Gene expression and oxalate oxidase activity of two germin isoforms induced by stress

Wheat germin is a homopentameric 125 kD glycoprotein mainly localized in the cell wall of monocots, and is a specific marker of the onset of growth in germinating seeds. The major objective of this study was to examine the expression and oxalate oxidase activity of two wheat germin isoforms: gf-2.8 and gf-3.8 in transgenic tobacco plants. The transgenic tobacco plants were created with different constructs: 1) one entire excision of gf-2.8 germin promoter and two partially deleted promoter sequences were used to generate 3 independent GUS constructs; 2) the whole gf-2.8 gene construct and the fusion with CaMV 35S promoter; 3) one entire excision of gf-3.8 germin gene and one partially deleted gf-3.8 promoter sequences were used to generate 2 independent GUS constructs; 4) the whole gf-3.8 gene and the fusion with CaMV 35S promoter. Hormonal treatment (auxin and gibberellin), salt treatment, heavy metals (Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, As) and Al induced high GUS activity in tobacco transformed with entire and one partially deleted of the gf-2.8 gene. The immunoblotting confirmed induction of gf-2.8 gene and its product expressed oxalate oxidase activity in tobacco transformed with the entire gf-2.8 construct. Neither nicotinic acid, salicylic acid, heat shock, cold nor UV-C have enhanced significant GUS activity and germin gf-2.8 synhesis and activity. The germin gf-3.8 constructs with GUS gene and with the entire gf-3.8 sequences gave non-positive response with factors mentioned above. It has been demonstrated that gf-3.8 germin isoform is present as a monomer (M_r 25 kD). The non-active gf-3.8 protein is synthetised in transgenic tobacco plants only under control of the CaMV 35S promoter. Consequently, among two germin isoforms, only the gf-2.8 protein seems to be regulated by hormonal, salt and heavy metal factors. The gf-2.8 oxalate oxidase activity could be then involved in general stress-induced signalling in plant.     

Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance

The wheat genome encodes a family of germin-like proteins that differ with respect to regulation and tissue specificity of expression of the corresponding genes. While germin exhibits oxalate oxidase (E.C. 1.2.3.4.) activity, the germin-like proteins (GLPs) have no known enzymatic activity. A role of oxalate oxidase in plant defence has been proposed, based on the capacity of the enzyme to produce H2O2, a reactive oxygen species. The role in defence of germin and other members of the germin-like gene family was functionally assessed in a transient assay system based on particle bombardment of wheat leaves. Transient expression of the pathogen-induced germin gf-2.8 gene, but not of the constitutively expressed HvGLP1 gene, reduced the penetration efficiency of Blumeria (syn. Erysiphe) graminis f.sp. tritici, the causal agent of wheat powdery mildew, on transformed cells. Two engineered germin-gf-2.8 genes and the TaGLP2a gene, which all encoded proteins without oxalate oxidase activity, also reduced the penetration efficiency of the fungus, demonstrating that oxalate oxidase activity is not required for conferring enhanced resistance. Instead, activity tagging experiments showed that in cells transiently expressing the germin gf-2.8 gene, the transgene product became insolubilized at sites of attempted fungal penetration where localised production of H2O2 was observed. Thus, germin and GLPs may play a structural role in cell-wall re-enforcement during pathogen attack.     

Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins

By screening approximately 10(6) plaques in a wheat DNA library with a "full-length" germin cDNA probe, two genomic clones were detected. When digested with EcoRI, one clone yielded a 2.8-kilobase pair fragment (gf-2.8) and the other yielded a 3.8-kilobase pair fragment (gf-3.8). By nucleotide sequencing, each of gf-2.8 and gf-3.8 was found to encode a complete sequence for germin and germin mRNA, and to contain appreciable amounts of 5'- and 3'-flanking sequences. The "cap" site in gf-2.8 was determined by primer extension and the corresponding site in gf-3.8 was deduced by analogy. The mRNA coding sequences in gf-2.8 and gf-3.8 are intronless and 87% homologous with one another. The 5'-flanking regions in gf-2.8 and gf-3.8 contain recognizable sites of what are probably cis-acting elements but there is otherwise little if any significant similarity between them. In addition to putative TATA and CAAT boxes in the 5'-flanking regions of gf-2.8 and gf-3.8, there are AT-rich inverted-repeats, GC boxes, long purine-rich sequences, two 19-base pair direct-repeat sequences in gf-2.8, and a remarkably long (200-base pair) inverted-repeat sequence (approximately 90% homology) in gf-3.8. An 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is reflected by a corresponding 7% difference between the corresponding 201-residue proteins. Most significantly, the same 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is allied with no change whatever in a central part (61-151) of the encoded polypeptide sequences. It seems likely that this central, strongly conserved core in the germins is of first importance in the biochemical involvements of the proteins. When an equivalence is assumed between like amino acids, the gf-2.8 and gf-3.8 germins show significant (approximately 44%) similarity to spherulins 1a and 1b of Physarum polycephalum, a similarity that increases to approximately 50% in the conserved core of germin. Near the middle (87-96) of the conserved core in the germins is a rare PH(I/T)HPRATEI decapeptide sequence which is shared by spherulins (1a and 1b) and germins (gf-2.8 and gf-3.8). These similarities are discussed in the context of evidence which can be interpreted to suggest that the biochemistry of germins and spherulins is involved with cellular, perhaps cell-wall responses to desiccation, hydration, and osmotic stress.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of Salt Stress on Germin Gene Expression in Barley Roots

Germin gene expression in barley (Hordeum vulgare L.) seedlings responds to developmental and environmental cues. During seed germination, germin mRNA levels were maximal 2 d after the start of imbibition in control seedlings and declined to low levels by 6 d. When seeds were sown in the presence of 200 mM NaCl, germin mRNA levels were also maximal after 2 d, but NaCl treatment, which slowed seedling growth, prolonged germin gene expression for an additional 1 d. In 4-d-old seedlings, germin mRNA levels were highest in roots and higher in the vascular transition region than in shoots. In roots of 6-d-old seedlings, germin gene expression was regulated by salt shock and plant growth regulators. Induced germin mRNA levels were maximal 8 h after treatment with NaCl, salicylate, methyl salicylate, or methyl jasmonate and 4 h after treatment with abscisic acid and indoleacetic acid. Like germin mRNA, dehydrin mRNA levels were maximal 8 h after NaCl treatment. In contrast, peroxidase mRNA levels declined to less than control levels within 30 min of treatment. Germin gene expression is regulated developmentally by salt stress and by treatments with plant hormones. Since germin is an oxalate oxidase, these result imply that oxalate has important roles in plant development and homeostasis.     

Overexpression of a wheat MYB transcription factor gene, TaMYB56-B, enhances tolerances to freezing and salt stresses in transgenic Arabidopsis

The MYB proteins play central roles in the stress response in plants. Our previous works identified a cold stress-related gene, TaMYB56, which encodes a MYB protein in wheat. In this study, we isolated the sequences of TaMYB56 genes, and mapped them to the wheat chromosomes 3B and 3D. The expression levels of TaMYB56-B and TaMYB56-D were strongly induced by cold stress, but slightly induced by salt stress in wheat. The detailed characterization of the Arabidopsis transgenic plants that overexpress TaMYB56-B revealed that TaMYB56-B is possibly involved in the responses of plant to freezing and salt stresses. The expression of some cold stress-responsive genes, such as DREB1A/CBF3 and COR15a, were found to be elevated in the TaMYB56-B-overexpressing Arabidopsis plants compared to wild-type. These results indicate that TaMYB56-B may act as a regulator in plant stress response.     

PCR—SSCP与测序技术相结合检测小麦耐盐突变体

根据位于小麦第四同源群上与耐盐有关的gf-2.8基因的编码区序列设计1对引物,分别以两个耐盐突变体及其亲本的总DNA为模板进行PCR扩增,在5个供试材料中均扩增出1条约685bp的目的条带,SSCP电泳显示突变体974915与其他供试材料之间存在差异。测序表明冀麦24和其耐盐突变体8901-17的扩增产物序列与gf-2.8基因的发表序列相同,这表明突变体8901-17的突变位点不在该基因上,而另一耐盐突变体974915的序列中则至少存在2个单碱基突变,有一处突变导致了氨基酸的变化,该突变位点位于gf-2.8基因的保守区域内。     

Cloning and sequence analysis of germin-like protein gene 2 promoter from Oryza sativa L. ssp. indica.

Germin and germin-like proteins (GLPs) are water soluble extracellular proteins reportedly expressed in response to some environmental and developmental signals. Some enzymatic activities have also been associated with germin/GLPs. However, their role in overall metabolism has not been fully understood. Significant insight into their function may also be gained by analysis of their promoter. During this study, about 1107 bp 5'region of OsRGLP2 gene was amplified, cloned and sequenced. The sequence analysis by BLAST showed that this promoter sequence has five common regions (CR1-CR5) of different sizes, which are repeated at 3-6 other locations in 30 kb region in which this gene driven by its promoter is located. Interestingly, all the genes driven by promoter harboring these common regions are GLPs/putative germins. Analysis of these common regions located on OsRGLP2 indicated presence of many elements including those for light responsiveness, dehydration and dark induced senescence, stresses (pathogen and salt), plant growth regulators, pollen specific expression and elements related to seed storage proteins. Analysis of the 30 kb germin/GLP clustered region by GenScan detected each gene to have a putative 40 bp promoter which contains TATA box and Dof factor which turned out to be a part of CR2.     

Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE

Plants have evolved sophisticated systems to cope with adverse environmental conditions such as cold, drought, and salinity. Although a number of stress response networks have been proposed, the role of plant apoplast in plant stress response has been ignored. To investigate the role of apoplastic proteins in the salt stress response, 10-day old rice plants were treated with 200mM NaCl for 1, 6 or 12h, and the soluble apoplast proteins of rice shoot stems were extracted for differential analysis, compared with untreated controls, by 2-D DIGE saturation labeling techniques. One hundred twenty-two significantly changed spots were identified by LC-MS/MS, and 117 spots representing 69 proteins have been identified. Of these proteins, 37 are apoplastic proteins according to the bioinformatic analysis. These proteins are mainly involved in the processes of carbohydrate metabolism, oxido-reduction, and protein processing and degradation. According to their functional categories and cluster analysis, a stress response model of apoplastic proteins has been proposed. These data indicate that the apoplast is important in plant stress signal reception and response.     

Germin Gene Expression Is Induced in Wheat Leaves by Powdery Mildew Infection

Germin gene expression is induced in wheat (Triticum aestivum L.) leaves by powdery mildew (Erysiphe graminis f. sp. tritici) infection. Germin is a protein marker for early cereal development and is an oxalate oxidase, an enzyme that catalyzes the conversion of oxalate to CO2 and H2O2. The induction of germin gene expression by powdery mildew infection is consistent with the importance of H2O2 to plant defense and identifies a mechanism for the elevation of H2O2 levels in wheat leaves. Germin mRNA levels increased 2 d after inoculation of seedlings with powdery mildew and continued to increase throughout an 8-d time course. The increase in accumulation of germin mRNA was accompanied by an increase in the germin oligomer, which reached maximal levels by d 6. An increase in oxalate oxidase activity paralleled germin oligomer accumulation. Germin gene expression was induced in a relatively resistant cultivar (Bobwhite) as well as in a susceptible cultivar (Cheyenne), suggesting that the induction of germin gene expression is an indicator of powdery mildew infection rather than cultivar resistance.     

Arabidopsis thaliana germin-like proteins: common and specific features point to a variety of functions

 Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germin's unusual biochemical properties and oxalate oxidase activity. Using specific antibodies, we have studied three GLPs (AtGER1, AtGER2 and AtGER3) in Arabidopsis thaliana (L.) Heynh. as well as in transgenic tobacco (Nicotiana tabacum L.) plants overexpressing these proteins. Like wheat (Triticum aestivum L.) germin, these Arabidopsis GLPs are associated with the extracellular matrix (ECM) and they also seem to exist as two glycosylated isoforms. However, none of them is an oxalate oxidase. Although GLPs display several conserved features, each has its specific characteristics. Both AtGER2 and AtGER3 are oligomeric proteins that share germin's resistance to pepsin and to dissociation by heat and SDS. In contrast, AtGER1 seems to exist as a monomer. The GLPs may interact with the ECM in a variety of ways, since each is efficiently extracted by different conditions. In addition, germins and GLPs all bind Cibacron Blue, a dye often but not exclusively used for the purification of enzymes having nucleotide cofactors. In the case of AtGER2, binding to the dye is so tight that it almost allows a one-step purification of this protein. The variety of sequences, expression patterns and biochemical features indicates that GLPs could be a class of receptors localized in the ECM and involved in physiological and developmental processes as well as stress response. Received: 28 June 1999 / Accepted: 6 December 1999     

Cellular desiccation and hydration: developmentally regulated proteins, and the maturation and germination of seed embryos.

Little more than a decade ago, 2-dimensional mapping of proteins and biochemical study of their allied coding elements (mRNA and DNA) were first used to probe possible changes in the embryo during seed germination. Because specification was of primary importance, our attention was initially directed toward the characterization of individual proteins and coding elements which, in preliminary surveys of the germinating wheat embryo, were found to be conspicuously subject to developmental regulation. Three of the proteins have become subjects of comprehensive investigations in this and other laboratories: the Em protein, the Ec protein, and germin. The Em and Ec proteins are encoded by the conserved mRNA 'stored' in the mature embryos of dry, field-ripened seeds but germin is encoded by the nascent mRNA formed after mature embryos are germinated in water. The Ec protein is the only bona fide Zn metallothionein yet found in higher plants. Studies of their biology and molecular biology suggest that the Em protein has a role in hormone-mediated (abscisic acid) cellular desiccation and that germin has a role in hormone-mediated (auxin) cellular hydration. It is projected that further studies of the Em protein may help elucidate the molecular basis for a loss of dessication tolerance during germination, and that further studies of germin may help elucidate the molecular basis of plant cell enlargement.     

The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two‐dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix‐assisted laser‐desorption ionisation time of flight mass spectrometry (MALDI ‐ TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two‐dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin‐like proteins or glucanases, and cell expansion, i.e. β‐D‐glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.     

Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 A resolution, comprises beta-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.