Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination.

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

Flowering Time-Regulated Genes in Maize Include the Transcription Factor ZmMADS1.

Flowering time (FTi) control is well examined in the long-day plant Arabidopsis (Arabidopsis thaliana), and increasing knowledge is available for the short-day plant rice (Oryza sativa). In contrast, little is known in the day-neutral and agronomically important crop plant maize (Zea mays). To learn more about FTi and to identify novel regulators in this species, we first compared the time points of floral transition of almost 30 maize inbred lines and show that tropical lines exhibit a delay in flowering transition of more than 3 weeks under long-day conditions compared with European flint lines adapted to temperate climate zones. We further analyzed the leaf transcriptomes of four lines that exhibit strong differences in flowering transition to identify new key players of the flowering control network in maize. We found strong differences among regulated genes between these lines and thus assume that the regulation of FTi is very complex in maize. Especially genes encoding MADS box transcriptional regulators are up-regulated in leaves during the meristem transition. ZmMADS1 was selected for functional studies. We demonstrate that it represents a functional ortholog of the central FTi integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) of Arabidopsis. RNA interference-mediated down-regulation of ZmMADS1 resulted in a delay of FTi in maize, while strong overexpression caused an early-flowering phenotype, indicating its role as a flowering activator. Taken together, we report that ZmMADS1 represents a positive FTi regulator that shares an evolutionarily conserved function with SOC1 and may now serve as an ideal stating point to study the integration and variation of FTi pathways also in maize.

Sequence and ionomic analysis of divergent strains of maize inbred line B73 with an altered growth phenotype.

Maize (Zea mays) is the most widely grown crop species in the world and a classical model organism for plant research. The completion of a high-quality reference genome sequence and the advent of high-throughput sequencing have greatly empowered re-sequencing studies in maize. In this study, plants of maize inbred line B73 descended from two different sets of seed material grown for several generations either in the field or in the greenhouse were found to show a different growth phenotype and ionome under phosphate starvation conditions and moreover a different responsiveness towards mycorrhizal fungi of the species Glomus intraradices (syn: Rhizophagus irregularis). Whole genome re-sequencing of individuals from both sets and comparison to the B73 reference sequence revealed three cryptic introgressions on chromosomes 1, 5 and 10 in the line grown in the greenhouse summing up to a total of 5,257 single-nucleotide polymorphisms (SNPs). Transcriptome sequencing of three individuals from each set lent further support to the location of the introgression intervals and confirmed them to be fixed in all sequenced individuals. Moreover, we identified >120 genes differentially expressed between the two B73 lines. We thus have found a nearly-isogenic line (NIL) of maize inbred line B73 that is characterized by an altered growth phenotype under phosphate starvation conditions and an improved responsiveness towards symbiosis with mycorrhizal fungi. Through next-generation sequencing of the genomes and transcriptomes we were able to delineate exact introgression intervals. Putative de novo mutations appeared approximately uniformly distributed along the ten maize chromosomes mainly representing G:C -> A:T transitions. The plant material described in this study will be a valuable tool both for functional studies of genes differentially expressed in both B73 lines and for research on growth behavior especially in response to symbiosis between maize and mycorrhizal fungi.

External application of gametophyte-specific ZmPMEI1 induces pollen tube burst in maize.

Regulated demethylesterification of homogalacturonan, a major component of plant cell walls, by the activity of pectin methylesterases (PMEs), plays a critical role for cell wall stability and integrity. Especially fast growing plant cells such as pollen tubes secrete large amounts of PMEs toward their apoplasmic space. PME activity itself is tightly regulated by its inhibitor named as PME inhibitor and is thought to be required especially at the very pollen tube tip. We report here the identification and functional characterization of PMEI1 from maize (ZmPMEI1). We could show that the protein acts as an inhibitor of PME but not of invertases and found that its gene is strongly expressed in both gametophytes (pollen grain and embryo sac). Promoter reporter studies showed gene activity also during pollen tube growth toward and inside the transmitting tract. All embryo sac cells except the central cell displayed strong expression. Weaker signals were visible at sporophytic cells of the micropylar region. ZmPMEI1-EGFP fusion protein is transported within granules inside the tube and accumulates at the pollen tube tip as well as at sites where pollen tubes bend and/or change growth directions. The female gametophyte putatively influences pollen tube growth behavior by exposing it to ZmPMEI1. We therefore simulated this effect by applying recombinant protein at different concentrations on growing pollen tubes. ZmPMEI1 did not arrest growth, but destabilized the cell wall inducing burst. Compared with female gametophyte secreted defensin-like ZmES4, which induces burst at the very pollen tube tip, ZmPMEI1-induced burst occurs at the subapical region. These findings indicate that ZmPMEI1 secreted by the embryo sac likely destabilizes the pollen tube wall during perception and together with other proteins such as ZmES4 leads to burst and thus sperm release.

OPTIMAS-DW: a comprehensive transcriptomics, metabolomics, ionomics, proteomics and phenomics data resource for maize.

BACKGROUND: Maize is a major crop plant, grown for human and animal nutrition, as well as a renewable resource for bioenergy. When looking at the problems of limited fossil fuels, the growth of the world's population or the world's climate change, it is important to find ways to increase the yield and biomass of maize and to study how it reacts to specific abiotic and biotic stress situations. Within the OPTIMAS systems biology project maize plants were grown under a large set of controlled stress conditions, phenotypically characterised and plant material was harvested to analyse the effect of specific environmental conditions or developmental stages. Transcriptomic, metabolomic, ionomic and proteomic parameters were measured from the same plant material allowing the comparison of results across different omics domains. A data warehouse was developed to store experimental data as well as analysis results of the performed experiments. DESCRIPTION: The OPTIMAS Data Warehouse (OPTIMAS-DW) is a comprehensive data collection for maize and integrates data from different data domains such as transcriptomics, metabolomics, ionomics, proteomics and phenomics. Within the OPTIMAS project, a 44K oligo chip was designed and annotated to describe the functions of the selected unigenes. Several treatment- and plant growth stage experiments were performed and measured data were filled into data templates and imported into the data warehouse by a Java based import tool. A web interface allows users to browse through all stored experiment data in OPTIMAS-DW including all data domains. Furthermore, the user can filter the data to extract information of particular interest. All data can be exported into different file formats for further data analysis and visualisation. The data analysis integrates data from different data domains and enables the user to find answers to different systems biology questions. Finally, maize specific pathway information is provided. CONCLUSIONS: With OPTIMAS-DW a data warehouse for maize was established, which is able to handle different data domains, comprises several analysis results that will support researchers within their work and supports systems biological research in particular. The system is available at http://www.optimas-bioenergy.org/optimas_dw.

Immunomodulation of plant function by in vitro selected single-chain Fv intrabodies.

In this chapter, we discuss and compare the different concepts and examples as well as present the basic protocols for applying intrabody-based approaches in plants for the investigation of cell functions and plant cell-pathogen interactions. The immunomodulation strategy, a molecular technique that allows to interfere with cellular metabolism, signal transduction pathways, or pathogen infectivity, is based on the ectopic expression of genes encoding specific recombinant antibodies. This needs basic prerequisites to be successfully applied as resources and techniques to isolate specific recombinant antibodies with sufficient binding parameters to bind and to block even low-concentrated targets or to compete successfully with substrates and ligands. Also techniques and constructs to efficiently transform plants and to target recombinant antibodies to selected compartments are important requirements. Basic protocols for all these techniques are provided.

Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots.

Transgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.

AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes.

Three members of the Arabidopsis sucrose transporter gene family, AtSUC6-AtSUC8 (At5g43610; At1g66570; At2g14670), share a high degree of sequence homology in their coding regions and even in their introns and in their 5'- and 3'-flanking regions. A fourth sucrose transporter gene, AtSUC9 (At5g06170), which is on the same branch of the AtSUC-phylogenetic tree, shows only slightly less sequence homology. Here we present data demonstrating that two genes from this subgroup, AtSUC6 and AtSUC7, encode aberrant proteins and seem to represent sucrose transporter pseudogenes, whereas AtSUC8 and AtSUC9 encode functional sucrose transporters. These results are based on analyses of splice patterns and polymorphic sites between these genes in different Arabidopsis ecotypes, as well as on functional analyses by cDNA expression in baker's yeast. For one of these genes, AtSUC7 (At1g66570), different, ecotype-specific splice patterns were observed in Wassilewskija (Ws), C24, Columbia wild type (Col-0) and Landsberg erecta (Ler). No incorrect splicing and no sequence polymorphism were detected in the cDNAs of AtSUC8 and AtSUC9, which encode functional sucrose transporters and are expressed in floral tissue. Finally, promoter-reporter gene plants and T-DNA insertion lines were analyzed for AtSUC8 and AtSUC9.

De-etiolated 1 and damaged DNA binding protein 1 interact to regulate Arabidopsis photomorphogenesis.

BACKGROUND: Plant development is exquisitely sensitive to light. Seedlings grown in the dark have a developmentally arrested etiolated phenotype, whereas in the light they develop leaves and complete their life cycle. Arabidopsis de-etiolated 1 (det1) mutants develop like light-grown seedlings even when grown in the dark. DET1 encodes a nuclear protein that appears to act downstream from multiple photoreceptors to regulate morphogenesis and gene expression in response to light. However, its function has remained unknown. RESULTS: We used microarrays to examine defects in transcription in dark-grown det1 seedlings. We found extensive changes in gene expression, including many of the transcriptional responses observed in light-treated wild-type seedlings. We used an epitope-tagging approach to determine the basis of DET1 function. GFP-DET1 rescues the det1 phenotype, is localized to the nucleus, and forms an approximately 350 kDa complex, which is required for full DET1 activity. We affinity-purified the DET1 complex and identified an approximately 120 kDa copurifying protein that is the plant homolog of UV-Damaged DNA Binding Protein 1 (DDB1), a protein implicated in the human disease xeroderma pigmentosa. A null mutation in Arabidopsis DDB1A results in no obvious phenotype on its own, yet it enhances the phenotype of a weak det1 allele. CONCLUSIONS: DET1 and DDB1 interact both biochemically and genetically. In animal cells, DDB1 interacts with histone acetyltransferase complexes. The DET1/DDB1 complex may regulate gene expression in response to light via recruitment of HAT activity. Thus, DET1, whose sequence is conserved in both animals and plants, may play a direct role in the regulation of many genes.