The reference genome and abiotic stress responses of the model perennial grass Brachypodium sylvaticum.

Perennial grasses are important forage crops and emerging biomass crops and have the potential to be more sustainable grain crops. However, most perennial grass crops are difficult experimental subjects due to their large size, difficult genetics, and/or their recalcitrance to transformation. Thus, a tractable model perennial grass could be used to rapidly make discoveries that can be translated to perennial grass crops. Brachypodium sylvaticum has the potential to serve as such a model because of its small size, rapid generation time, simple genetics, and transformability. Here, we provide a high-quality genome assembly and annotation for B. sylvaticum, an essential resource for a modern model system. In addition, we conducted transcriptomic studies under 4 abiotic stresses (water, heat, salt, and freezing). Our results indicate that crowns are more responsive to freezing than leaves which may help them overwinter. We observed extensive transcriptional responses with varying temporal dynamics to all abiotic stresses, including classic heat-responsive genes. These results can be used to form testable hypotheses about how perennial grasses respond to these stresses. Taken together, these results will allow B. sylvaticum to serve as a truly tractable perennial model system.

Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.

Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

CRISPR-Cas9 Gene Editing of the Sal1 Gene Family in Wheat.

The highly conserved Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted. Precise gene editing techniques were used to generate Sal1 mutants in hexaploid bread wheat. The CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) Cas9 system with three guide RNAs (gRNAs) was used to inactivate six Sal1 homologous genes within the Bobwhite wheat genome. The resulting mutant wheat plants with all their Sal1 genes disabled had slimmer stems, had a modest reduction in biomass and senesced more slowly in water limiting conditions, but did not exhibit improved yield under drought conditions. Our results show that multiplexed gRNAs enabled effective targeted gene editing of the Sal1 gene family in hexaploid wheat. These Sal1 mutant wheat plants will be a resource for further research studying the function of this gene family in wheat.

Tissue-specific expression of Ruby in Mexican lime (C. aurantifolia) confers anthocyanin accumulation in fruit.

Tissue specific promoters are important tools for the precise genetic engineering of crop plants. Four fruit-preferential promoters were examined for their ability to confer a novel fruit trait in transgenic Mexican lime (Citrus aurantifolia). The Ruby transcription factor activates fruit anthocyanin accumulation within Moro blood orange and has been shown to function in activating anthocyanin accumulation in heterologous plant species. Although the CitVO1, CitUNK, SlE8, and PamMybA promoters were previously shown to confer strong fruit-preferential expression in transgenic tomato, they exhibited no detectable expression in transgenic Mexican lime trees. In contrast, the CitWax promoter exhibited high fruit-preferential expression of Ruby, conferring strong anthocyanin accumulation within the fruit juice sac tissue and moderate activity in floral/reproductive tissues. In some of the transgenic trees with high levels of flower and fruit anthocyanin accumulation, juvenile leaves also exhibited purple coloration, but the color disappeared as the leaves matured. We show that the CitWax promoter enables the expression of Ruby to produce anthocyanin colored fruit desired by consumers. The production of this antioxidant metabolite increases the fruits nutritional value and may provide added health benefits.

Efficient Gene Stacking in Rice Using the GAANTRY System.

Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.

Gene Assembly in Agrobacterium via Nucleic Acid Transfer Using Recombinase Technology (GAANTRY).

Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.

Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology.

BACKGROUND: Promoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development. RESULTS: In this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity. CONCLUSIONS: Among the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.

Transgene stacking in potato using the GAANTRY system.

OBJECTIVE: GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 '10-stack' strain to generate transgenic potato plants. RESULTS: The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that 'backbone' DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes.

A versatile and robust Agrobacterium-based gene stacking system generates high-quality transgenic Arabidopsis plants.

Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site-specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5-kbp T-DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T-DNA left border, and only 8% contained sequence from outside the T-DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high-quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.

Salt tolerance of two perennial grass Brachypodium sylvaticum accessions.

KEY MESSAGE: We studied the salt stress tolerance of two accessions isolated from different areas of the world (Norway and Tunisia) and characterized the mechanism(s) regulating salt stress in Brachypodium sylvaticum Osl1 and Ain1. Perennial grasses are widely grown in different parts of the world as an important feedstock for renewable energy. Their perennial nature that reduces management practices and use of energy and agrochemicals give these biomass crops advantages when dealing with modern agriculture challenges such as soil erosion, increase in salinized marginal lands and the runoff of nutrients. Brachypodium sylvaticum is a perennial grass that was recently suggested as a suitable model for the study of biomass plant production and renewable energy. However, its plasticity to abiotic stress is not yet clear. We studied the salt stress tolerance of two accessions isolated from different areas of the world and characterized the mechanism(s) regulating salt stress in B. sylvaticum Osl1, originated from Oslo, Norway and Ain1, originated from Ain-Durham, Tunisia. Osl1 limited sodium transport from root to shoot, maintaining a better K/Na homeostasis and preventing toxicity damage in the shoot. This was accompanied by higher expression of HKT8 and SOS1 transporters in Osl1 as compared to Ain1. In addition, Osl1 salt tolerance was accompanied by higher abundance of the vacuolar proton pump pyrophosphatase and Na+/H+ antiporters (NHXs) leading to a better vacuolar pH homeostasis, efficient compartmentation of Na+ in the root vacuoles and salt tolerance. Although preliminary, our results further support previous results highlighting the role of Na+ transport systems in plant salt tolerance. The identification of salt tolerant and sensitive B. sylvaticum accessions can provide an experimental system for the study of the mechanisms and regulatory networks associated with stress tolerance in perennials grass.

Escherichia coli O157:H7 Converts Plant-Derived Choline to Glycine Betaine for Osmoprotection during Pre- and Post-harvest Colonization of Injured Lettuce Leaves.

Plant injury is inherent to the production and processing of fruit and vegetables. The opportunistic colonization of damaged plant tissue by human enteric pathogens may contribute to the occurrence of outbreaks of foodborne illness linked to produce. Escherichia coli O157:H7 (EcO157) responds to physicochemical stresses in cut lettuce and lettuce lysates by upregulation of several stress response pathways. We investigated the tolerance of EcO157 to osmotic stress imposed by the leakage of osmolytes from injured lettuce leaf tissue. LC-MS analysis of bacterial osmoprotectants in lettuce leaf lysates and wound washes indicated an abundant natural pool of choline, but sparse quantities of glycine betaine and proline. Glycine betaine was a more effective osmoprotectant than choline in EcO157 under osmotic stress conditions in vitro. An EcO157 mutant with a deletion of the betTIBA genes, which are required for biosynthesis of glycine betaine from imported choline, achieved population sizes twofold lower than those of the parental strain (P < 0.05) over the first hour of colonization of cut lettuce in modified atmosphere packaging (MAP). The cell concentrations of the betTIBA mutant also were 12-fold lower than those of the parental strain (P < 0.01) when grown in hypertonic lettuce lysate, indicating that lettuce leaf cellular contents provide choline for osmoprotection of EcO157. To demonstrate the utilization of available choline by EcO157 for osmoadaptation in injured leaf tissue, deuterated (D-9) choline was introduced to wound sites in MAP lettuce; LC-MS analysis revealed the conversion of D9-choline to D-9 glycine betaine in the parental strain, but no significant amounts were observed in the betTIBA mutant. The EcO157 ΔbetTIBA-ΔotsBA double mutant, which is additionally deficient in de novo synthesis of the compatible solute trehalose, was significantly less fit than the parental strain after their co-inoculation onto injured lettuce leaves and MAP cut lettuce. However, its competitive fitness followed a different time-dependent trend in MAP lettuce, likely due to differences in O2 content, which modulates betTIBA expression. Our study demonstrates that damaged lettuce leaf tissue does not merely supply EcO157 with substrates for proliferation, but also provides the pathogen with choline for its survival to osmotic stress experienced at the site of injury.

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

Novel R2R3-MYB transcription factors from Prunus americana regulate differential patterns of anthocyanin accumulation in tobacco and citrus.

The level of anthocyanins in plants vary widely among cultivars, developmental stages and environmental stimuli. Previous studies have reported that the expression of various MYBs regulate anthocyanin pigmentation during growth and development. Here we examine the activity of 3 novel R2R3-MYB transcription factor (TF) genes, PamMybA.1, PamMybA.3 and PamMybA.5 from Prunus americana. The anthocyanin accumulation patterns mediated by CaMV double35S promoter (db35Sp) controlled expression of the TFs in transgenic tobacco were compared with citrus-MoroMybA, Arabidopsis-AtMybA1 and grapevine-VvMybA1 transgenics during their entire growth cycles. The db35Sp-PamMybA.1 and db35Sp-PamMybA.5 constructs induced high levels of anthocyanin accumulation in both transformed tobacco calli and the regenerated plants. The red/purple color pigmentation induced in the PamMybA.1 and PamMybA.5 lines was not uniformly distributed, but appeared as patches in the leaves, whereas the flowers showed intense uniform pigmentation similar to the VvMybA1 expressing lines. MoroMybA and AtMybA1 showed more uniform pink coloration in both vegetative and reproductive tissues. Plant morphology, anthocyanin content, seed viability, and transgene inheritance were examined for the PamMybA.5 transgenic plants and compared with the controls. We conclude that these TFs alone are sufficient for activating anthocyanin production in plants and may be used as visible reporter genes for plant transformation. Evaluating these TFs in a heterologous crop species such as citrus further validated that these genes can be useful for the metabolic engineering of anthocyanin production and cultivar enhancement.

PIECE 2.0: an update for the plant gene structure comparison and evolution database.

PIECE (Plant Intron Exon Comparison and Evolution) is a web-accessible database that houses intron and exon information of plant genes. PIECE serves as a resource for biologists interested in comparing intron-exon organization and provides valuable insights into the evolution of gene structure in plant genomes. Recently, we updated PIECE to a new version, PIECE 2.0 (http://probes.pw.usda.gov/piece or http://aegilops.wheat.ucdavis.edu/piece). PIECE 2.0 contains annotated genes from 49 sequenced plant species as compared to 25 species in the previous version. In the current version, we also added several new features: (i) a new viewer was developed to show phylogenetic trees displayed along with the structure of individual genes; (ii) genes in the phylogenetic tree can now be also grouped according to KOG (The annotation of Eukaryotic Orthologous Groups) and KO (KEGG Orthology) in addition to Pfam domains; (iii) information on intronless genes are now included in the database; (iv) a statistical summary of global gene structure information for each species and its comparison with other species was added; and (v) an improved GSDraw tool was implemented in the web server to enhance the analysis and display of gene structure. The updated PIECE 2.0 database will be a valuable resource for the plant research community for the study of gene structure and evolution.

Draft Genome Sequence of Agrobacterium rhizogenes Strain NCPPB2659.

This work reports the draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, composed of one circular chromosome, the pRi2659 virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild-type strain causes hairy root disease in dicots and has been used to make transgenic hairy root cultures and composite plants (nontransgenic shoots with transgenic roots). Disarmed variants of the strain have been used to produce stable transgenic monocot and dicot plants.

Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum.

The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.

AIM: a comprehensive Arabidopsis interactome module database and related interologs in plants.

Systems biology analysis of protein modules is important for understanding the functional relationships between proteins in the interactome. Here, we present a comprehensive database named AIM for Arabidopsis (Arabidopsis thaliana) interactome modules. The database contains almost 250,000 modules that were generated using multiple analysis methods and integration of microarray expression data. All the modules in AIM are well annotated using multiple gene function knowledge databases. AIM provides a user-friendly interface for different types of searches and offers a powerful graphical viewer for displaying module networks linked to the enrichment annotation terms. Both interactive Venn diagram and power graph viewer are integrated into the database for easy comparison of modules. In addition, predicted interologs from other plant species (homologous proteins from different species that share a conserved interaction module) are available for each Arabidopsis module. AIM is a powerful systems biology platform for obtaining valuable insights into the function of proteins in Arabidopsis and other plants using the modules of the Arabidopsis interactome. Database URL:http://probes.pw.usda.gov/AIM

Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase.

BACKGROUND: Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated. RESULTS: A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used. CONCLUSION: The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.

NetVenn: an integrated network analysis web platform for gene lists.

Many lists containing biological identifiers, such as gene lists, have been generated in various genomics projects. Identifying the overlap among gene lists can enable us to understand the similarities and differences between the data sets. Here, we present an interactome network-based web application platform named NetVenn for comparing and mining the relationships among gene lists. NetVenn contains interactome network data publically available for several species and supports a user upload of customized interactome network data. It has an efficient and interactive graphic tool that provides a Venn diagram view for comparing two to four lists in the context of an interactome network. NetVenn also provides a comprehensive annotation of genes in the gene lists by using enriched terms from multiple functional databases. In addition, it allows for mapping the gene expression data, providing information of transcription status of genes in the network. The power graph analysis tool is integrated in NetVenn for simplified visualization of gene relationships in the network. NetVenn is freely available at http://probes.pw.usda.gov/NetVenn or http://wheat.pw.usda.gov/NetVenn.

The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon.

The grass species Brachypodium distachyon has emerged as a model system for the study of gene structure and function in temperate cereals. As a first demonstration of the utility of Brachypodium to study wheat gene promoter function, we transformed it with a T-DNA that included the uidA reporter gene under control of a wheat High-Molecular-Weight Glutenin Subunit (HMW-GS) gene promoter and transcription terminator. For comparison, the same expression cassette was introduced into wheat by biolistics. Histochemical staining for ß-glucuronidase (GUS) activity showed that the wheat promoter was highly expressed in the endosperms of all the seeds of Brachypodium and wheat homozygous plants. It was not active in any other tissue of transgenic wheat, but showed variable and sporadic activity in a minority of styles of the pistils of four homozygous transgenic Brachypodium lines. The ease of obtaining transgenic Brachypodium plants and the overall faithfulness of expression of the wheat HMW-GS promoter in those plants make it likely that this model system can be used for studies of other promoters from cereal crop species that are difficult to transform.