RY Elements Research Articles

FaLEC2 repressing FaLOX2 promoter involved in the metabolism of LOX-derived volatiles during strawberry ripening

• FaLOX2 expression level was negatively correlated with hexanal content. • RNA-Seq identified 43 B3 TFs, including LAV (3), RAV (18), ARF (13), and REM (9). • FaLEC2 expression level was negatively correlated with ( E,Z )-2,6-nonadienal content. • FaLEC2 transcriptionally repressed the activity of FaLOX2 promoter. Lipoxygenase (LOX) pathway is the key and the most important pathway for the metabolism of fruit volatile. However, the transcription mechanism of the LOX pathway is still obscure in non-climacteric fruit. In order to identify the transcription factor involved in LOX-derived volatile metabolism, RNA sequencing and metabolome were used to profile strawberry ( Fragaria × ananassa Duch.). Correlation analysis shows that the expression of FaLOX2 was negatively correlated with the hexanal content ( r = −0.985). The RY element that specifically bound by the B3 domain transcription factors, are found in FaLOX2 promoter. B3 domain-containing transcription factors including LEAFY COTYLEDON2 (LEC2)–ABI3–VAL (LAV, 3), RELATED TO ABI3 and VP1 (RAV, 18), AUXIN RESPONSE FACTOR (ARF, 13), and REPRODUCTIVE MERISTEM (REM, 9) are identified. It is noticed that FaLEC2 expression is negatively correlated with ( E,Z )-2,6-nonadienal content ( r = −1.000). Yeast one-hybrid analysis and dual-luciferase analysis suggest that FaLEC2 transcriptionally represses the activity of FaLOX2 promoter. Moreover, FaLEC2 expression level is negatively correlated with FaLOX2 expression ( r = −0.956). Here, our demonstration that FaLEC2 trans-represses the activity of the FaLOX2 promoter provides evidence that FaLEC2 directly controls a transcriptional program for LOX-derived volatile metabolism in strawberries.

Characterization and expression quantitative trait loci analysis of TaABI4, a pre-harvest sprouting related gene in wheat

AbstractPre-harvest sprouting (PHS) induced by the absence of seed dormancy causes a severe reduction in crop yield and flour quality. In this study, we isolated and characterized TaABI4, an ABA-responsive transcription factor that participates in regulating seed germination in wheat. Sequence analysis revealed that TaABI4 has three homologues, located on chromosomes 1A/1B/1D. TaABI4 contains a conserved AP2 domain, and AP2-associated, LRP and potential PEST motifs. Putative cis-acting regulatory elements (CE1-like box, W-box, ABRE elements and RY elements) were identified in the TaABI4 promoter region that showed high conservation in 17 wheat cultivars and wheat-related species. Expression profiling of TaABI4 indicated that it is a seed-specific gene accumulating during the middle stages of seed development. Transcript accumulation of TaABI4 in wheat cultivar Chuanmai 32 (CM32, PHS susceptible) was 5.07-fold and 1.39-fold higher than that in synthetic hexaploidy wheat SHW-L1 (PHS resistant) at 15 and 20 DPA, respectively. Six expression quantitative trait loci (eQTL) of TaABI4 on chromosomes 2A, 2D, 3B and 4A were characterized based on the accumulated transcripts of TaABI4 in SHW-L1 and CM32-derived recombinant inbred lines. These QTLs explained 10.7 to 46.1% of the trait variation with 4.53–10.59 of LOD scores, which contain genes that may affect the expression of TaABI4.

Seed-specific activity of the Arabidopsis β-glucosidase 19 promoter in transgenic Arabidopsis and tobacco.

The promoter of the Arabidopsis thaliana β-glucosidase 19 gene directs GUS expression in a seed-specific manner in transgenic Arabidopsis and tobacco. In the present study, an 898-bp putative promoter of theArabidopsis β-glucosidase 19 (AtBGLU19) gene was cloned. The bioinformatics analysis of the cis-acting elements indicated that this putative promoter contains many seed-specific elements, such as RY elements. The features of this promoter fragment were evaluated for the capacity to direct the β-glucuronidase (GUS) reporter gene in transgenic Arabidopsis and tobacco. Histochemical and fluorometric GUS analyses of transgenic Arabidopsis plants revealed that the AtBGLU19 promoter directed strong GUS activity in late-maturing seeds and dry seeds, whereas no GUS expression was observed in other organs. The results indicated that the AtBGLU19 promoter was able to direct GUS expression in a seed-specific manner in transgenic Arabidopsis. In tobacco, the intensity of the staining and the level of GUS activity were considerably higher in the seeds than in the other tissues. These results further confirmed that the AtBGLU19 promoter is seed specific and can be used to control transgene expression in a heterologous plant system.

Analysis of the Function of the Alfalfa Mslea-D34 Gene in Abiotic Stress Responses and Flowering Time.

A novel late embryogenesis abundant (LEA) gene, MsLEA-D34, was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression (OE) and RNA interference (RNAi) of the gene in Arabidopsis and in hairy roots of alfalfa, as well as via analyzing key genes related to MsLEA-D34 during developmental phases in alfalfa. The results showed that MsLEA-D34 was a typical intrinsically disordered protein with a high capability for protein protection. Overexpression of MsLEA-D34 increased plant tolerance to osmotic and salt stresses, and caused Arabidopsis early flowering under drought and well-watered conditions. Overexpressing MsLEA-D34 induced up-regulation of FLOWERING LOCUS T (FT) and GIGANTEA (GI) at the flowering phase of Arabidopsis and hairy roots of alfalfa, but only FT was down-regulated in MsLEA-D34-RNAi lines. A positive effect of MsLEA-D34 on FT accumulation was demonstrated in alfalfa hairy roots. An ABA-responsive element (ABRE)-binding transcription factor (MsABF2), a novel transcription factor cloned from alfalfa, directly bound to the RY element in the MsLEA-D34 promoter and activated MsLEA-D34 expression. The above results indicate that MsLEA-D34 can regulate abiotic stress response in plants and influence flowering time of Arabidopsis.

HSI2/VAL1 and HSL1/VAL2 function redundantly to repress DOG1 expression in Arabidopsis seeds and seedlings.

Summary DELAY OF GERMINATION1 (DOG1) is a primary regulator of seed dormancy. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination in Arabidopsis. B3 domain‐containing transcriptional repressors HSI2/VAL1 and HSL1/VAL2 silence seed dormancy and enable the subsequent germination and seedling growth. However, the roles of HSI2 and HSL1 in regulation of DOG1 expression and seed dormancy remain elusive.Seed dormancy was analysed by measurement of maximum germination percentage of freshly harvested Arabidopsis seeds. In vivo protein–protein interaction analysis, ChIP‐qPCR and EMSA were performed and suggested that HSI2 and HSL1 can form dimers to directly regulate DOG1.HSI2 and HSL1 dimers interact with RY elements at DOG1 promoter. Both B3 and PHD‐like domains are required for enrichment of HSI2 and HSL1 at the DOG1 promoter. HSI2 and HSL1 recruit components of polycomb‐group proteins, including CURLY LEAF (CLF) and LIKE HETERCHROMATIN PROTEIN 1 (LHP1), for consequent deposition of H3K27me3 marks, leading to repression of DOG1 expression.Our findings suggest that HSI2‐ and HSL1‐dependent histone methylation plays critical roles in regulation of seed dormancy during seed germination and early seedling growth.

HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition from Seed Maturation to Vegetative Growth in Arabidopsis.

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

PCR amplification and bioinformatics assessment of promoters of PBF-DOF (DNA binding with one finger) genes of finger millet

The Dof (DNAbinding with one finger) family is a plant specific transcription factor known to be involved with regulating diverse functions in plants and have been extensively studied in many crops. The Dof transcription factor regulating gene expression by interacting with Cis-regulatory elements namely prolamin box (P box), GCN4, AACA and ACGT motifs present in the promoters of seed storage protein genes is known as PBF (Prolamin-box Binding Factor) Dof transcription factor. A set of 15 primers were designed by considering approximately 1.5 kb upstream and 500bp downstream sequences of full length Dof genes from TSS of cereals like rice, wheat and sorghum available in databases. These primers were used for PCR amplification of putative promoters of Dof genes of finger millet along with few cereals and millets. Furthermore, based on the presence of expected size amplicon with different sets of primers tested, a total of 6 bands of expected size representing putative promoters of PBF-Dof genes of rice, sorghum, barnyard, finger millets (PRM-1 PRM-801, PRM-701) were eluted, sequenced and subjected to in silico investigation. The bioinformatics based characterization revealed uniform presence TSS and numerous seed storage protein specific motifs like DPBF motif, RY element, SKN1 motif, GCN4 motif, E-Box confirming the promoters of respective PBF-Dof genes of cereals and millets. Further, validation by cloning in promoter probe vector is required for confirmation of temporal and spatial expression associated with seed storage protein genes.

Abscisic acid-induced gene expression in the liverwort Marchantia polymorpha is mediated by evolutionarily conserved promoter elements.

Abscisic acid (ABA) is a phytohormone widely distributed among members of the land plant lineage (Embryophyta), regulating dormancy, stomata closure and tolerance to environmental stresses. In angiosperms (Magnoliophyta), ABA-induced gene expression is mediated by promoter elements such as the G-box-like ACGT-core motifs recognized by bZIP transcription factors. In contrast, the mode of regulation by ABA of gene expression in liverworts (Marchantiophyta), representing one of the earliest diverging land plant groups, has not been elucidated. In this study, we used promoters of the liverwort Marchantia polymorpha dehydrin and the wheat Em genes fused to the β-glucuronidase (GUS) reporter gene to investigate ABA-induced gene expression in liverworts. Transient assays of cultured cells of Marchantia indicated that ACGT-core motifs proximal to the transcription initiation site play a role in the ABA-induced gene expression. The RY sequence recognized by B3 transcriptional regulators was also shown to be responsible for the ABA-induced gene expression. In transgenic Marchantia plants, ABA treatment elicited an increase in GUS expression in young gemmalings, which was abolished by simultaneous disruption of the ACGT-core and RY elements. ABA-induced GUS expression was less obvious in mature thalli than in young gemmalings, associated with reductions in sensitivity to exogenous ABA during gametophyte growth. In contrast, lunularic acid, which had been suggested to function as an ABA-like substance, had no effect on GUS expression. The results demonstrate the presence of ABA-specific response mechanisms mediated by conserved cis-regulatory elements in liverworts, implying that the mechanisms had been acquired in the common ancestors of embryophytes.

Profiles of embryonic nuclear protein binding to the proximal promoter region of the soybean β-conglycinin α subunit gene.

β-Conglycinin, a major component of seed storage protein in soybean, comprises three subunits: α, α' and β. The expression of genes for these subunits is strictly controlled during embryogenesis. The proximal promoter region up to 245bp upstream of the transcription start site of the α subunit gene sufficiently confers spatial and temporal control of transcription in embryos. Here, the binding profile of nuclear proteins in the proximal promoter region of the α subunit gene was analysed. DNase I footprinting analysis indicated binding of proteins to the RY element and DNA regions including box I, a region conserved in cognate gene promoters. An electrophoretic mobility shift assay (EMSA) using different portions of box I as a probe revealed that multiple portions of box I bind to nuclear proteins. In addition, an EMSA using nuclear proteins extracted from embryos at different developmental stages indicated that the levels of major DNA-protein complexes on box I increased during embryo maturation. These results are consistent with the notion that box I is important for the transcriptional control of seed storage protein genes. Furthermore, the present data suggest that nuclear proteins bind to novel motifs in box I including 5'-TCAATT-3' rather than to predicted cis-regulatory elements.

The rice GERMINATION DEFECTIVE 1, encoding a B3 domain transcriptional repressor, regulates seed germination and seedling development by integrating GA and carbohydrate metabolism

It has been shown that seed development is regulated by a network of transcription factors in Arabidopsis including LEC1 (LEAFY COTYLEDON1), L1L (LEC1-like) and the B3 domain factors LEC2, FUS3 (FUSCA3) and ABI3 (ABA-INSENSITIVE3); however, molecular and genetic regulation of seed development in cereals is poorly understood. To understand seed development and seed germination in cereals, a large-scale screen was performed using our T–DNA mutant population, and a mutant germination-defective1 (gd1) was identified. In addition to the severe germination defect, the gd1 mutant also shows a dwarf phenotype and abnormal flower development. Molecular and biochemical analyses revealed that GD1 encodes a B3 domain-containing transcription factor with repression activity. Consistent with the dwarf phenotype of gd1, expression of the gibberelic acid (GA) inactivation gene OsGA2ox3 is increased dramatically, accompanied by reduced expression of GA biosynthetic genes including OsGA20ox1, OsGA20ox2 and OsGA3ox2 in gd1, resulting in a decreased endogenous GA4 level. Exogenous application of GA not only induced GD1 expression, but also partially rescued the dwarf phenotype of gd1. Furthermore, GD1 binds to the promoter of OsLFL1, a LEC2/FUS3-like gene of rice, via an RY element, leading to significant up-regulation of OsLFL1 and a large subset of seed maturation genes in the gd1 mutant. Plants over-expressing OsLFL1 partly mimic the gd1 mutant. In addition, expression of GD1 was induced under sugar treatment, and the contents of starch and soluble sugar are altered in the gd1 mutant. These data indicate that GD1 participates directly or indirectly in regulating GA and carbohydrate homeostasis, and further regulates rice seed germination and seedling development.

Arabidopsis CYP82C4 expression is dependent on Fe availability and circadian rhythm, and correlates with genes involved in the early Fe deficiency response

Under conditions of reduced iron availability, most frequent in calcareous soils, plants induce the “Fe Deficiency Response” to improve root Fe uptake. The transcription factor FIT is essential for such a response in strategy I plants, such as Arabidopsis thaliana. From microarray analysis of Arabidopsis roots, it is known that three different cytochrome P450 genes, CYP82C4, CYP82C3 and CYP71B5 are up-regulated under Fe deficiency through a FIT-dependent pathway. We show that, out of these three P450 genes, only CYP82C4 strongly correlates with genes involved in metal uptake/transport. The CYP82C4 promoter, unlike those of CYP82C3 and CYP71B5, contains several IDE1-like sequences ( iron deficiency-responsive element) as well as an RY element. While confirming that the CYP82C4 transcript accumulates in Fe-deficient Arabidopsis seedlings, with circadian fluctuations in a light-dependent way, we also demonstrate that such accumulation is suppressed under Fe excess. Full suppression of CYP82C4 expression, as observed in the atc82c4-1 KO mutant, is associated with longer roots at the seedling stage. We propose that CYP82C4 is involved in the early Fe deficiency response, possibly through an IDE1-like mediated pathway.

Dual regulation of iron deficiency response mediated by the transcription factor IDEF1

Higher plants respond to fluctuating Fe availability by regulating the expression of genes involved in Fe homeostasis. Transcriptional responses to Fe deficiency in plants are mediated via various interactions between cis-acting elements and trans-acting factors. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing the CATGC sequence within the Fe deficiency-responsive cis-acting element IDE1. We recently presented evidence that IDEF1 mediates two-phase responses to Fe deficiency. During the early stages of Fe deficiency, the majority of known Fe uptake/utilization-related genes are positively regulated by IDEF1. In subsequent stages, IDEF1-mediated regulation of these Fe uptake/utilization-related genes are less obvious. In turn, expression of several Fe deficiency-induced genes encoding late embryogenesis abundant proteins is increasingly regulated by IDEF1 at the subsequent stages. We propose a dual function of IDEF1 for Fe deficiency response, namely, (1) the coordinated transactivation of Fe utilization-related genes via CATGC-containing IDE1-like elements, especially at the early stage, and (2) the transactivation of seed maturation-related genes via RY elements, especially during the subsequent stages of Fe deficiency. IDEF1 appears to have evolved to mediate an interface between Fe deficiency-inducible and seed maturation-related gene expression.

The cowpea trypsin inhibitor promoter drives expression in response to cellular maturation in Arabidopsis thaliana.

The bowman-birk type trypsin inhibitors accumulate in high concentration in legume and cereal seeds, especially during seed maturation and are considered to be involved in insect tolerance. The 5' flanking sequences of the trypsin inhibitor was isolated from cowpea genomic DNA using anchor PCR. Analysis of sequences showed presence of seed specific RY elements and also other elements associated with seed development such as abscisic acid responsive elements (ABA responsive elements; ABRE) and dehydration responsive elements (DRE). Spatial and temporal control of the promoter driven expression pattern was analyzed using gus as reporter. Expression was found to occur both in embryo and endosperm; starting from torpedo stage of embryogenesis and continuing till the stage of final maturation i.e. bent cotyledon stage. Additional expression analyses showed that the promoter actually drives expression in tissues like leaves, roots, stipules, etc., but followed a specific pattern. Comparative analysis of expression in seeds and other organs indicated that the promoter driven expression is in response to cellular maturation.

The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes

Higher plants maintain iron homeostasis by regulating the expression of iron (Fe)-related genes in accordance with Fe availability. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing CATGC sequences within the Fe deficiency-responsive cis-acting element IDE1. To investigate the function of IDEF1 in detail, we analyzed the response to Fe deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression. Fe-deficiency treatment in hydroponic culture revealed that IDEF1 knock-down plants are susceptible to early-stage Fe deficiency, in contrast to IDEF1-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the IDEF1 expression level was positively correlated with the level of induction of the Fe utilization-related genes OsIRO2, OsYSL15, OsIRT1, OsYSL2, OsNAS1, OsNAS2, OsNAS3 and OsDMAS1, just after the onset of Fe starvation. However, this overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit over-representation of CATGC and IDE1-like elements within the proximal promoter regions. These results indicate the existence of early and subsequent responses to Fe deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1-regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in Fe-deficient roots and/or leaves in an IDEF1-dependent manner, suggesting a possible function of seed maturation-related genes in Fe-deficient vegetative organs.

The RY/Sph element mediates transcriptional repression of maturation genes from late maturation to early seedling growth

In orthodox seeds, the transcriptional activator ABI3 regulates two major stages in embryo maturation: a mid-maturation (MAT) stage leading to accumulation of storage compounds, and a late maturation (LEA) stage leading to quiescence and desiccation tolerance. Our aim was to elucidate mechanisms for transcriptional shutdown of MAT genes during late maturation, to better understand phase transition between MAT and LEA stages. Using transgenic and transient approaches in Nicotiana, we examined activities of two ABI3-dependent reporter genes driven by multimeric RY and abscisic acid response elements (ABREs) from a Brassica napus napin gene, termed RY and ABRE, where the RY reporter requires ABI3 DNA binding. Expression of RY peaks during mid-maturation and drops during late maturation, mimicking the MAT gene program, and in Arabidopsis thaliana RY elements are over-represented in MAT, but not in LEA, genes. The ABI3 transactivation of RY is inhibited by staurosporine, by a PP2C phosphatase, and by a repressor of maturation genes, VAL1/HSI2. The RY element mediates repression of MAT genes, and we propose that transcriptional shutdown of the MAT program during late maturation involves inhibition of ABI3 DNA binding by dephosphorylation. Later, during seedling growth, VAL1/HSI2 family repressors silence MAT genes by binding RY elements.

Efficient LEC2 activation of OLEOSIN expression requires two neighboring RY elements on its promoter

As the main structural protein of oil body, OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 domain transcription factor leafy cotyledon2 (LEC2) plays an important role in regulating seed development and seed-specific gene expression. Here, we first report how seed-specific B3 domain transcription factor leafy cotyledon2 (LEC2) efficiently activates OLEOSIN expression. The central promoter region of OLEOSIN, responsible for seed specificity and LEC2 activation, was determined by 5'-deletion analysis. Binding experiments in yeast cells and electrophoretic mobility shift assays showed that LEC2 specifically bound to two conserved RY elements in this region. In transient expression assays, mutation in either RY element dramatically reduced LEC2 activation of OLEOSIN promoter activity, while double mutation abolished it. Analysis of the distribution of RY elements in seed-specific genes activated by LEC2 also supported the idea that genes containing neighboring RY elements responded strongly to LEC2 activation. Therefore, we conclude that two neighboring RY elements are essential for efficient LEC2 activation of OLEOSIN expression. These findings will help us better utilize seed-specific promoter activity.

A Nuclear Gene Encoding the Iron-Sulfur Subunit of Mitochondrial Complex II Is Regulated by B3 Domain Transcription Factors during Seed Development in Arabidopsis

Mitochondrial complex II (succinate dehydrogenase) is part of the tricarboxylic acid cycle and the respiratory chain. Three nuclear genes encode its essential iron-sulfur subunit in Arabidopsis (Arabidopsis thaliana). One of them, SUCCINATE DEHYDROGENASE2-3 (SDH2-3), is specifically expressed in the embryo during seed maturation, suggesting that SDH2-3 may have a role as the complex II iron-sulfur subunit during embryo maturation and/or germination. Here, we present data demonstrating that three abscisic acid-responsive elements and one RY-like enhancer element, present in the SDH2-3 promoter, are involved in embryo-specific SDH2-3 transcriptional regulation. Furthermore, we show that ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON2, three key B3 domain transcription factors involved in gene expression during seed maturation, control SDH2-3 expression. Whereas ABI3 and FUS3 interact with the RY element in the SDH2-3 promoter, the abscisic acid-responsive elements are shown to be a target for bZIP53, a member of the basic leucine zipper (bZIP) family of transcription factors. We show that group S1 bZIP53 protein binds the promoter as a heterodimer with group C bZIP10 or bZIP25. To the best of our knowledge, the SDH2-3 promoter is the first embryo-specific promoter characterized for a mitochondrial respiratory complex protein. Characterization of succinate dehydrogenase activity in embryos from two homozygous sdh2-3 mutant lines permits us to conclude that SDH2-3 is the major iron-sulfur subunit of mature embryo complex II. Finally, the absence of SDH2-3 in mutant seeds slows down their germination, pointing to a role of SDH2-3-containing complex II at an early step of germination.

Analysis of the activity of a γ-gliadin promoter in transgenic wheat and characterization of gliadin synthesis in wheat by MALDI-TOF during grain development

The wheat grain is the most important organ for human food and therefore is the target for much research focused on modifying its composition to improve nutritional and functional components. Genetic transformation provides a precise tool to alter the composition of wheat grain by expressing new genes or by down-regulating groups of proteins encoded by multigene families such as gliadins, which contain clusters of epitopes that are active groups in triggering celiac disease. For such work, specific promoters are required to express such constructs in the wheat endosperm. In the present study we report the isolation and characterization of a γ-gliadin promoter from transgenic wheat, and the analysis of gliadin synthesis during grain development in bread wheat by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI/TOF MS). The γ-gliadin promoter fragment was isolated from bread wheat by genome walking and was re-introduced, driving the expression of the gusA gene, by particle bombardment, giving fifteen independent transgenic lines. Detailed analysis of the sequence of the 885 bp promoter fragment showed that it contains three prolamin boxes but only one is conserved according to the consensus sequence reported. The AACA/TA motif is present twice in published γ-gliadin promoter sequences. The RY element i.e., CATGCAT or CATGCAC, is also present twice in the published promoter. Transgenic lines were classified as high, medium, and low expressers. The expression of the gusA gene was found only in the seeds of the transgenic lines. GUS staining was first detected in the outer endosperm of the lobes, and then it extended to the whole outer endosperm. GUS staining was not found in the aleurone layer nor in the embryo. The qRT-PCR data confirmed the data obtained by GUS staining. The expression of the gusA gene determined by qRT-PCR for the high expresser line (B281) was 4 and 8 times higher than that of medium (B282) and low (B286) expresser lines, respectively. MALDI/TOF-MS showed that gliadins exhibited different patterns of synthesis during the course of seed maturation. Thus, gliadins with masses higher than 36,000 Da were synthesised within the first 12 days post anthesis while those with masses lower than 36,000 Da were synthesised later. Results of GUS staining, qRT-PCR and MALDI/TOF-MS showed that the γ-gliadin promoter reported in this work could be a good candidate to downregulate wheat gliadins.

Transient embryo-specific expression of anthocyanin in wheat

Three barley tissue-specific promoters (Dhn12, Itr1, and Ltp1) were transcriptionally fused to the C1 and/or the B-peru genes involved in anthocyanin biosynthesis. Transient expression of these constructs was examined in different wheat tissues. Constructs with tissue-specific promoters were only active in embryos. A green fluorescent protein (GFP) driven by the actin promoter was used as an internal standard to monitor the effectiveness of each bombardment. Further, normalization of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments, and the intensity of anthocyanin pigmentation quantified by image analysis allowed for a rapid and accurate evaluation of different promoters. Compared to CaMV35S promoter, tissue-specific promoters were more effective in directing anthocyanin production, specifically in wheat embryos, and the C1 gene was more effective than B-peru. Among the tissue-specific promoters, the Ltp1 was superior to ltr1 and Dhn12 in combination with C1 and/or B-peru gene(s). Analysis of nucleotide sequences of all three tissue-specific promoters revealed the presence of G-box, E-box, and RY elements, which might trigger embryo-specific expression in wheat.

AtGA3ox2, a Key Gene Responsible for Bioactive Gibberellin Biosynthesis, Is Regulated during Embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.