Martin Kreis

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.

The classification and nomenclature of wheat gluten proteins: A reassessment

The nomenclature, relationships and classification of wheat gluten proteins are discussed in relation to recently-reported amino acid sequences of individual components. Although all gliadin and glutenin components show some degree of sequence relationship, three groups of closely related proteins can be recognised. These are high molecular weight (HMW) prolamins (HMW subunits of glutenin), the S-poor prolamins (ω-gliadins) and the S-rich prolamins. The latter group includes gtiadins (α- β-, γ-) and glutenins (LMW subunits). It is concluded that the classical division into gliadins and glutenins is based on a secondary characteristic, the formation of interor intra- molecular disulphide bonds, rather than on homology of the primary amino acid sequences.

Molecular evolution of the seed storage proteins of barley, rye and wheat

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.

Expression of the Arabidopsis thaliana invertase gene family

Abstract. Cell-wall and vacuolar invertases (β-d-fructofuranosidase, EC 3.2.1.26) from Arabidopsis thaliana (L.) Heynh. are encoded by at least four genes, namely Atβfruct1, Atβfruct2, Atβfruct3 and Atβfruct4. Different A. thaliana organs from four developmental stages and under different environmental conditions were analyzed for invertase gene expression. Our results clearly show that both the cell-wall and vacuolar invertase genes are expressed in a development and organ-specific manner. No transcripts of the cell-wall invertase gene Atβfruct1 were found in the cotyledons; however, relatively high levels were detected in the leaves of mature plants. The expression of the second cell-wall gene Atβfruct2 was found to be flower-specific, conversely no expression of Atβfruct1 was detected in flowers. The vacuolar gene Atβfruct3 shows a distinctly different regulation of expression from Atβfruct1. Northern and reverse transcriptase-polymerase chain reaction analyses revealed the presence of transcripts in the cotyledons and only low levels in leaves, roots and flower buds. The second vacuolar invertase gene, Atβfruct4, was found to be expressed in leaves of very young plants, but no transcripts were detected in the leaves of mature flowering plants. In order to investigate the respective roles of invertases and sucrose synthase, a comparative analysis of the expression of these genes was carried out. The present study shows that cell-wall and vacuolar invertase genes are differentially regulated by environmental factors.

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.

Arabidopsis kinome: after the casting

Arabidopsis thaliana is used as a favourite experimental organism for many aspects of plant biology. We capitalized on the recently available Arabidopsis genome sequence and predicted proteome, to draw up a genome-scale protein serine/threonine kinase (PSTK) inventory. The PSTKs represent about 4% of the A. thaliana proteome. In this study, we provide a description of the content and diversity of the non-receptor PSTKs. These kinases have crucial functions in sensing, mediating and coordinating cellular responses to an extensive range of stimuli. A total of 369 predicted non receptor PSTKs were detailed: the Raf superfamily, the CMGC, CaMK, AGC and STE families, as well as a few small clades and orphan sequences. An extensive relationship analysis of these kinases allows us to classify the proteins in superfamilies, families, sub-families and groups. The classification provides a better knowledge of the characteristics shared by the different clades. We focused on the MAP kinase module elements, with particular attention to their docking sites for protein-protein interaction and their biological function. The large number of A. thaliana genes encoding kinases might have been achieved through successive rounds of gene and genome duplications. The evolution towards an increasing gene number suggests that functional redundancy plays an important role in plant genetic robustness.

Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes.

We have identified cDNA clones coding for the major sulphur‐rich and sulphur‐poor groups of barley storage proteins (the B‐ and C‐hordeins, respectively). Hybridization studies have revealed unexpected homologies between B‐ and C‐hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C‐hordein‐related sequences within, or closely associated with, B‐hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B‐hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline‐rich, sulphur‐poor), which is homologous to C‐hordein sequences, and domain 2 (non‐repetitive, proline‐poor, sulphur‐rich), which makes up two‐thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B‐ and C‐hordein mRNAs have an asymmetric base composition (>80% C‐A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro‐Gln‐Gln‐Pro‐Phe‐Pro‐Gln‐Gln. We discuss the evolutionary implications of the domain structure of the B‐hordeins and the unusual relationship between the two groups of barley storage proteins.

Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (hordeum vulgare)

We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.

Functional analysis of the upstream regions of a silent and an expressed member of a family of wheat seed protein genes in transgenic tobacco

The Glu-1Ay gene of bread wheat is a silent member of a family of six endosperm-specific storage protein genes that encodes the high molecular weight (HMW) subunits of glutenin. We previously proposed that the lack of expression of this gene may be due to an 85bp deletion 315bps upstream of the transcription start site. To test this hypothesis we have carried out functional analyses of the 5′-flanking regions of the silent Glu-1Ay gene and an expressed gene of the same family, Glu-1Dx5, in transgenic tobacco plants. They were linked to the reporter gene encoding β-glucuronidase (Gus) and the chimaeric gene constructs were stably integrated into tobacco using Agrobacterium tumefaciens-mediated transformation. The difference in expression of the Glu-1Ay and Glu-1Dx5 genes was maintained in the transgenic plants. Plants transformed with constructs containing upstream sequences of the Glu-1Dx5 gene showed greatly enhanced enzyme activity in the endosperm, the 277 bps immediately upstream of the transcription start site being sufficient for temporal and tissue-specific regulation. In contrast plants transformed with constructs containing an equivalent or longer fragment from the Glu-1Ay gene or this fragment linked to further upstream sequences from a second expressed HMW subunit gene, Glu-1By9 (replacing part of the deleted region) showed no significant increase in Gus activity in any tissue. We conclude that the Glu-1Ay gene is transcriptionally inactive and is silenced by mutations in the 280 bps immediately upstream of the transcription start site and not by the 85-bp deletion or any other changes further upstream. Comparison of this region in seven alleles and homealleles reveals only ten single base pair substitutions unique to the silent gene.

The 5' flanking region of a barley B hordein gene controls tissue and developmental specific CAT expression in tobacco plants.

The 549 base pairs of the 5′ flanking region of a barley seed storage protein (B1 hordein) gene were linked to the reporter gene encoding chloramphenicol acetyl transferase (CAT). The chimaeric gene was transferred into tobacco plants using Agrobacterium tumefaciens. CAT enzyme activity was detected in the seeds, but not in the leaves, of the transgenic plants. Furthermore, enzyme activity was found only in the endosperm, and only from fifteen days after pollination. In contrast, the constitutive 19S promoter from cauliflower mosaic virus (CaMV) directed the expression of the CAT gene in the leaves as well as in both the endosperm and embryo and at all stages in seed development.