Multicellular organisms produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are other classes restricted to the plant kingdom. These include thionins, lipid transfer proteins and snakins. In earlier work, we identified several divergent classes of small putatively secreted cysteine-rich peptides (CRPs) in legumes [Graham et al. (2004)Plant Physiol. 135, 1179-97]. Here, we built sequence motif models for each of these classes of peptides, and iteratively searched for related sequences within the comprehensive UniProt protein dataset, the Institute for Genomic Research's 33 plant gene indices, and the entire genomes of the model dicot, Arabidopsis thaliana, and the model monocot and crop species, Oryza sativa (rice). Using this search strategy, we identified approximately 13,000 plant genes encoding peptides with common features: (i) an N-terminal signal peptide, (ii) a small divergent charged or polar mature peptide with conserved cysteines, (iii) a similar intron/exon structure, (iv) spatial clustering in the genomes studied, and (v) overrepresentation in expressed sequences from reproductive structures of specific taxa. The identified genes include classes of defensins, thionins, lipid transfer proteins, and snakins, plus other protease inhibitors, pollen allergens, and uncharacterized gene families. We estimate that these classes of genes account for approximately 2-3% of the gene repertoire of each model species. Although 24% of the genes identified were not annotated in the latest Arabidopsis genome releases (TIGR5, TAIR6), we confirmed expression via RT-PCR for 59% of the sequences attempted. These findings highlight limitations in current annotation procedures for small divergent peptide classes.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2007.03136.x

Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants.

Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.

OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis

GRAS proteins belong to a plant-specific transcription factor family. Currently, 33 GRAS members including a putative expressed pseudogene have been identified in the Arabidopsis genome. With a reverse genetic approach, we have constructed a "phenome-ready unimutant collection" of the GRAS genes in Arabidopsis thaliana. Of this collection, we focused on loss-of-function mutations in 23 novel GRAS members. Under standard conditions, homozygous mutants have no obvious morphological phenotypes compared with those of wild-type plants. Expression analysis of GRAS genes using quantitative real-time RT-PCR (qRT-PCR), microarray data mining, and promoter::GUS reporter fusions revealed their tissue-specific expression patterns. Our analysis of protein-protein interaction and subcellular localization of individual GRAS members indicated their roles as transcription regulators. In our yeast two-hybrid (Y2H) assay, we confirmed the protein-protein interaction between SHORT-ROOT (SHR) and SCARECROW (SCR). Furthermore, we identified a new SHR-interacting protein, SCARECROW-LIKE23 (SCL23), which is the most closely related to SCR. Our large-scale analysis provides a comprehensive evaluation on the Arabidopsis GRAS members, and also our phenome-ready unimutant collection will be a useful resource to better understand individual GRAS proteins that play diverse roles in plant growth and development.

Large-scale analysis of the GRAS gene family in Arabidopsis thaliana.

The sequencing of large, complex genomes has become routine, but understanding how sequences relate to biological function is less straightforward. Although much attention is focused on how to annotate genomic features such as developmental enhancers and non-coding RNAs, there is still no higher eukaryote for which we know the correct exon-intron structure of at least one ORF for each gene. Despite this uncomfortable truth, genome annotation has made remarkable progress since the first drafts of the human genome were analysed. By combining several computational and experimental methods, we are now closer to producing complete and accurate gene catalogues than ever before.

Steady progress and recent breakthroughs in the accuracy of automated genome annotation

In support of the international effort to obtain a reference sequence of the bread wheat genome and to provide plant communities dealing with large and complex genomes with a versatile, easy-to-use online automated tool for annotation, we have developed the TriAnnot pipeline. Its modular architecture allows for the annotation and masking of transposable elements, the structural, and functional annotation of protein-coding genes with an evidence-based quality indexing, and the identification of conserved non-coding sequences and molecular markers. The TriAnnot pipeline is parallelized on a 712 CPU computing cluster that can run a 1-Gb sequence annotation in less than 5 days. It is accessible through a web interface for small scale analyses or through a server for large scale annotations. The performance of TriAnnot was evaluated in terms of sensitivity, specificity, and general fitness using curated reference sequence sets from rice and wheat. In less than 8 h, TriAnnot was able to predict more than 83% of the 3,748 CDS from rice chromosome 1 with a fitness of 67.4%. On a set of 12 reference Mb-sized contigs from wheat chromosome 3B, TriAnnot predicted and annotated 93.3% of the genes among which 54% were perfectly identified in accordance with the reference annotation. It also allowed the curation of 12 genes based on new biological evidences, increasing the percentage of perfect gene prediction to 63%. TriAnnot systematically showed a higher fitness than other annotation pipelines that are not improved for wheat. As it is easily adaptable to the annotation of other plant genomes, TriAnnot should become a useful resource for the annotation of large and complex genomes in the future.

https://hal.inria.fr/hal-00753407/document

TriAnnot: A Versatile and High Performance Pipeline for the Automated Annotation of Plant Genomes.

Background
Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.

https://link.springer.com/content/pdf/10.1186%2F1746-4811-6-18.pdf

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Several lines of evidence support the existence of novel genes and other transcribed units which have not yet been annotated in the Arabidopsis genome. Two gene prediction programs which make use of comparative genomic analysis, Twinscan and EuGene, have recently been deployed on the Arabidopsis genome. The ability of these programs to make use of sequence data from other species has allowed both Twinscan and EuGene to predict over 1000 genes that are intergenic with respect to the most recent annotation release. A high throughput RACE pipeline was utilized in an attempt to verify the structure and expression of these novel genes. 1,071 un-annotated loci were targeted by RACE, and full length sequence coverage was obtained for 35% of the targeted genes. We have verified the structure and expression of 378 genes that were not present within the most recent release of the Arabidopsis genome annotation. These 378 genes represent a structurally diverse set of transcripts and encode a functionally diverse set of proteins. We have investigated the accuracy of the Twinscan and EuGene gene prediction programs and found them to be reliable predictors of gene structure in Arabidopsis. Several hundred previously un-annotated genes were validated by this work. Based upon this information derived from these efforts it is likely that the Arabidopsis genome annotation continues to overlook several hundred protein coding genes.

/pdf/experimental-validation-of-novel-genes-predicted-in-the-un-3c3b1yv8ki.pdf

Experimental validation of novel genes predicted in the un-annotated regions of the Arabidopsis genome

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High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Experimental validation of novel genes predicted in the un-annotated regions of the Arabidopsis genome