Both limiting and toxic soil concentrations of the essential micronutrient boron represent major limitations to crop production worldwide. We identified Bot1, a BOR1 ortholog, as the gene responsible for the superior boron-toxicity tolerance of the Algerian barley landrace Sahara 3771 (Sahara). Bot1 was located at the tolerance locus by high-resolution mapping. Compared to intolerant genotypes, Sahara contains about four times as many Bot1 gene copies, produces substantially more Bot1 transcript, and encodes a Bot1 protein with a higher capacity to provide tolerance in yeast. Bot1 transcript levels identified in barley tissues are consistent with a role in limiting the net entry of boron into the root and in the disposal of boron from leaves via hydathode guttation.

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca 2+ transporters, CAX1 (Ca 2+ /H + -antiporter), ACA4, and ACA11 (Ca 2+ -ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO 2 assimilation, and leaf growth rate; increased transcript abundance of other Ca 2+ transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca 2+ ], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca 2+ ] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

Assaying relative and absolute levels of gene expression in a diverse series of tissues is a central step in the process of characterizing gene function and a necessary component of almost all publications describing individual genes or gene family members. However, throughout the literature, such studies lack consistency in genotype, tissues analyzed, and growth conditions applied, and, as a result, the body of information that is currently assembled is fragmented and difficult to compare between different studies. The development of a comprehensive platform for assaying gene expression that is available to the entire research community provides a major opportunity to assess whole biological systems in a single experiment. It also integrates detailed knowledge and information on individual genes into a unified framework that provides both context and resource to explore their contributions in a broader biological system. We have established a data set that describes the expression of 21,439 barley genes in 15 tissues sampled throughout the development of the barley cv. Morex grown under highly controlled conditions. Rather than attempting to address a specific biological question, our experiment was designed to provide a reference gene expression data set for barley researchers; a gene expression atlas and a comparative data set for those investigating genes or regulatory networks in other plant species. In this paper we describe the tissues sampled and their transcriptomes, and provide summary information on genes that are either specifically expressed in certain tissues or show correlated expression patterns across all 15 tissue samples. Using specific examples and an online tutorial, we describe how the data set can be interrogated for patterns and levels of barley gene expression and how the resulting information can be used to generate and/or test specific biological hypotheses.

A large proportion of the nitrate (NO3 −) acquired by plants from soil is actively transported via members of the NRT families of NO3 − transporters. In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes (maize, rice, sorghum, Brachypodium). We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO3 − transporters and NO3 − transport in grass crop species.

SummaryHere, we report the transcript profile of wheat egg cells and proembryos, just after the first cell division. Microdissected female gametophytes of wheat were used to isolate eggs and two-celled proembryos to construct cell type-specific cDNA libraries. In total, 1197 expressed sequence tags (ESTs) were generated. Analysis of these ESTs revealed numerous novel transcripts. In egg cells, 17.6% of the clustered ESTs represented novel transcripts, while 11.4% novel clusters were identified in the two-celled proembryo. Functional classification of sequences with similarity to previously characterized proteins indicates that the unfertilized egg cell has a higher metabolic activity and protein turnover than previously thought. Transcript composition of two-celled proembryos was significantly distinct from egg cells, reflecting DNA replication as well as high transcriptional and translational activity. Several novel transcripts of the egg cell are specific for this cell. In contrast, some fertilization induced novel mRNAs are abundant also in sporophytic tissues indicating a more general role in plant growth and development. The potential functions of genes based on similarity to known genes involved in developmental processes are discussed. Our analysis has identified numerous genes with potential roles in embryo sac function such as signaling, fertilization or induction of embryogenesis.

BackgroundAnnotations that describe the function of sequences are enormously important to researchers during laboratory investigations and when making computational inferences. However, there has been little investigation into the data quality of sequence function annotations. Here we have developed a new method of estimating the error rate of curated sequence annotations, and applied this to the Gene Ontology (GO) sequence database (GOSeqLite). This method involved artificially adding errors to sequence annotations at known rates, and used regression to model the impact on the precision of annotations based on BLAST matched sequences.ResultsWe estimated the error rate of curated GO sequence annotations in the GOSeqLite database (March 2006) at between 28% and 30%. Annotations made without use of sequence similarity based methods (non-ISS) had an estimated error rate of between 13% and 18%. Annotations made with the use of sequence similarity methodology (ISS) had an estimated error rate of 49%.ConclusionWhile the overall error rate is reasonably low, it would be prudent to treat all ISS annotations with caution. Electronic annotators that use ISS annotations as the basis of predictions are likely to have higher false prediction rates, and for this reason designers of these systems should consider avoiding ISS annotations where possible. Electronic annotators that use ISS annotations to make predictions should be viewed sceptically. We recommend that curators thoroughly review ISS annotations before accepting them as valid. Overall, users of curated sequence annotations from the GO database should feel assured that they are using a comparatively high quality source of information.

Boron (B) toxicity is a significant limitation to cereal crop production in a number of regions worldwide. Here we describe the cloning of a gene from barley (Hordeum vulgare), underlying the chromosome 6H B toxicity tolerance quantitative trait locus. It is the second B toxicity tolerance gene identified in barley. Previously, we identified the gene Bot1 that functions as an efflux transporter in B toxicity-tolerant barley to move B out of the plant. The gene identified in this work encodes HvNIP2;1, an aquaporin from the nodulin-26-like intrinsic protein (NIP) subfamily that was recently described as a silicon influx transporter in barley and rice (Oryza sativa). Here we show that a rice mutant for this gene also shows reduced B accumulation in leaf blades compared to wild type and that the mutant protein alters growth of yeast (Saccharomyces cerevisiae) under high B. HvNIP2;1 facilitates significant transport of B when expressed in Xenopus oocytes compared to controls and to another NIP (NOD26), and also in yeast plasma membranes that appear to have relatively high B permeability. We propose that tolerance to high soil B is mediated by reduced expression of HvNIP2;1 to limit B uptake, as well as by increased expression of Bot1 to remove B from roots and sensitive tissues. Together with Bot1, the multifunctional aquaporin HvNIP2;1 is an important determinant of B toxicity tolerance in barley.

BackgroundMicroRNAs are important components of the regulatory network of biological systems and thousands have been discovered in both animals and plants. Systematic investigations performed in species with sequenced genomes such as Arabidopsis, rice, poplar and Brachypodium have provided insights into the evolutionary relationships of this class of small RNAs among plants. However, miRNAs from barley, one of the most important cereal crops, remain unknown.ResultsWe performed a large scale study of barley miRNAs through deep sequencing of small RNAs extracted from leaves of two barley cultivars. By using the presence of miRNA precursor sequences in related genomes as one of a number of supporting criteria, we identified up to 100 miRNAs in barley. Of these only 56 have orthologs in wheat, rice or Brachypodium that are known to be expressed, while up to 44 appear to be specifically expressed in barley.ConclusionsOur study, the first large scale investigation of small RNAs in barley, has identified up to 100 miRNAs. We demonstrate that reliable identification of miRNAs via deep sequencing in a species whose genome has not been sequenced requires a more careful analysis of sequencing errors than is commonly performed. We devised a read filtering procedure for dealing with errors. In addition, we found that the use of a large dataset of almost 35 million reads permits the use of read abundance distributions along putative precursor sequences as a practical tool for isolating miRNAs in a large background of reads originating from other non-coding and coding RNAs. This study therefore provides a generic approach for discovering novel miRNAs where no genome sequence is available.