The Brachypodium distachyon DREB transcription factor BdDREB-39 confers oxidative stress tolerance in transgenic tobacco.

Key message BdDREB-39 is a DREB/CBF transcription factor, localized in the nucleus with transactivation activity, and BdDREB-39-overexpressing transgenic yeasts and tobacco enhanced the tolerance to oxidative stress.Abstract The DREB/CBF transcription factors are generally recognized to play an important factor in plant growth, development and response to various abiotic stresses. However, the mechanism of DREB/CBFs in oxidative stress response is largely unknown. This study isolated a DREB/CBF gene BdDREB-39 from Brachypodium distachyon (B. distachyon). Multiple sequence alignment and phylogenetic analysis showed that BdDREB-39 was closely related to the DREB proteins of oats, barley, wheat and rye and therefore its study can provide a reference for the excavation and genetic improvement of BdDREB-39 or its homologs in its closely related species. The transcript levels of BdDREB-39 were significantly up-regulated under H2O2 stress. BdDREB-39 was localised in the nucleus and functioned as a transcriptional activator. Overexpression of BdDREB-39 enhanced H2O2 tolerance in yeast. Transgenic tobaccos with BdDREB-39 had higher germination rates, longer root, better growth status, lesser reactive oxygen species (ROS) and malondialdehyde (MDA), and higher superoxide dismutase (SOD) and peroxidase (POD) activities than wild type (WT). The expression levels of ROS-related and stress-related genes were improved by BdDREB-39. In summary, these results revealed that BdDREB-39 can improve the viability of tobacco by regulating the expression of ROS and stress-related genes, allowing transgenic tobacco to accumulate lower levels of ROS and reducing the damage caused by ROS to cells. The BdDREB-39 gene has the potential for developing plant varieties tolerant to stress.

Three near-complete genome assemblies reveal substantial centromere dynamics from diploid to tetraploid in Brachypodium genus.

BACKGROUND: Centromeres are critical for maintaining genomic stability in eukaryotes, and their turnover shapes genome architectures and drives karyotype evolution. However, the co-evolution of centromeres from different species in allopolyploids over millions of years remains largely unknown. RESULTS: Here, we generate three near-complete genome assemblies, a tetraploid Brachypodium hybridum and its two diploid ancestors, Brachypodium distachyon and Brachypodium stacei. We detect high degrees of sequence, structural, and epigenetic variations of centromeres at base-pair resolution between closely related Brachypodium genomes, indicating the appearance and accumulation of species-specific centromere repeats from a common origin during evolution. We also find that centromere homogenization is accompanied by local satellite repeats bursting and retrotransposon purging, and the frequency of retrotransposon invasions drives the degree of interspecies centromere diversification. We further investigate the dynamics of centromeres during alloploidization process, and find that dramatic genetics and epigenetics architecture variations are associated with the turnover of centromeres between homologous chromosomal pairs from diploid to tetraploid. Additionally, our pangenomes analysis reveals the ongoing variations of satellite repeats and stable evolutionary homeostasis within centromeres among individuals of each Brachypodium genome with different polyploidy levels. CONCLUSIONS: Our results provide unprecedented information on the genomic, epigenomic, and functional diversity of highly repetitive DNA between closely related species and their allopolyploid genomes at both coarse and fine scale.

Group VII ethylene response factors forming distinct regulatory loops mediate submergence responses.

Group VII ethylene response factors (ERFVIIs), whose stability is oxygen concentration-dependent, play key roles in regulating hypoxia response genes in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) during submergence. To understand the evolution of flooding tolerance in cereal crops, we evaluated whether Brachypodium distachyon ERFVII genes (BdERFVIIs) are related to submergence tolerance. We found that three BdERFVIIs, BdERF108, BdERF018, and BdERF961, form a feedback regulatory loop to mediate downstream responses. BdERF108 and BdERF018 activated the expression of BdERF961 and PHYTOGLOBIN 1 (PGB1), which promoted nitric oxide turnover and preserved ERFVII protein stability. The activation of PGB1 was subsequently counteracted by increased BdERF961 accumulation through negative feedback regulation. Interestingly, we found that OsERF67, the orthologue of BdERF961 in rice, activated PHYTOGLOBIN (OsHB2) expression and formed distinct regulatory loops during submergence. Overall, the divergent regulatory mechanisms exhibited by orthologs collectively offer perspectives for the development of submergence-tolerant crops.

Subgenomic Stability of Progenitor Genomes During Repeated Allotetraploid Origins of the Same Grass Brachypodium hybridum.

Both homeologous exchanges and homeologous expression bias are generally found in most allopolyploid species. Whether homeologous exchanges and homeologous expression bias differ between repeated allopolyploid speciation events from the same progenitor species remains unknown. Here, we detected a third independent and recent allotetraploid origin for the model grass Brachypodium hybridum. Our homeologous exchange with replacement analyses indicated the absence of significant homeologous exchanges in any of the three types of wild allotetraploids, supporting the integrity of their progenitor subgenomes and the immediate creation of the amphidiploids. Further homeologous expression bias tests did not uncover significant subgenomic dominance in different tissues and conditions of the allotetraploids. This suggests a balanced expression of homeologs under similar or dissimilar ecological conditions in their natural habitats. We observed that the density of transposons around genes was not associated with the initial establishment of subgenome dominance; rather, this feature is inherited from the progenitor genome. We found that drought response genes were highly induced in the two subgenomes, likely contributing to the local adaptation of this species to arid habitats in the third allotetraploid event. These findings provide evidence for the consistency of subgenomic stability of parental genomes across multiple allopolyploidization events that led to the same species at different periods. Our study emphasizes the importance of selecting closely related progenitor species genomes to accurately assess homeologous exchange with replacement in allopolyploids, thereby avoiding the detection of false homeologous exchanges when using less related progenitor species genomes.

Multiple origins, one evolutionary trajectory: gradual evolution characterizes distinct lineages of allotetraploid Brachypodium.

The "genomic shock" hypothesis posits that unusual challenges to genome integrity such as whole genome duplication may induce chaotic genome restructuring. Decades of research on polyploid genomes have revealed that this is often, but not always the case. While some polyploids show major chromosomal rearrangements and derepression of transposable elements in the immediate aftermath of whole genome duplication, others do not. Nonetheless, all polyploids show gradual diploidization over evolutionary time. To evaluate these hypotheses, we produced a chromosome-scale reference genome for the natural allotetraploid grass Brachypodium hybridum, accession "Bhyb26." We compared 2 independently derived accessions of B. hybridum and their deeply diverged diploid progenitor species Brachypodium stacei and Brachypodium distachyon. The 2 B. hybridum lineages provide a natural timecourse in genome evolution because one formed 1.4 million years ago, and the other formed 140 thousand years ago. The genome of the older lineage reveals signs of gradual post-whole genome duplication genome evolution including minor gene loss and genome rearrangement that are missing from the younger lineage. In neither B. hybridum lineage do we find signs of homeologous recombination or pronounced transposable element activation, though we find evidence supporting steady post-whole genome duplication transposable element activity in the older lineage. Gene loss in the older lineage was slightly biased toward 1 subgenome, but genome dominance was not observed at the transcriptomic level. We propose that relaxed selection, rather than an abrupt genomic shock, drives evolutionary novelty in B. hybridum, and that the progenitor species' similarity in transposable element load may account for the subtlety of the observed genome dominance.

Genome Wide Identification and Annotation of NGATHA Transcription Factor Family in Crop Plants.

The NGATHA (NGA) transcription factor (TF) belongs to the ABI3/VP1 (RAV) transcriptional subfamily, a subgroup of the B3 superfamily, which is relatively well-studied in Arabidopsis. However, limited data are available on the contributions of NGA TF in other plant species. In this study, 207 NGA gene family members were identified from a genome-wide search against Arabidopsis thaliana in the genome data of 18 dicots and seven monocots. The phylogenetic and sequence alignment analyses divided NGA genes into different clusters and revealed that the numbers of genes varied depending on the species. The phylogeny was followed by the characterization of the Solanaceae (tomato, potato, capsicum, tobacco) and Poaceae (Brachypodium distachyon, Oryza sativa L. japonica, and Sorghum bicolor) family members in comparison with A. thaliana. The gene and protein structures revealed a similar pattern for NGA and NGA-like sequences, suggesting that both are conserved during evolution. Promoter cis-element analysis showed that phytohormones such as abscisic acid, auxin, and gibberellins play a crucial role in regulating the NGA gene family. Gene ontology analysis revealed that the NGA gene family participates in diverse biological processes such as flower development, leaf morphogenesis, and the regulation of transcription. The gene duplication analysis indicates that most of the genes are evolved due to segmental duplications and have undergone purifying selection pressure. Finally, the gene expression analysis implicated that the NGA genes are abundantly expressed in lateral organs and flowers. This analysis has presented a detailed and comprehensive study of the NGA gene family, providing basic knowledge of the gene, protein structure, function, and evolution. These results will lay the foundation for further understanding of the role of the NGA gene family in various plant developmental processes.

Brachypodium: 20 years as a grass biology model system; the way forward?

It has been 20 years since Brachypodium distachyon was suggested as a model grass species, but ongoing research now encompasses the entire genus. Extensive Brachypodium genome sequencing programmes have provided resources to explore the determinants and drivers of population diversity. This has been accompanied by cytomolecular studies to make Brachypodium a platform to investigate speciation, polyploidisation, perenniality, and various aspects of chromosome and interphase nucleus organisation. The value of Brachypodium as a functional genomic platform has been underscored by the identification of key genes for development, biotic and abiotic stress, and cell wall structure and function. While Brachypodium is relevant to the biofuel industry, its impact goes far beyond that as an intriguing model to study climate change and combinatorial stress.

Loss of PROTEIN TARGETING TO STARCH 2 has variable effects on starch synthesis across organs and species.

Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops.

MiR398-regulated antioxidants contribute to Bamboo mosaic virus accumulation and symptom manifestation.

Virus infections that cause mosaic or mottling in leaves commonly also induce increased levels of reactive oxygen species (ROS). However, how ROS contributes to symptoms is less well documented. Bamboo mosaic virus (BaMV) causes chlorotic mosaic symptoms in both Brachypodium distachyon and Nicotiana benthamiana. The BaMV △CPN35 mutant with an N-terminal deletion of its coat protein gene exhibits asymptomatic infection independently of virus titer. Histochemical staining of ROS in mock-, BaMV-, and BaMV△CPN35-infected leaves revealed that hydrogen peroxide (H2O2) accumulated solely in BaMV-induced chlorotic spots. Moreover, exogenous H2O2 treatment enhanced yellowish chlorosis in BaMV-infected leaves. Both BaMV and BaMV△CPN35 infection could induce the expression of Cu/Zu superoxide dismutase (CSD) antioxidants at messenger RNA and protein level. However, BaMV triggered the abundant accumulation of full-length NbCSD2 preprotein (prNbCSD2, without transit peptide cleavage), whereas BaMV△CPN35 induced a truncated prNbCSD2. Confocal microscopy showed that majority of NbCSD2-green fluorescent protein (GFP) predominantly localized in the cytosol upon BaMV infection, but BaMV△CPN35 infection tended to cause NbCSD2-GFP to remain in chloroplasts. By 5'-RNA ligase-mediated rapid amplification of cDNA ends, we validated CSDs are the targets of miR398 in vivo. Furthermore, BaMV infection increased the level of miR398, while the level of BaMV titer was regulated positively by miR398 but negatively by CSD2. In contrast, overexpression of cytosolic form NbCSD2, impairing the transport into chloroplasts, greatly enhanced BaMV accumulation. Taken together, our results indicate that induction of miR398 by BaMV infection may facilitate viral titer accumulation, and cytosolic prNbCSD2 induction may contribute to H2O2 accumulation, resulting in the development of BaMV chlorotic symptoms in plants.

Ammonium nutrition interacts with iron homeostasis in Brachypodium distachyon.

Most plant species develop stress symptoms when exposed to high ammonium (NH4+) concentrations. The root is the first organ in contact with high NH4+ and therefore the first barrier to cope with ammonium stress. In this work, we focused on root adaptation to ammonium nutrition in the model plant Brachypodium distachyon. Proteome analysis revealed changes associated with primary metabolism, cell wall remodelling, and redox homeostasis. In addition, it showed a strong induction of proteins related to methionine (Met) metabolism and phytosiderophore (PS) synthesis in ammonium-fed plants. In agreement with this, we show how ammonium nutrition impacts Met/S-adenosyl-Met and PS metabolic pathways together with increasing root iron content. Nevertheless, ammonium-fed plants displayed higher sensitivity to iron deficiency, suggesting that ammonium nutrition triggers impaired iron utilization and root to shoot transport, which entailed an induction in iron-related responses. Overall, this work demonstrates the importance of iron homeostasis during ammonium nutrition and paves a new way to better understand and improve ammonium use efficiency and tolerance.

Genes involved in mRNA surveillance are induced in Brachypodium distachyon under cadmium toxicity.

BACKGROUND: Cd accumulation in plant cells results in dramatic problems including oxidative stress and inhibition of vital enzymes. It also affects mineral uptakes by disrupting membrane permeability. Interaction among Cd and other plant nutrient elements changes the nutritional contents of crops and reduces their yield. METHODS AND RESULTS: In the present study, Cd stress in Brachypodium distachyon led to the upregulation of some heavy metal transport genes (influx or efflux) encoding cation-efflux proteins, heavy metal-associated proteins and NRAMP proteins. The Arabidopsis orthologs of the differentially expressed B. distachyon genes (DEGs) under Cd toxicity were identified, which exhibited Bradi4g26905 was an ortholog of AtALY1-2. Detailed co-expression network and gene ontology analyses found the potential involvement of the mRNA surveillance pathway in Cd tolerance in B. distachyon. These genes were shown to be downregulated by sulfur (S) deficiency. CONCLUSIONS: This is the first transcriptomic study investigating the effect of Cd toxicity in B. distachyon, a model plant for genomic studies in Poaceae (Gramineae) species. The results are expected to provide valuable information for more comprehensive research related to heavy metal toxicity in plants.

In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon.

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca2+. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the "off signal" afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5'NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca2+ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.

Programmed Cell Death in Developing Brachypodium distachyon Grain.

The normal developmental sequence in a grass grain entails the death of several maternal and filial tissues in a genetically regulated process termed programmed cell death (PCD). The progression and molecular aspects of PCD in developing grains have been reported for domesticated species such as barley, rice, maize and wheat. Here, we report a detailed investigation of PCD in the developing grain of the wild model species Brachypodium distachyon. We detected PCD in developing Brachypodium grains using molecular and histological approaches. We also identified in Brachypodium the orthologs of protease genes known to contribute to grain PCD and surveyed their expression. We found that, similar to cereals, PCD in the Brachypodium nucellus occurs in a centrifugal pattern following anthesis. However, compared to cereals, the rate of post-mortem clearance in the Brachypodium nucellus is slower. However, compared to wheat and barley, mesocarp PCD in Brachypodium proceeds more rapidly in lateral cells. Remarkably, Brachypodium mesocarp PCD is not coordinated with endosperm development. Phylogenetic analysis suggests that barley and wheat possess more vacuolar processing enzymes that drive nucellar PCD compared to Brachypodium and rice. Our expression analysis highlighted putative grain-specific PCD proteases in Brachypodium. Combined with existing knowledge on grain PCD, our study suggests that the rate of nucellar PCD moderates grain size and that the pattern of mesocarp PCD influences grain shape.

Duplicated antagonistic EPF peptides optimize grass stomatal initiation.

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.

BdFAR4, a root-specific fatty acyl-coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon.

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.

A novel wheat ASR gene, TaASR2D, enhances drought tolerance in Brachypodium distachyon.

Abscisic acid-, stress-, and ripening-induced (ASR) proteins play an important role in protecting plants against adverse environmental conditions. Here, we identified 24 ASR genes in the wheat genome and analyzed their characteristics. Among these, five ASR genes highly induced by abscisic acid (ABA) and polyethylene glycol were cloned and further characterized. The TaASR genes were expressed in response to different abiotic stresses and ABA and were found to be localized in the nucleus and plasma membrane of transformed tobacco cells. Brachypodium distachyon transgenic plants overexpressing TaASR2D showed enhanced drought tolerance by regulating leaf transpiration. The expression levels of stress-related and ABA-responsive genes were higher in transgenic plants than in wild-type plants under drought stress conditions. Moreover, overexpression of TaASR2D increased the levels of both endogenous ABA and hydrogen peroxide in response to drought stress, and these plants showed hypersensitivity to exogenous ABA at the germination stage. Furthermore, plants overexpressing TaASR2D showed increased stomatal closure. Further analysis revealed that TaASR2D interacts with ABA biosynthesis and stress-related proteins in yeast and tobacco plants. Collectively, these findings indicate that TaASR2D plays an important role in the response of plants to drought stress by regulating the ABA biosynthesis pathway and redox homeostasis system.

The Role of Brachypodium distachyon Wall-Associated Kinases (WAKs) in Cell Expansion and Stress Responses.

The plant cell wall plays a critical role in signaling responses to environmental and developmental cues, acting as both the sensing interface and regulator of plant cell integrity. Wall-associated kinases (WAKs) are plant receptor-like kinases located at the wall-plasma membrane-cytoplasmic interface and implicated in cell wall integrity sensing. WAKs in Arabidopsis thaliana have been shown to bind pectins in different forms under various conditions, such as oligogalacturonides (OG)s in stress response, and native pectin during cell expansion. The mechanism(s) WAKs use for sensing in grasses, which contain relatively low amounts of pectin, remains unclear. WAK genes from the model monocot plant, Brachypodium distachyon were identified. Expression profiling during early seedling development and in response to sodium salicylate and salt treatment was undertaken to identify WAKs involved in cell expansion and response to external stimuli. The BdWAK2 gene displayed increased expression during cell expansion and stress response, in addition to playing a potential role in the hypersensitive response. In vitro binding assays with various forms of commercial polysaccharides (pectins, xylans, and mixed-linkage glucans) and wall-extracted fractions (pectic/hemicellulosic/cellulosic) from both Arabidopsis and Brachypodium leaf tissues provided new insights into the binding properties of BdWAK2 and other candidate BdWAKs in grasses. The BdWAKs displayed a specificity for the acidic pectins with similar binding characteristics to the AtWAKs.

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors.

Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution.

A triticale tapetal non-specific lipid transfer protein (nsLTP) is translocated to the pollen cell wall.

KEY MESSAGE: A Triticeae type III non-specific lipid transfer protein (nsLTP) was shown for the first time to be translocated from the anther tapetum to the pollen cell wall. Two anther-expressed non-specific lipid transfer proteins (nsLTPs) were identified in triticale (× Triticosecale Wittmack). LTPc3a and LTPc3b contain a putative signal peptide sequence and eight cysteine residues in a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C pattern. These proteins belong to the type III class of nsLTPs which are expressed exclusively in the inflorescence of angiosperms. The level of LTPc3 transcript in the anther was highest at the tetrad and uninucleate microspore stages, and absent in mature pollen. In situ hybridization showed that LTPc3 was expressed in the tapetal layer of the developing triticale anther. The expression of the LTPc3 protein peaked at the uninucleate microspore stage, but was also found to be associated with the mature pollen. Accordingly, an LTPc3a::GFP translational fusion expressed in transgenic Brachypodium distachyon first showed activity in the tapetum, then in the anther locule, and later on the mature pollen grain. Altogether, these results represent the first detailed characterization of a Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation.