Karen Skriver

Gene expression in response to abscisic acid and osmotic stress.

Abscisic acid (ABA) was discovered in the 1950s to be a phytohormone affecting leaf abscision and bud dormancy. It was soon characterized as a sesquiterpene derived from mevalonate although certain steps of its biosynthesis in plants are still unknown (Li and Walton, 1987; Zeevaart and Creelman, 1988). Continuing work on ABA has shown that it mediates various developmental and physiological processes that affect the agronomic performance of crop plants (Austin et al., 1982; Ramagopal, 1987). These proc? esses include embryo maturation and germination as well as the response of vegetative tissues to osmotic stress (Singh et al., 1987; Zeevaart and Creelman, 1988). ABA levels increase in tissues subjected to osmotic stress by desiccation, salt, or cold (Henson, 1984; Mohapatra et al., 1988). Under these conditions, specific genes are ex? pressed that can also be induced in unstressed tissues by the application of exogenous ABA (Singh et al., 1987; Gomez et al., 1988; Mundy and Chua, 1988). Some of these genes are also expressed during the normal embryogenic program when seeds desiccate and embryos be? come dormant (Dure et al., 1981). Although different sets of ABA-responsive genes exhibit different patterns of de? velopmental and tissue-specific expression, some of them appear to be part of a general reaction to osmotic stress. This system is a normal part of the embryogenic program but is inducible in vegetative tissues at other times in the plant life cycle. Several ABA-responsive genes have now been isolated (Baker et al., 1988; Gomez et al., 1988; Marcotte et al., 1988; Mundy and Chua, 1988; Vilardell et al., 1990; Yamaguchi-Shinozaki et al., 1990). A major goal of the research discussed below is to understand the role these genes play in osmotic stress and desiccation tolerance.

NESbase version 1.0: a database of nuclear export signals

Protein export from the nucleus is often mediated by a Leucine-rich Nuclear Export Signal (NES). NESbase is a database of experimentally validated Leucine-rich NESs curated from literature. These signals are not annotated in databases such as SWISS-PROT, PIR or PROSITE. Each NESbase entry contains information of whether NES was shown to be necessary and/or sufficient for export, and whether the export was shown to be mediated by the export receptor CRM1. The compiled information was used to make a sequence logo of the Leucine-rich NESs, displaying the conservation of amino acids within a window of 25 residues. Surprisingly, only 36% of the sequences used for the logo fit the widely accepted NES consensus L-x(2,3)-[LIVFM]-x(2,3)-L-x-[LI]. The database is available online at http://www.cbs.dtu.dk/databases/NESbase/.

Structure, function and networks of transcription factors involved in abiotic stress responses.

Transcription factors (TFs) are master regulators of abiotic stress responses in plants. This review focuses on TFs from seven major TF families, known to play functional roles in response to abiotic stresses, including drought, high salinity, high osmolarity, temperature extremes and the phytohormone ABA. Although ectopic expression of several TFs has improved abiotic stress tolerance in plants, fine-tuning of TF expression and protein levels remains a challenge to avoid crop yield loss. To further our understanding of TFs in abiotic stress responses, emerging gene regulatory networks based on TFs and their direct targets genes are presented. These revealed components shared between ABA-dependent and independent signaling as well as abiotic and biotic stress signaling. Protein structure analysis suggested that TFs hubs of large interactomes have extended regions with protein intrinsic disorder (ID), referring to their lack of fixed tertiary structures. ID is now an emerging topic in plant science. Furthermore, the importance of the ubiquitin-proteasome protein degradation systems and modification by sumoylation is also apparent from the interactomes. Therefore; TF interaction partners such as E3 ubiquitin ligases and TF regions with ID represent future targets for engineering improved abiotic stress tolerance in crops.

Ligand mimicry? Plant-parasitic nematode polypeptide with similarity to CLAVATA3

The importance of peptides in plant intercellular signaling has become apparent during the past decade. Among recently identified peptide signals is CLAVATA3 (CLV3), which is involved in cell-fate determination in the shoot apical meristem of Arabidopsis. There is evidence that CLV3 is a ligand for CLAVATA1 (CLV1), a receptor kinase with an extracellular domain containing leucine-rich repeats (LRRs) [1xSignaling through the CLAVATA1 receptor complex. DeYoung, B.J. and Clark, S.E. Plant Mol. Biol. 2001; 46: 505–513CrossRef | PubMed | Scopus (27)See all References][1]. The Arabidopsis genome contains a large gene family, called CLE for CLAVATA3/ESR-related, encoding polypeptides with similarity to CLV3. These small polypeptides are characterized by a short, C-terminal motif and an N-terminal signal peptide or signal anchor [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. Similar sequences are encoded by expressed sequence tag (EST) clones from various plants.Using motif-based database search methods, we have discovered sequence similarity between the CLE polypeptides and an esophageal gland cell polypeptide from Heterodera glycines, the soybean cyst nematode (Fig. 1Fig. 1) . The sequence similarity between the plant polypeptide family and the H. glycines polypeptide, here referred to as HgCLE, was evident from a position-specific iterative BLAST (PSI-BLAST) [3xGapped BLAST and PSI-BLAST: a new generation of protein database search programs. Altschul, S.F. et al. Nucleic Acids Res. 1997; 25: 3389–3402CrossRef | PubMed | Scopus (44567)See all References][3] database search with the CLV3 sequence. Furthermore, the motif-discovery tool MEME [4xFitting a mixture model by expectation maximization to discover motifs in biopolymers. Bailey, T.L. and Elkan, C. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1994; 2: 28–36PubMedSee all References][4] was applied to the CLE sequences, and the resulting file was used as input to the MAST algorithm [5xCombining evidence using p-values: application to sequence homology searches. Bailey, T.L. and Gribskov, M. Bioinformatics. 1998; 14: 48–54CrossRef | PubMedSee all References][5] in a database search. The high-scoring sequences were CLV3-like plant polypeptides, but also included HgCLE.Fig. 1Alignment of the esophageal gland cell polypeptide from Heterodera glycines (GenBank Accession number: AAG21331, here referred to as HgCLE) with CLAVATA3 and Arabidopsis CLE sequences. Also included in the alignment is the sequence encoded by wheat EST clone BE401912, exemplifying a sequence encoding multiple CLE motifs. CLE1–CLE27 were identified by J. Mark Cock and Sheila McCormick [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. CLE40 and CLE41 are included in the annotation of the Arabidopsis genome performed by The Institute for Genomic Research, Rockville, MD, USA (http://www.tigr.org); the sequences are encoded by loci At5g12990 and At3g24770, respectively. Residues highlighted in red are common to at least half of the sequences, and residues highlighted in blue are chemically similar in more than half of the sequences or similar to the dominating residue. The last line shows the consensus residues. Predicted signal peptides are underlined. CLE16, CLE25, CLE26 and CLE27 are predicted to contain an N-terminal signal anchor instead of a signal peptide [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. The three occurrences of the conserved motif in the sequence encoded by EST clone BE401912 are highlighted in gray.View Large Image | Download PowerPoint SlideH. glycines is a sedentary plant-parasitic nematode. The infective juvenile stage penetrates the root and migrates to a site near the vascular tissue to establish a permanent feeding site. The nematode induces the transformation of plant cells into metabolically active feeding cells. Secretions from the esophageal gland cells of the nematode are released through the stylet, a mouth spear that is used to pierce plant cell walls. These secretions are thought to contain the substances that cause the transformation of root cells by altering gene expression in the cells [6xNematode parasitism genes. Davis, E.L. et al. Annu. Rev. Phytopathol. 2000; 38: 365–396CrossRef | PubMed | Scopus (186)See all References][6].HgCLE is a hypothetical polypeptide predicted from cDNA sequences cloned from esophageal gland cells [7xSignal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Wang, X. et al. Mol. Plant–Microbe Interact. 2001; 14: 536–544CrossRef | PubMedSee all References, 8xIdentification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Gao, B. et al. Mol. Plant–Microbe Interact. 2001; 14: 1247–1254CrossRef | PubMedSee all References]. The cDNA clone hybridized to genomic DNA of H. glycines, and expression of the gene was specifically detected in the dorsal esophageal gland cell of parasitic stages of H. glycines [7xSignal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Wang, X. et al. Mol. Plant–Microbe Interact. 2001; 14: 536–544CrossRef | PubMedSee all References][7]. The dorsal gland cell is the predominate gland of the parasitic stages of H. glycines [6xNematode parasitism genes. Davis, E.L. et al. Annu. Rev. Phytopathol. 2000; 38: 365–396CrossRef | PubMed | Scopus (186)See all References][6]. HgCLE contains the C-terminal motif and the N-terminal signal peptide that characterize the CLE sequences (Fig. 1Fig. 1). Predicted signal peptide cleavage produces a polypeptide of 117 amino acids, but further processing cannot be excluded. It has yet to be determined whether the CLE polypeptides are processed subsequent to signal peptide cleavage [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. Indeed, several EST clones from wheat encode sequences containing multiple CLE motifs (Fig. 1Fig. 1). The striking resemblance to a polyprotein precursor could suggest that the conserved motif constitutes the mature CLE peptides.Based on the similarity between a bioactive plant peptide and a plant-parasite polypeptide, we hypothesize that the nematode has co-opted the plant signaling peptide for parasitic modification of host plant cells. HgCLE might thus function as a ligand for a host LRR receptor kinase, possibly imitating or inhibiting the function of an endogenous peptide ligand. This hypothesis concurs with a model proposed by David McKenzie Bird [9xManipulation of host gene expression by root-knot nematodes. Bird, D.M. J. Parasitol. 1996; 82: 881–888CrossRef | PubMed | Scopus (52)See all References][9] to explain the development of nematode-induced feeding sites. Moreover, it has been demonstrated that a low-molecular-weight peptide component of potato cyst nematode secretions can co-stimulate the proliferation of tobacco protoplasts in the presence of phytohormones [10xNaturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells. Goverse, A. et al. Mol. Plant–Microbe Interact. 1999; 12: 872–881CrossRef | PubMedSee all References][10]. It would be interesting to determine the effects of HgCLE in a plant protoplast proliferation assay. Furthermore, double-stranded RNA-mediated interference of HgCLE gene expression could be applied to investigate the role of HgCLE in the plant–parasite interaction [11xIngestion of double-stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference. Urwin, P.E. et al. Mol. Plant–Microbe Interact. 2002; 15: 747–752CrossRef | PubMedSee all References][11].Database searches revealed no sequences from Caenorhabditis elegans or other non-plant organisms with the characteristics of HgCLE or of the plant CLE sequences. The intriguing similarity between host and parasite sequences could be an example of adaptive molecular mimicry, defined by Roger Hall [12xMolecular mimicry. Hall, R. Adv. Parasitol. 1994; 34: 81–132CrossRef | PubMedSee all References][12] as ‘a parasite molecule mimics a host molecule for a biological reason’. Although lateral gene transfer influences the evolution of archeal and bacterial genomes, the transfer of genes between two multicellular eukaryotes is not expected to be common [13xPhylogenetic classification and the universal tree. Doolittle, W.F. Science. 1999; 284: 2124–2128CrossRef | PubMed | Scopus (998)See all References][13]. Considering the simplicity of the features shared by HgCLE and the CLE sequences, a more likely explanation of the sequence similarity is convergent evolution. If indeed this is an example of ligand mimicry, it emphasizes the importance of peptide ligands in plant biology and constitutes a convergence of research in nematode parasitism and peptide signaling.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin‐immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G]CGT as ATAF1 consensus binding sequences. Co‐expression analysis across publicly available microarray experiments identified 25 genes co‐expressed with ATAF1. The promoter regions of ATAF1 co‐expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP‐qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

Structure and expression of the barley lipid transfer protein gene Ltp1.

We have characterized a gene (Ltp1) encoding a barley lipid transfer protein. Northern blot analysis showed that Ltp1 mRNA accumulates specifically in the aleurone layer of developing and germinating seeds. Southern blot analysis indicated that LTP1 protein is encoded by a single gene in barley. Sequence analysis of Ltp1 showed that it contains an open reading frame of 351 bp interrupted by a single intron of 133 bp. Transient expression assays indicated that 702 bp of the 5′ upstream region of Ltp1 is sufficient to direct aleurone-specific expression during late seed development and early germination.

Senescence-associated Barley NAC (NAM, ATAF1,2, CUC) Transcription Factor Interacts with Radical-induced Cell Death 1 through a Disordered Regulatory Domain

Background: Plant NAC transcription factors (TF) are important regulators of senescence. Results: Senescence-associated barley HvNAC013 uses intrinsic disorder in transcriptional activation and interactions. Conclusion: Radical induced cell death 1 exploits the intrinsic disorder of HvNAC013 and other TFs for interactions without structure induction in HvNAC013. Significance: This first structural characterization of NAC intrinsic disorder may reveal general features of important TF regulatory interactions. Senescence in plants involves massive nutrient relocation and age-related cell death. Characterization of the molecular components, such as transcription factors (TFs), involved in these processes is required to understand senescence. We found that HvNAC005 and HvNAC013 of the plant-specific NAC (NAM, ATAF1,2, CUC) TF family are up-regulated during senescence in barley (Hordeum vulgare). Both HvNAC005 and HvNAC013 bound the conserved NAC DNA target sequence. Computational and biophysical analyses showed that both proteins are intrinsically disordered in their large C-terminal domains, which are transcription regulatory domains (TRDs) in many NAC TFs. Using motif searches and interaction studies in yeast we identified an evolutionarily conserved sequence, the LP motif, in the TRD of HvNAC013. This motif was sufficient for transcriptional activity. In contrast, HvNAC005 did not function as a transcriptional activator suggesting that an involvement of HvNAC013 and HvNAC005 in senescence will be different. HvNAC013 interacted with barley radical-induced cell death 1 (RCD1) via the very C-terminal part of its TRD, outside of the region containing the LP motif. No significant secondary structure was induced in the HvNAC013 TRD upon interaction with RCD1. RCD1 also interacted with regions dominated by intrinsic disorder in TFs of the MYB and basic helix-loop-helix families. We propose that RCD1 is a regulatory protein capable of interacting with many different TFs by exploiting their intrinsic disorder. In addition, we present the first structural characterization of NAC C-terminal domains and relate intrinsic disorder and sequence motifs to activity and protein-protein interactions.

Widespread occurrence of a highly conserved RING-H2 zinc finger motif in the model plant Arabidopsis thaliana

Several novel Arabidopsis thaliana proteins containing a RING‐H2 zinc finger motif were predicted after database searches. Alignment of 29 RING‐H2 finger sequences shows that the motif is strikingly conserved in otherwise unrelated proteins. Only short, non‐conserved polar/charged sequences distinguish these domains. The RING‐H2 domain is most often present in multi‐domain structures, a number of which are likely to contain a membrane‐spanning region or an additional zinc finger. However, there are several small (126–200 residues) proteins consisting of an N‐terminal domain, rich in aliphatic residues, and a C‐terminal RING‐H2 domain. Reverse‐transcription PCR suggests that the RING‐H2 genes are widely expressed at low levels.

A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana

Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domains sequence. The developed methodology, including the application of complementary functional genomics filters, makes it possible to translate, for each TF, protein binding microarray data into a set of high-quality target genes. With this approach, we confirm NAC target genes reported from independent in vivo analyses. We emphasize that candidate target gene sets together with the workflow associated with functional modules offer a strong resource to unravel the regulatory potential of NAC genes and that this workflow could be used to study other families of transcription factors.