GUS Reporter Gene Research Articles

Type-B cytokinin response regulators link hormonal stimuli and molecular responses during the transition from endo- to ecodormancy in apple buds.

Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. The final step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genome-wide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identified ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profiles, determined during dormancy cycle under field and artificially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfillment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple.

Genome-Wide Identification of Dickeya solani Transcriptional Units Up-Regulated in Response to Plant Tissues From a Crop-Host Solanum tuberosum and a Weed-Host Solanum dulcamara.

Dickeya solani is a Gram-negative bacterium able to cause disease symptoms on a variety of crop and ornamental plants worldwide. Weeds including Solanum dulcamara (bittersweet nightshade) growing near agricultural fields have been reported to support populations of soft rot bacteria in natural settings. However, little is known about the specific interaction of D. solani with such weed plants that may contribute to its success as an agricultural pathogen. The aim of this work was to assess the interaction of D. solani with its crop plant (Solanum tuberosum) and an alternative (S. dulcamara) host plant. From a collection of 10,000 Tn5 transposon mutants of D. solani IPO2222 carrying an inducible, promotorless gusA reporter gene, 210 were identified that exhibited plant tissue-dependent expression of the gene/operon into which the Tn5 insertion had occurred. Thirteen Tn5 mutants exhibiting the greatest plant tissue induction of such transcriptional units in S. tuberosum or S. dulcamara as measured by qRT-PCR were assessed for plant host colonization, virulence, and ability to macerate plant tissue, as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, motility, chemotaxis toward plant extracts, biofilm formation, growth under anaerobic conditions and quorum sensing. These 13 transcriptional units encode proteins involved in bacterial interactions with plants, with functions linked to cell envelope structure, chemotaxis and carbon metabolism. The selected 13 genes/operons were differentially expressed in, and thus contributed preferentially to D. solani fitness in potato and/or S. dulcamara stem, leaf, and root tissues.

Effects of 4-bromodiphenyl ether on the adventitious root differentiation of tissue culture seedlings of Triarrhena sacchariflora

Polybrominated diphenyl ethers (PBDEs) have been highly concerned as environmental organic pollutants. 4-Bromodiphenyl ether (BDE-3) is one of the lowest brominated diphenyl ethers used as a representative of monobromobiphenyl ethers for exploring the environmental behavior and toxicology of PBDEs. The root differentiation is the critical response to the exposure of persistent organic pollutants (POPs) for understanding the phytoremediation mechanism. In this article, the adventitious root differentiation of tissue cultural seedlings of Triarrhena sacchariflora was observed under BDE-3 exposure conditions. The results showed that 3.0 mg·L−1 BDE-3 had significant positive effects on the adventitious root differentiation and the biomass, in which the higher dosage of 30.0 mg·L−1 showed inhibition effects and symptoms of injury of seedlings. The endogenous IAA/ZR ratio was at higher level than the controls and coincident with the adventitious roots differentiation in the same treatment of 3.0 mg·L−1 BDE-3. However, the responses of coleoptile elongation of Zea mays and the DR5::GUS reporter gene of Arabidopsis thaliana to BDE-3 exposure were all negative comparing with the positive controls. It could be inferred from the results that the BDE-3 may not be an auxin-like substance, and its hormesis on the adventitious root differentiation of T. sacchariflora might result from changes of endogenous IAA/ZR due to the action of BDE-3. In our knowledge, it was the first report concerning the BDE-3 hormesis on the adventitious root differentiation of T. sacchariflora tissue culture seedlings. The findings are meaningful to the further understanding of the BDE-3 effects on the plant endogenous signal substances that regulate the plant growth and development which are closely related to the phytoremediation of POPs.

Molecular basis for neofunctionalization of duplicated E3 ubiquitin ligases underlying adaptation to drought tolerance in Arabidopsis thaliana.

Multigene families in plants expanded from ancestral genes via gene duplication mechanisms constitute a significant fraction of the coding genome. Although most duplicated genes are lost over time, many are retained in the genome. Clusters of tandemly arrayed genes are commonly found in the plant genome where they can promote expansion of gene families. In the present study, promoter fusion to the GUS reporter gene was used to examine the promoter architecture of duplicated E3 ligase genes that are part of group C in the Arabidopsis thaliana ATL family. Acquisition of gene expression by AtATL78, possibly generated from defective AtATL81 expression, is described. AtATL78 expression was purportedly enhanced by insertion of a TATA box within the core promoter region after a short tandem duplication that occurred during evolution of Brassicaceae lineages. This gene is associated with an adaptation to drought tolerance of A. thaliana. These findings also suggest duplicated genes could serve as a reservoir of tacit genetic information, and expression of these duplicated genes is activated upon acquisition of core promoter sequences. Remarkably, drought transcriptome profiling in response to rehydration suggests that ATL78-dependent gene expression predominantly affects genes with root-specific activities.

Characterization of a novel LmSAP gene promoter from Lobularia maritima: Tissue specificity and environmental stress responsiveness.

Halophyte Lobularia maritima LmSAP encodes an A20AN1 zinc-finger stress-associated protein which expression is up-regulated by abiotic stresses and heavy metals in transgenic tobacco. To deepen our understanding of LmSAP function, we isolated a 1,147 bp genomic fragment upstream of LmSAP coding sequence designated as PrLmSAP. In silico analyses of PrLmSAP revealed the presence of consensus CAAT and TATA boxes and cis-regulatory elements required for abiotic stress, phytohormones, pathogen, and wound responses, and also for tissue-specific expression. The PrLmSAP sequence was fused to the β-glucuronidase (gusA) reporter gene and transferred to rice. Histochemical GUS staining showed a pattern of tissue-specific expression in transgenic rice, with staining observed in roots, coleoptiles, leaves, stems and floral organs but not in seeds or in the root elongation zone. Wounding strongly stimulated GUS accumulation in leaves and stems. Interestingly, we observed a high stimulation of the promoter activity when rice seedlings were exposed to NaCl, PEG, ABA, MeJA, GA, cold, and heavy metals (Al3+, Cd2+, Cu2+ and Zn2+). These results suggest that the LmSAP promoter can be a convenient tool for stress-inducible gene expression and is a potential candidate for crop genetic engineering.

Constitutive Contribution by the Rice OsHKT1;4 Na+ Transporter to Xylem Sap Desalinization and Low Na+ Accumulation in Young Leaves Under Low as High External Na+ Conditions.

HKT Na+ transporters correspond to major salt tolerance QTLs in different plant species and are targets of great interest for breeders. In rice, the HKT family is composed of seven or eight functional genes depending on cultivars. Three rice HKT genes, OsHKT1;1, OsHKT1;4 and OsHKT1;5, are known to contribute to salt tolerance by reducing Na+ accumulation in shoots upon salt stress. Here, we further investigate the mechanisms by which OsHKT1;4 contributes to this process and extend this analysis to the role of this transporter in plants in presence of low Na+ concentrations. By analyzing transgenic rice plants expressing a GUS reporter gene construct, we observed that OsHKT1;4 is mainly expressed in xylem parenchyma in both roots and leaves. Using mutant lines expressing artificial microRNA that selectively reduced OsHKT1;4 expression, the involvement of OsHKT1;4 in retrieving Na+ from the xylem sap in the roots upon salt stress was evidenced. Since OsHKT1;4 was found to be also well expressed in the roots in absence of salt stress, we extended the analysis of its role when plants were subjected to non-toxic Na+ conditions (0.5 and 5 mM). Our finding that the transporter, expressed in Xenopus oocytes, displayed a relatively high affinity for Na+, just above 1 mM, provided first support to the hypothesis that OsHKT1;4 could have a physiological role at low Na+ concentrations. We observed that progressive desalinization of the xylem sap along its ascent to the leaf blades still occurred in plants grown at submillimolar Na+ concentration, and that OsHKT1;4 was involved in reducing xylem sap Na+ concentration in roots in these conditions too. Its contribution to tissue desalinization from roots to young mature leaf blades appeared to be rather similar in the whole range of explored external Na+ concentrations, from submillimolar to salt stress conditions. Our data therefore indicate that HKT transporters can be involved in controlling Na+ translocation from roots to shoots in a much wider range of Na+ concentrations than previously thought. This asks questions about the roles of such a transporter-mediated maintaining of tissue Na+ content gradients in non-toxic conditions.

Identification of Elements Responsible for Maternally-Silenced Imprinted Gene Expression of Upward Curly Leaf1, an F-box Protein Gene that Regulates Curly Leaf in Arabidopsis

Upward Curly Leaf 1 (UCL1) is an Arabidopsis thaliana E3 ligase that targets the Curly Leaf (CLF) SET-domain polycomb-group (PcG) protein for degradation via the ubiquitin-26S proteasome system. UCL1 is a paternally-expressed imprinted gene in the endosperm. To precisely locate the promoter elements required for UCL1 imprinting pattern, various gene constructs were created in which the imprinting control region (ICR), endosperm-specific expression (ENSE) element, and/or the linker sequence were altered. By fusing these constructs with a GUS reporter gene, GUS expression patterns were monitored after reciprocal crosses with wild-type Columbia-0 allowing the determination of parent-of-origin expression. Analysis of publicly-available data on the UCL1 promoter region facilitated the search for allele-specific DNA and H3K27 methylation patterns. Overall, three promoter elements are required for maternal repression of UCL1; the ICR sequence located from − 2.5 to − 2.4 kb upstream of the translation start site, a differentially methylated region 2 (DMR2) that overlaps the short ATLINE1-1 transposable element in the linker region, and a minimal 271 bp ENSE element. In addition, DNA methylation patterns in the DMR2 contribute to the repression of the maternal UCL1 allele. Our findings would help to understand how parent-of-origin epigenetic patterns are created and maintained in the endosperm.

Genetic engineering system for syngas-utilizing acetogen, Eubacterium limosum KIST612

Acetogens are capable of fixing inorganic gases to organic acids with/without bio-alcohols via the Wood-Ljungdahl pathway. Because of their potential to produce value-added metabolites, acetogens have received attention in the field of biorefinery. Eubacterium limosum KIST612 is one of the promising acetogens, which has been well studied for its syngas utilizing ability, and its genomic data are available. A strain-specific genetic engineering system for maximal benefits has not yet been studied in the strain, though. In the present study, we first developed a foreign gene expression system using a native constitutive promoter and the CRISPR/Cas9-based gene editing system for KIST612. Transcriptomics-based candidate selection of native promoter and GUS reporter gene assay enabled the identification of promoter of rubredoxin oxidoreductase (Prbo) as a strong constitutive promoter in the strain. Application of Cas9-mediated genome editing was able to effectively delete the pyrF gene in the chromosome of KIST612.

Contribution of NtZIP1-like, NtZIP11 and a WAK-pectin based mechanism to the formation of Zn-related lesions in tobacco leaves

Previous studies on tobacco (Nicotiana tabacum var. Xanthi) have shown that in the presence of 200 μM Zn, lesions develop on the leaf blades from Zn accumulating mesophyll cells. This was interpreted as a tolerance mechanism against high Zn, protecting non-accumulating photosynthesizing cells. Underlying this phenomenon is the distinct capacity of mesophyll cells for Zn accumulation. To learn more about the involved mechanisms, here we searched for two key elements. First, for Zn transporters contributing to Zn accumulation in groups of mesophyll cells. Tissue-specific expression of two ZIP transporters previously identified in the leaves of tobacco plants exposed to high Zn was examined. Transgenic tobacco lines expressing GUS reporter gene under 2156 bp and 1922 bp of the promoter region of NtZIP1-like and NtZIP11, respectively, were used for analysis. Promoter activity of both genes in the leaves of high Zn-exposed plants was detected in groups of mesophyll cells, which correlates with the pattern of Zn accumulation. Second, we searched for putative components signalling the status of the cell wall. The WAKs/WAKLs and pectin fractions were the focus in the study, as it was known that upon binding pectin, WAK/WAKLs directly signals from the cell wall to the cytoplasm. Pectin microarray identified rhamnogalacturonan I (RG I) as the only pectin fraction that increased in the apoplast at high Zn (relative to control conditions), and immunocytochemistry demonstrated its presence in groups of cells. This was accompanied by increased expression of NtWAK2P-2 (putative NtWAK2), NtWAK4P-4, and NtWAK4P-2. Based on these data, a hypothesis was forwarded that RG I generated in the cell wall upon exposure to high Zn might bind to the extracellular domain of NtWAK2P-2, NtWAK4P-4, and/or NtWAK4P-2, which subsequently could transmit a signal on Zn status into the cytoplasm. It was also considered that regulation of NtZIP1-like and NtZIP11 expression by Zn availability might depend on the interaction between the RG I and/or NtWAK2P-2-, NtWAK4P-4, NtWAK4P-2.

Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation

Мета. Дослідити експресію генів gus та gfp в калюсних експлантах амфідиплоїдної пшениці спельти ( Triticum spelta L.) після Agrobacterium -опосередкованої генетичної трансформації. Методи. Для трансформації було обрано сорт пшениці спельти озимої ‘Європа’. Як експланти використовували калюси, отримані зі зрілих зародків. Прекультивацію калюсів здійснювали на живильному середовищі МС (Мурасіге–Скуга), доповненому 2 мг/л 2,4-Д (2,4-дихлорфеноксиоцтова кислота) та 10 мг/л нітратом срібла. Для генетичної трансформації використовували Agrobacterium tumefaciens Conn., штам GV3101, та генетичну конструкцію з репортерними генами gus (ген бета-глюкуронідази (-glucuronidase)) та gfp (ген зеленого флюоресцентного білка (Green Fluorescent Protein, GFP)). Калюси трансформували шляхом інокуляції з агробактеріями та вакуумною інфільтрацією. Далі їх ко-культивували на середовищі МС із 2 мг/л 2,4-Д та 10 мг/л AgNO 3 , але без антибіотиків. Експресію гена gus перевіряли за допомогою гістохімічного, а гена gfp – візуального аналізу (флуоресценція білка GFP в УФ світлі). Рівні експресії генів gfp та gus оцінювали за допомогою програмного забезпечення ImajeJ. Інтеграцію генів gfp та gus в геном спельти перевіряли методом ПЛР . Результати. Генетична трансформація калюсних експлан­тів спельти шляхом їхньої інокуляції в живильному середовищі з агробактеріями та вакуумною інфільтрацією відбувалась з різною частотою. Рівень експресії гена gus за вакуумної інфільтрації становив 4,66 ± 0,74%, за інокуляції – 4,00 ± 0,91%; а гена gfp за вакуумної інфільтрації – 3,66 ± 0,74%, за інокуляції – 4,66 ± 1,39%. Рівень експресії гена gfp був вищим у разі використання інокуляції з агробактеріями, а гена gus – при ва­ку­умній інфільтрації. За допомогою ПЛР-аналізу було під­тверджено інтеграцію генів gfp та gus в геном калюсів спельти. Довжина ПЛР продукту із праймерами до гена gus становила 240 п. н., а до гена gfp – 717 п. н. Висновки. Використання методів вакуумної інфільтрації та інокуляції для генетичної трансформації спельти дали різні результати. Частота генетичної трансформації коливалась від 3,66 до 4,66%. Agrobacterium -опосередкована генетична трансформація амфідиплоїдної пшениці спельти дозволяє дослідити експресію репортерних генів gus та gfp за використання калюсних експлантів, отриманих із зрілих зародків.

Identification of the Core Pollen-Specific Regulation in the Rice OsSUT3 Promoter.

The regulatory mechanisms of pollen development have potential value for applications in agriculture, such as better understanding plant reproductive regularity. Pollen-specific promoters are of vital importance for the ectopic expression of functional genes associated with pollen development in plants. However, there is a limited number of successful applications using pollen-specific promoters in genetic engineering for crop breeding and hybrid generation. Our previous work led to the identification and isolation of the OsSUT3 promoter from rice. In this study, to analyze the effects of different putative regulatory motifs in the OsSUT3 promoter, a series of promoter deletions were fused to a GUS reporter gene and then stably introduced into rice and Arabidopsis. Histochemical GUS analysis of transgenic plants revealed that p385 (from −385 to −1) specifically mediated maximal GUS expression in pollen tissues. The S region (from −385 to −203) was the key region for controlling the pollen-specific expression of a downstream gene. The E1 (−967 to −606), E2 (−202 to −120), and E3 (−119 to −1) regions enhanced ectopic promoter activity to different degrees. Moreover, the p385 promoter could alter the expression pattern of the 35S promoter and improve its activity when they were fused together. In summary, the p385 promoter, a short and high-activity promoter, can function to drive pollen-specific expression of transgenes in monocotyledon and dicotyledon transformation experiments.

Two promoter regions confer heat-induced activation of SlDREBA4 in Solanum lycopersicum

Dehydration-responsive element binding (DREB) transcription factors activate the expression of downstream functional genes in combination with a dehydration-responsive element (DRE), and thereby improve the resistance of plants to abiotic stresses such as heat. However, the upstream regulatory mechanism of DREB genes under heat is unclear. A DREBA4 subfamily transcription factor (SlDREBA4), which is heat-responsive and improves heat resistance, was isolated from Solanum lycopersicum ‘Microtom’. In this study, promoter truncation experiments were performed to verify changes in β-glucuronidase (GUS) enzyme activity and GUS gene expression levels in transgenic plants with different lengths of promoter fragments under heat and to identify specific regions in the promoter that respond to heat. Our results showed that the GUS reporter gene was constitutively expressed in tissues of the full-length promoter transgenic ‘Microtom’ plants, with higher expression in conducting tissues of root, stem, and leaf, as well as sepals of flowers and fruits. Under heat treatment, GUS enzyme activity and GUS gene expression levels in tissues of the full-length promoter transgenic plants increased. Promoter deletion analysis identified two positive regulatory regions (−1095 to −730 bp and −162 to −38 bp) responsible for the promoter’s response to heat. These results indicated that the heat shock element (HSE) and MYC recognition sequences may cooperate in heat-induced activation of SlDREBA4 promoter.

Functional analysis of the promoter of the MdFRK2 gene encoding a high-affinity fructokinase in apple (malus × domestica)

Our previous studies have revealed that the MdFRK2 gene is a key gene in determining fructokinase (FRK) activity and fructose metabolism. However, the transcriptional regulation mechanism of MdFRK2 is largely unknown. In this study, we cloned the 1730 bp 5′ flanking region of the MdFRK2 gene (MdFRK2P) from ‘Gala’ apple plants and constructed the pBI121-MdFRK2P expression vector by fusing the putative promoter with the GUS reporter gene and stably transforming it into Arabidopsis thaliana. Subsequent GUS staining indicated that MdFRK2 promoter expression was extremely high in active sink tissues (shoot tips). Bioinformatics analysis showed a large number of cis-acting elements associated with plant hormone regulation, light responsiveness and abiotic stress located within the 1730 bp 5′ regulatory region. To identify the critical regulatory elements that control the transcription of the MdFRK2 gene, four different lengths of 5′ deletions (-1730, -1500, -1200 and -600 bp) were fused to the GUS gene and transformed into tobacco plants. The -600 bp deletion had the highest activity. Furthermore, the MdFRK2 promoter region was strongly induced by exogenous fructose, exogenous glucose and drought stress. These findings provide essential information for studying the transcriptional regulation mechanism of MdFRK2.

Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces

BackgroundClostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq.ResultsHundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding β-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. β-glucuronidase expression was then measured in cells grown in liquid or on plates. β-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces.ConclusionsThis study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.

Characterization of Protease Inhibitor Promoter from Black Gram (Vigna mungo L.) and gus Gene Expression in Transgenic Tobacco Plants

Like other legumes, black gram is also known to be source of plant protease inhibitors. Plant protease inhibitors (PIs) are small proteins which represent a class of well studied compounds of plant defense genes and are abundant in storage organs such as seeds and tubers. The protease inhibitor proteins serve various functions in the growth and developmental processes. Like other legumes black gram (Vigna mungo L.) is also known to be a source of plant protease inhibitors and considered to be involved in insect tolerance. In this study, promoter sequence consisting of 0.75 fragments of a 52 upstream flanking region preceding a protease inhibitor gene of black gram was cloned from genomic clones and its sequence is characterized. The spatial and temporal control of the 0.75 fragment promoter sequence was also further investigated for its regulation on expression of the â-glucuronidase (gus) reporter gene in transgenic tobacco. Analysis of gus activities showed that the 0.75 kb flanking fragment of the protease inhibitor gene actually drives the gus reporter gene expression in ovary, pistil and seeds of tobacco. In another case, promoter sequence consisting of 1.3 kb fragment of a 52 flanking region of black gram protease inhibitor gene was isolated by Genome Walker strategy and promoter sequence was analyzed in-silico by PLACE and PlantCARE database.

Ectopic Expression of PAP1 Leads to Anthocyanin Accumulation and Novel Floral Color in Genetically Engineered Goldenrod (Solidago canadensis L.)

Floral pigmentation is of major importance to the ornamental industry, which is constantly searching for cultivars with novel colors. Goldenrod (Solidago canadensis) has monochromatic yellow carotenoid-containing flowers that cannot be modified using classical breeding approaches due to a limited gene pool. To generate Solidago with novel colors through metabolic engineering, we first developed a procedure for its regeneration and transformation. Applicability of different cytokinins for adventitious regeneration was examined in the commercial cv. Tara, with zeatin yielding higher efficiency than 6-benzylaminopurine or thidiazuron. A comparison of regeneration of commercial cvs. Tara, Golden Glory and Ivory Glory revealed Tara to be the most potent, with an efficiency of 86% (number of shoots per 100 leaf explants). Agrobacterium-based transformation efficiency was highest for cv. Golden Glory (5 independent transgenic shoots per 100 explants) based on kanamycin selection and the GUS reporter gene. In an attempt to promote anthocyanin biosynthesis, we generated transgenic Solidago expressing snapdragon (Antirrhinum majus) Rosea1 and Delila, as well as Arabidopsis thaliana PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) transcription factors. Transgenic cv. Golden Glory expressing cauliflower mosaic virus 35S-driven PAP1 generated red flowers that accumulated delphinidin and its methylated derivatives, as compared to control yellow flowers in the GUS-expressing plants. The protocol described here allows efficient engineering of Solidago for novel coloration and improved agricultural traits.

Diverse transcription factors control monoterpene synthase expression in lavender (Lavandula).

We cloned eight transcription factors that activate lavender monoterpene synthase promoters. In this study, we employed the Yeast One-Hybrid (Y1H) assay system to identify transcription factors that control promoters for two Lavandula × intermedia monoterpene synthase genes, linalool synthase (LiLINS) and 1,8-cineole synthase (LiCINS). The bait sequences used in the assay were either a 768-bp LiLINS, or a 1087-bp LiCINS promoter. The prey included proteins expressed in L. × intermedia floral tissue. The assay identified 96 sequences encoding proteins that interacted with one or both promoters. To explore the nature of this interaction, the LiLINS and LiCINS promoter fragments were each fused to the E. coli gusA (GUS) reporter gene. The constructs were separately transformed into tobacco (Nicotiana benthamiana) leaves co-expressing individually a subset of ten representative transcription factors (TFs) predicted to control these promoters. Six TFs induced expression from both promoters, two activated LiCINS promoter alone, and two did not induce expression from either promoter. The TFs identified in this study belong to various groups including those containing conserved domains typical of MYB, bZIP, NAC, GeBP and SBP-related proteins.

Identification of an Embryonic Cell-Specific Region within the Pineapple SERK1 Promoter.

Plant tissue culture methods, such as somatic embryogenesis, are attractive alternatives to traditional breeding methods for plant propagation. However, they often suffer from limited efficiency. Somatic embryogenesis receptor kinase (SERK)1 is a marker gene of early somatic embryogenesis in several plants, including pineapple. It can be selectively induced and promotes a key step in somatic embryogenesis. We investigated the embryonic cell-specific transcriptional regulation of AcSERK1 by constructing a series of vectors carrying the GUS (Beta-glucuronidase) reporter gene under the control of different candidate cis-regulatory sequences. These vectors were transfected into both embryonic and non-embryonic callus, and three immature embryo stages and the embryonic-specific activity of the promoter fragments was analyzed. We found that the activity of the regulatory sequence of AcSERK1 lacking −983 nt ~−880 nt, which included the transcription initiation site, was significantly reduced in the embryonic callus of pineapple, accompanied by the loss of embryonic cell-specific promoter activity. Thus, this fragment is an essential functional segment with highly specific promoter activity for embryonic cells, and it is active only from the early stages of somatic embryo development to the globular embryo stage. This study lays the foundation for identifying mechanisms that enhance the efficiency of somatic embryogenesis in pineapple and other plants.

The ScRALF3 secreted peptide is involved in sporophyte to gametophyte signalling and affects pollen mitosis I.

Signalling events through small peptides are essential in multiple aspects of plant reproduction. The ScRALF3 Solanum chacoense Rapid Alkalinization Factor (RALF) peptide was previously shown to regulate multiple aspects of cell-cell communication between the surrounding sporophytic tissue and the female gametophyte during ovule development. We analysed the global expression pattern of ScRALF3 with GUS reporter gene under control of the ScRALF3 promoter and validated it with in situ hybridisation. To better understand the role of ScRALF3 we used three different RNA interference (RNAi) lines that reduced the expression of ScRALF3 during pollen development. Both expression methods showed the presence of ScRALF3 in different tissues, including stigma, style, vascular tissues and during stamen development. Down-regulation of ScRALF3 expression through RNAi showed drastic defects in early stages of pollen development, mainly on the first mitosis. These results suggest that the ScRALF3 secreted peptide regulates the transition from sporogenesis to gametogenesis in both male and female gametophytes.

Comprehensive construction strategy of bidirectional green tissue-specific synthetic promoters.

SummaryBidirectional green tissue‐specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue‐specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequence selection and the bidirectional green tissue‐specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium‐mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activities in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiencies specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters, and we successfully created four bidirectional green tissue‐specific synthetic promoters. It is the first report on bidirectional green tissue‐specific promoters that could be efficiently applied in genetic engineering.