase from Oryza sativa

An invertase (β-fructofuranosidase, EC 3.2.1.26) from aerial parts of 17-day-old Oryza sativa plants has been purified to homogeneity by gel filtration on Sephadex G-150 and adsorption on brushite. The invertase is a dimeric glycoprotein ( M r 98 000) composed of two identical subunits ( M r 46 000). The pI is ca 6 and the activation energy is 8250 cal mol −1 over 30° and 24 000 cal mol −1 below this temperature. The enzyme shows a high specificity for sucrose ( K m 6.6 × 10 −3M) and its activity is modulated by fructose. Glucose is a classical non-competitive inhibitor and fructose produces a complex non-linear competitive inhibition. Proteins are activators of the invertase, but they do not suppress the inhibitory action of the reaction products. The in vitro spontaneous complex formation among invertases and proteins in higher plants suggests that invertases are functioning complexed with some protein in vivo.

Enzymatic cell wall proteins in higher plants

Abstract The entire life of the plant cell is intimately related to the biogenesis of cell wall and growing attention has been devoted in last years to the understanding of the biochemical events that lead to the synthesis and cross-linking of the major wall components. In this context it is now clearly assessed that proteins play a key role in the architecture and chemo-rheological properties of growing cell wall thus participating in important ontogenetics events such as growth and differentiation. Wall proteins are mostly glycoprotein accounting up to almost 20% wall dry weight and many of them are structural proteins. Here we are dealing with enzymatic proteins occurring in the cell wall and their physiological significance.

Light‐dependent phosphorylation of D1 reaction centre protein of photosystem II: hypothesis for the functional role in vivo

Reversible phosphorylation of the D1 reaction centre protein of photosystem II (PSII) occurs in thylakoid membranes of higher plants. The significance of D1 protein phosphorylation in the function of PSII is not yet clear. This paper summarizes the data implying that phosphorylation of D1 protein in higher plants is involved in the regulation of the repair cycle of photoinhibited PSII centres. Photoinhibition of PSII, D1 protein phosphorylation and degradation have been studied in vivo in higher plant leaves acclimated to different growth irradiances. It is shown that photoinhibitory illumination induces maximal phosphorylation of the D1 protein. Under these conditions D1 turnover is also saturated. We postulate that phosphorylation retards the degradation of damaged D1 protein under conditions where rapid replacement by a new D1 copy is not possible. This would protect PSII from total disassembly and degradation of all PSII subunits. We conclude that the phosphorylation of D1 protein and the regulation of D1 protein degradation may have evolved together. Furthermore, these characteristics seem to be related to the highly organized structure of higher‐plant type thylakoid membranes, since the capability to phosphorylate D1 protein is restricted to seed plants.

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50-100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a lambda gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.

Characterization of alanine-rich, hydroxyproline-containing cell wall proteins and their application for identifying Pythium species

Cell wall glycoproteins of six Pythium species (P. iwayamai, P. paddicum, P. okanoganense, P. graminicola, P. vanterpoolii and P. volutum) that cause snow rot of winter cereals were characterized and examined for their applicability for species identification. Glycoproteins of approx. 25–40 kDa constituted a major component among cell wall proteins of each species. Their electrophoretic patterns, detected with Coomassie brilliant blue, lectin and antibody, showed sufficient interspecific polymorphism and intraspecific stability to identify and classify these six species. These species were divided into three classes by the types of sugar chains in the glycoprotein, inferred from their reactivity to specific lectins. This classification was verified from their N-terminal primary structure. An antibody for a deglycosylated glycoprotein of P. iwayamai reacted with all the distinctive glycoproteins of these six species, indicating that they have common epitope(s). Amino acid analyses also revealed their common characteristics of especially high content of alanine and presence of a significant amount of hydroxyproline. The amino acid compositions of these alanine-rich, hydroxyproline-containing cell wall proteins were similar to those of arabinogalactan proteins in higher plants. This suggests a homologous role(s) of these cell wall glycoproteins in both Pythium spp. and higher plants.

Characterization of genes encoding the light-harvesting proteins in diatoms: biogenesis of the fucoxanthin chlorophylla/c protein complex

In chromophytic algae the major light-harvesting complex is the fucoxanthin chlorophylla/c protein complex. Recently, we have cloned several highly related cDNA and genomic sequences encoding the fucoxanthin chlorophylla/c proteins from the diatomPhaeodactylum tricornutum. These genes are clustered on the nuclear genome. The sequences of the fucoxanthin chlorophylla/c proteins as deduced from the gene sequences have some similarity to the chlorophylla/b proteins associated with light-harvesting complexes of higher plants and green algae. Like the chlorophylla/b proteins of higher plants, the fucoxanthin chlorophylla/c proteins are synthesized as higher-molecular weight precursors in the cytoplasm of the cell and are transported into the plastids. However, the mode of transport into diatom plastids is very different from the mechanism involved in transporting proteins into the chloroplasts of higher plants and green algae. We focus here on the characteristics of the fucoxanthin chlorophylla/c proteins, the mode of transport of these proteins into plastids, the arrangement of the genes encoding these proteins, and efforts to utilize these genes to develop a DNA transformation system for diatoms.