Anne Berna

Germins and germin-like proteins : Plant do-all proteins. But what do they do exactly?

Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant proteins. The name germin was given because the first described member of the family, wheat germin, was discovered in germinating wheat grains. However, it is now known that proteins from this family exist in all organs and developmental stages and that several are also involved in the response to various stress conditions. It is thus increasingly obvious that germins and GLPs participate in many processes that are important for plant development and defence. However, their exact participation in these processes generally remains obscure so the main challenge now is to determine the precise activities and functions of all germins and GLPs. Molecular and biochemical studies are contributing to a better understanding of this protein family and much data has accumulated in the last 2 years. All species possess a wide range of GLPs and each GLP gene is subjected to a tight regulation. All germins and GLPs are glycoproteins somehow associated with the extracellular matrix. More specifically, three classes of functions are starting to be recognized for these proteins: some possess an enzymatic activity (oxalate oxidase or superoxide dismutase), others seem to be structural proteins while some others act as receptors. To explain the diversity and ubiquitousness of germins and GLPs, we propose that most, if not all, of them carry out a combination of different functions.

Regulation by biotic and abiotic stress of a wheat germin gene encoding oxalate oxidase, a H2O2-producing enzyme.

Germins and germin-like proteins (GLPs) constitute a ubiquitous family of plant proteins that seem to be involved in many developmental and stress-related processes. Wheat germin has been extensively studied at the biochemical level: it is found in the apoplast and the cytoplasm of germinating embryo cells and it has oxalate oxidase activity (EC 1.2.3.4). Germin synthesis can also be induced in adult wheat leaves by auxins and by a fungal pathogen but it remains to be determined whether the same gene is involved in developmental, hormonal and stress response. In this work, we have studied the expression of one of the wheat germin genes, named gf-2.8, in wheat as well as in transgenic tobacco plants transformed with either this intact gene or constructs with GUS driven by its promoter. This has allowed us to demonstrate that expression of this single gene is both developmentally and pathogen- regulated. In addition, we show that expression of the wheat gf-2.8 germin gene is also stimulated by some abiotic stresses, especially the heavy metal ions Cd2+, Cu2+ and Co2+. Several chemicals involved in stress signal transduction pathways were also tested: only polyamines were shown to stimulate expression of this gene. Because regulation of the wheat gf-2.8 germin gene is complex and because its product results in developmental and stress-related release of hydrogen peroxide in the apoplast, it is likely that it plays an important role in several aspects of plant growth and defence mechanisms.

REGULATED EXPRESSION OF A WHEAT GERMIN GENE IN TOBACCO : OXALATE OXIDASE ACTIVITY AND APOPLASTIC LOCALIZATION OF THE HETEROLOGOUS PROTEIN

Wheat (Triticum aestivum) germin is a homopentameric glycoprotein whose synthesis is allied with seed germination. Germin pentamers show an unusual resistance to dissociation and possess an oxalate oxidase (OxO) activity. In order to increase our knowledge of germin gene expression, the function(s) of germin during development and possible uses in plant genetic engineering, an in vivo expression system is required. To this end, a gene for germin, named gf-2.8, was studied by expressing either promoter-GUS fusions or the intact gene in transgenic tobacco (Nicotiana tabacum) plants. Heterologous gene transcription was monitored in vitro and in vivo by GUS or OxO activity and was found to occur in developing seeds and in seedlings. This transcription was stimulated by auxins, as would be expected because of the presence of putative auxin-responsive elements in the promoter of the gf-2.8 gene. Auxin stimulation also extended to young leaves since OxO activity could be detected in treated but not in untreated leaves. The biochemical characteristics of wheat germin were also conserved in a transgenic host: the OxO activity was present under the form of a doublet co-migrating with germin G and G′ isoforms. Also, germin distributed between a soluble and an apoplastic fractions despite the fact that wheat cell wall substantially differs from tobacco cell wall. Therefore, tobacco constitutes a suitable host for in vivo studies of this monocotyledon gene.

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

Abstract. Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germins 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 germins 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.

A Non-structural Protein of Alfalfa Mosaic Virus in the Walls of Infected Tobacco Cells

Summary The 32000 mol. wt. (32K) non-structural protein of alfalfa mosaic virus, P3, has previously been detected in a crude membrane fraction of infected tobacco leaves, where it accumulated transiently at the beginning of the infection period. We show here, by immunoblotting with an antiserum to a synthetic peptide corresponding to the C terminus of the protein, that the majority of P3 is in the cell wall fraction where it remains throughout infection, both at 25 °C and at 10 °C. The cell wall-associated, P3-related material is heterogeneous and contains polypeptide species of slightly lower electrophoretic mobility than the major in vitro translation product of RNA 3, which suggests that P3 may be post-translationally modified.

In situ location of an alfalfa mosaic virus non-structural protein in plant cell walls: correlation with virus transport

Summary The 32000 mol. wt. non-structural protein (P3) of alfalfa mosaic virus (AlMV) has previously been shown to accumulate in the cell wall fraction of tobacco leaves infected with AlMV. We now report the ultrastructural location of this protein. P3 was visualized immunocytochemically in the middle lamella of the walls of those parenchymal or epidermal cells which had just been reached by the infection front and in which viral multiplication had just begun. P3 was not found when AlMV had accumulated to high levels in infected cells. These findings support the concept that P3 is involved in the spread of viral infection from cell to cell, i.e. is the transport factor of AlMV.

Ring up the curtain on DING proteins

DING proteins have a characteristic DINGGG‐ or closely related N‐terminal sequence. One is found in human synovial fluid, and may be associated with rheumatoid arthritis. Other examples have receptor or signalling roles in various human and animal cells, or are involved in biomineralisation, and several of them bind to phytochemicals. As plant DING proteins have recently been discovered, we hypothesise that the DING protein‐phytochemical association may represent one aspect of a ubiquitous receptor‐linked signalling system. Several microbial proteins related to DING proteins have phosphatase activity, which may relate to biomineralisation in eukaryotic systems. Plant DING proteins and their microbial relatives may elicit allergic responses leading to arthritic disease.

cDNA sequence, genomic organization and differential expression of three Arabidopsis genes for germin/oxalate oxidase-like proteins

Wheat germin is a protein expressed during germination which possesses an oxalate oxidase activity. Germin-type oxalate oxidases have been extensively studied in monocotyledons (wheat and barley) where they are thought to have important functions for development, stress response and defence against pathogens. In contrast, almost nothing is known about the germin-like proteins found in dicotyledons, gymnosperms and myxomycetes. In this work, cDNA clones for three genes (ATGER1, ATGER2 and ATGER3) encoding germin-like proteins, initially characterized as expressed sequence tags (ESTs), from Arabidopsis thaliana cDNA libraries were further characterized. In addition, we isolated and sequenced a Brassica napus cDNA which was strongly homologous to the cDNA for ATGER1. Sequence analysis and secondary structure predictions of the proteins encoded by these cDNAs showed that they possess all the characteristic features of members of the germin family and of the germin/seed globulins/sucrose binding protein superfamily. Sequence comparisons and mapping demonstrated the existence of at least two different gene families in the A. thaliana genome encoding a minimum of three genes for germins. These three genes have been mapped in three different location on the Arabidopsis genome. By northern blot hybridizations we found that these genes are differentially regulated. ATGER1 was expressed during germination, like wheat germin, but also in leaves whereas ATGER2 transcripts were exclusively found in developing embryos, like wheat pseudo-germin. ATGER3 mRNAs were found in leaves and flowers and their abundance was shown to vary during the circadian cycle.

Kinetics of accumulation of the three non-structural proteins of alfalfa mosaic virus in tobacco plants

Summary Antisera to synthetic peptides corresponding to the C-termini of two non-structural proteins (NSP) of alfalfa mosaic virus (AIMV), P1 (126K) and P3 (32K), were prepared and characterized. These antisera, together with one which had previously been made against the C-terminus of P2 (90K), enabled us to detect the three NSPs in a crude membrane fraction from AlMV-infected tobacco leaves. The accumulation of these proteins at 25 °C and at 10 °C was followed as a function of time after inoculation, and their amounts were compared with viral replicase activity. All three proteins accumulated at the beginning of the infection period and then disappeared, P3 more rapidly than the other two. There was a good correlation between replicase activity and the amounts of P1 and P2, but not the amount of P3. These results are consistent with the notion that P1 and P2 are part of the replication complex. Although much less coat protein was made in inoculated leaves at 10 °C than at 25 °C, the maximum amounts of the three NSPs and the maximum replicase activity were at least as high at 10 °C as at 25 °C. Thus, 10 °C is not a restrictive temperature for the assembly of a functional replication complex.

Involvement of the Phospholipid Sterol Acyltransferase1 in Plant Sterol Homeostasis and Leaf Senescence

Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging.