Patrick J. Gulick

This study investigated the role of secreted peptides and receptors in root hair development. The secreted peptides EPF1, EPF2, EPFL4, EPFL5, EPFL6, and EPFL9 and their putative receptors ERECTA (ER), ERECTA-LIKE1 (ERL1), ERL2, and TMM were found to mediate the development of the root hairs in Arabidopsis under control conditions and in response to nitrate deficiency. EPF2, EPFL4, EPFL5, and EPFL6 act as positive regulators of root hair density, whereas EPFL9 functions as a negative regulator for root hair density and root hair length. The receptor ERL2 was found to be a positive regulator of root hair density and ERL1 was found to be a positive regulator of both root hair density and root hair length. Nitrogen starvation caused a small and statistically insignificant increase in root hair density in wild type plants but led to significantly greater increases in root hair density in epf mutant genotypes. In contrast, the EPFL9 RNA interference (EPFL9i) line showed a significant decrease, indicating that EPF2, EPFL4, EPFL5 and EPFL6 act as negative regulators of root hair density in response to nitrate starvation, whereas EPFL9 functions as a positive regulator. Nitrate starvation also led to significant increases in root hair length in the wild type and in nine of the epf single, double and triple mutant lines but caused a significant decrease in the EPFL9i line. Analysis indicated that EPF2, EPFL4, EPFL5 are negative regulators, and EPFL9 is a positive regulator, of root hair length increase in response to nitrogen starvation. Our findings provide insight into the role of members of EPF secreted peptides and ER family receptors in regulating Arabidopsis root hair development.

Pirins are nuclear bicupin proteins, encoded by genes that are one of several gene families that comprise the cupin superfamily in plants. Pirin genes have been implicated in stress response pathways studied in Arabidopsis and At-Pirin1 has been shown to interact with the heterotrimeric G-protein alpha subunit (GPA1). The aim of this study was to identify the members of the Pirin gene family in Triticum aestivum, to correct their annotations in the whole genome, and gain an insight into their tissue-specific expression as well as their response to abiotic and biotic stresses. The Pirin gene family in T. aestivum is comprised of 18 genes that represent six paralogous gene copies, each having an A, B, and D homeolog. Expression analysis of the Pirin genes in T. aestivum Illumina RNA-seq libraries, which included sampling from differing tissue types as well as abiotic and biotic stresses, indicates that the members of the Pirin gene family have specialized expression and play a role in stress responses. Pirin gene families are also identified in other monocots including Aegilops tauschii, Hordeum vulgare, Brachypodium distachyon, Oryza sativa, Zea mays, Sorghum bicolor, and the dicot Arabidopsis thaliana.

This study characterizes the heterotrimeric G protein gene families in Triticum aestivum, their tissue-specific expression patterns during development and in response to biotic and abiotic stress conditions. There are three Gα genes, three Gβ and 12 Gγ genes, totaling 18 genes encoding heterotrimeric G proteins in the hexaploid wheat genome. Each haploid genome of the hexaploid T. aestivum has a single gene encoding the α subunit of the heterotrimeric G protein complex, GA1, a single Gβ and four Gγ genes. Each gene has three homeologous copies in the A, B and D genomes. The physical interaction between the Gβ (Gpb) and two Gγ subunits, Gpg1 and Gpg2, was shown through bimolecular fluorescence complementation (BiFC). The gene expression in response to biotic and abiotic stresses showed both up-regulation and down-regulation of members of the gene families. Gγ2-B and Gγ2-D are significantly upregulated during heat stress, GA1-D is upregulated by cold stress and Gγ1-A and Gγ1-D were upregulated by Fusarium graminearum inoculation in a F. graminearum resistant cultivar. This suggests that these members may play roles in biotic and abiotic signaling pathways and the roles of these genes within these pathways need further investigation.

Key message A stress induced calcium-binding protein, RD20/CLO3 interacts with the alpha subunit of the heterotrimeric G-protein complex in Arabidopsis and affects etiolation and leaf morphology. Abstract Heterotrimeric G proteins and calcium signaling have both been shown to play a role in the response to environmental abiotic stress in plants; however, the interaction between calcium-binding proteins and G-protein signaling molecules remains elusive. We investigated the interaction between the alpha subunit of the heterotrimeric G-protein complex, GPA1, of Arabidopsis thaliana with the calcium-binding protein, the caleosin RD20/CLO3, a gene strongly induced by drought, salt and abscisic acid. The proteins were found to interact in vivo by bimolecular fluorescent complementation (BiFC); the interaction was localized to the endoplasmic reticulum and to oil bodies within the cell. The constitutively GTP-bound GPA1 (GPA1^QL) also interacts with RD20/CLO3 as well as its EF-hand mutant variations and these interactions are localized to the plasma membrane. The N-terminal portion of RD20/CLO3 was found to be responsible for the interaction with GPA1 and GPA1^QL using both BiFC and yeast two-hybrid assays. RD20/CLO3 contains a single calcium-binding EF-hand in the N-terminal portion of the protein; disruption of the calcium-binding capacity of the protein obliterates interaction with GPA1 in in vivo assays and decreases the interaction between the caleosin and the constitutively active GPA1^QL. Analysis of rd20/clo3 mutants shows that RD20/CLO3 plays a key role in the signaling pathway controlling hypocotyl length in dark grown seedlings and in leaf morphology. Our findings indicate a novel role for RD20/CLO3 as a negative regulator of GPA1.

Peptide signaling has emerged as a key component of plant growth and development, including stomatal patterning, which is crucial for plant productivity and survival. Although exciting progress has been made in understanding EPIDERMAL PATTERNING FACTOR (EPF) signaling in Arabidopsis, the mechanisms by which EPF peptides control different stomatal patterns and morphologies in grasses are poorly understood. Here, by examining expression patterns, overexpression transgenics and cross-species complementation, the antagonistic stomatal ligands orthologous to Arabidopsis AtEPF2 and AtSTOMAGEN/AtEPFL9 peptides were identified in Triticum aestivum (wheat) and the grass model organism Brachypodium distachyon. Application of bioactive BdEPF2 peptides inhibited stomatal initiation, but not the progression or differentiation of stomatal precursors in Brachypodium. Additionally, the inhibitory roles of these EPF peptides during grass stomatal development were suppressed by the contrasting positive action of the BdSTOMAGEN peptide in a dose-dependent manner. These results not only demonstrate how conserved EPF peptides that control different stomatal patterns exist in nature, but also suggest new strategies to improve crop yield through the use of plant-derived antagonistic peptides that optimize stomatal density on the plant epidermis.

Numerous quantitative trait loci (QTLs) have been mapped in tetraploid and hexaploid wheat and wheat relatives, mostly with simple sequence repeat (SSR) or single nucleotide polymorphism (SNP) markers. To conduct meta-analysis of QTLs requires projecting them onto a common genomic framework, either a consensus genetic map or genomic sequence. The latter strategy is pursued here. Of 774 QTLs mapped in wheat and wheat relatives found in the literature, 585 (75.6%) were successfully projected onto the Aegilops tauschii pseudomolecules. QTLs mapped with SNP markers were more successfully projected (92.2%) than those mapped with SSR markers (66.2%). The QTLs were not distributed homogeneously along chromosome arms. Their frequencies increased in the proximal-to-distal direction but declined in the most distal regions and were weakly correlated with recombination rates along the chromosome arms. Databases for projected SSR markers and QTLs were constructed and incorporated into the Ae. tauschii JBrowse. To facilitate meta-QTL analysis, eight clusters of QTLs were used to estimate standard deviations (ô) of independently mapped QTLs projected onto the Ae. tauschii genome sequence. The standard deviations ô were modeled as an exponential decay function of recombination rates along the Ae. tauschii chromosomes. We implemented four hypothesis tests for determining the membership of query QTLs. The hypothesis tests and estimation procedure for were implemented in a web portal for meta-analysis of projected QTLs. Twenty-one QTLs for fusarium head blight resistance mapped on wheat chromosomes 3A, 3B, and 3D were analyzed to illustrate the use of the portal for meta-QTL analyses.

Members of the Early Salt Induced 3 (Esi3/RCI2/PMP3) gene family in plants have been shown to be induced in response to both biotic and abiotic stresses and to enhance stress tolerance in both transgenic plants and Saccharomyces cerevisiae. Esi3 was first identified as a salt stress induced gene in the salt tolerant wild wheat grass, Lophopyrum elongatum, and subsequently homologous genes in many other species were found to be members of the gene family. These include Arabidopsis thaliana and Oryza sativa where they are referred to as Rare Cold Inducible 2 (RCI2), and Zea mays where they are referred to as Plasma Membrane Protein 3 (PMP3). This study characterizes the Esi3 family members in Triticum aestivum and explores the tissue specific expression patterns of the gene family members as well as their response to a variety of environmental stresses. The Esi3 gene family was found to have a total of 29 family members comprised of ten paralogous groups in the hexaploid T. aestivum. Each paralogous group contains three homeologous copies, one in each of the A, B and D genomes with the exception of Esi3–2 which is missing the B copy. The genes of the Esi3 gene family were also identified in four other monocot species, Aegilops tauschii, Hordeum vulgare, Secale cereale and Sorghum bicolor, and were confirmed or corrected for Brachypodium distachyon, Oryza sativa and Zea mays, as well as the dicot Arabidopsis thaliana. Gene expression of the Esi3s was analyzed using tissue-specific, abiotic and biotic stress RNA-Seq 454 sequence libraries and Affymetrix microarray data for T. aestivum. Members of nearly all paralogous groups of the Esi3 genes in T. aestivum have altered gene expression in response to abiotic or biotic stress conditions. In addition, there are modest differences in gene expression among homeologous members of the gene family. This suggests that the Esi3 gene family plays an important role in the plants response to the stresses presented in this study. The Esi3–9 in T. aestivum has a unique N terminal extension placing it into Group III, a new group for the Esi3/RCI2/PMP3 gene family.

One of the most important evolutionary processes in plants is polyploidization. The combination of two or more genomes in one organism often initially leads to changes in gene expression and extensive genomic reorganization, compared to the parental species. Hexaploid triticale (x Triticosecale) is a synthetic hybrid crop species generated by crosses between T. turgidum and Secale cereale. Because triticale is a recent synthetic polyploid it is an important model for studying genome evolution following polyploidization. Molecular studies have demonstrated that genomic sequence changes, consisting of sequence elimination or loss of expression of genes from the rye genome, are common in triticale. High-throughput DNA sequencing allows a large number of genes to be surveyed, and transcripts from the different homeologous copies of the genes that have high sequence similarity can be better distinguished than hybridization methods previously employed. The expression levels of 23,503 rye cDNA reference contigs were analyzed in 454-cDNA libraries obtained from anther, root and stem from both triticale and rye, as well as in five 454-cDNA data sets created from triticale seedling shoot, ovary, stigma, pollen and seed tissues to identify the classes of rye genes silenced or absent in the recent synthetic hexaploid triticale. Comparisons between diploid rye and hexaploid triticale detected 112 rye cDNA contigs (~0.5%) that were totally undetected by expression analysis in all triticale tissues, although their expression was relatively high in rye tissues. Non-expressed rye genes were found to be strikingly less similar to their closest BLASTN matches in the wheat genome or in the other Triticum genomes than a test set of 200 random rye genes. Genes that were not detected in the RNA-seq data were further characterized by testing for their presence in the triticale genome by PCR using genomic DNA as a template. Genes with low similarity between rye sequences and their closest matches in the Triticum genome have a higher probability to be repressed or absent in the allopolyploid genome.