The sucrose synthase-1 promoter from Citrus sinensis directs expression of the β-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides −410 to −268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides −268 to −103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

GUS expression driven by constitutive and phloem-specific promoters in citrus hybrid US-802

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.     

Sucrose carrier RcSCR1 is involved in sucrose retrieval, but not in sucrose unloading in growing hypocotyls of Ricinus communis L

The transport of assimilates from source to sink tissues is mediated by the phloem. Along the vascular system the phloem changes its physiological function from loading phloem to transport and unloading phloem. Sucrose carrier proteins have been identified in the transport phloem, but it is unclear whether the physiological role of these transporters is phloem unloading of sucrose or retrieval of apoplasmic sucrose back into the sieve element/companion cell complex. Here, we describe the dynamic expression of the Ricinus communis sucrose carrier RcSCR1 in the hypocotyl at different sink strengths. Our results indicate that phloem unloading in castor bean is not catalysed by the phloem loader RcSCR1. However, this sucrose carrier represents the molecular basis of the sucrose retrieval mechanism along the transport phloem, which is dynamically adjusted to the sink strength. As a consequence, we assume that other release carrier(s) exist in sink tissues, such as the hypocotyl, in R. communis.     

Arabidopsis SUC1 loads the phloem in suc2 mutants when expressed from the SUC2 promoter

Active loading of sucrose into phloem companion cells (CCs) is an essential process in apoplastic loaders, such as Arabidopsis or tobacco (Nicotiana sp.), and is even used by symplastic loaders such as melon (Cucumis melo) under certain stress conditions. Reduction of the amount or complete removal of the transporters catalysing this transport step results in severe developmental defects. Here we present analyses of two Arabidopsis lines, suc2-4 and suc2-5, that carry a null allele of the SUC2 gene which encodes the Arabidopsis phloem loader. These lines were complemented with constructs expressing either the Arabidopsis SUC1 or the Ustilago maydis srt1 cDNA from the SUC2 promoter. Both SUC1 and Srt1 are energy-dependent sucrose/H(+) symporters and differ in specific kinetic properties from the SUC2 protein. Transgene expression was confirmed by RT-PCRs, the subcellular localization of Srt1 in planta with an Srt1-RFP fusion, and the correct CC-specific localization of the recombinant proteins by immunolocalization with anti-Srt1 and anti-SUC1 antisera. The transport capacity of Srt1 was studied in Srt1-GFP expressing Arabidopsis protoplasts. Although both proteins were found exclusively in CCs, only SUC1 complemented the developmental defects of suc2-4 and suc2-5 mutants. As SUC1 and Srt1 are well characterized, this result provides an insight into the properties that are essential for sucrose transporters to load the phloem successfully.     

Molecular characterization of the PpMADS1 gene from peach

PpMADS1, a member of the euAP1 clade of the class A genes, was previously cloned from peach. In this study, PpMADS1 was constitutively expressed in Arabidopsis thaliana to study its function in plant development. The transgenic A. thaliana plants containing 35S::PpMADS1 showed severe phenotype variation including early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, production of higher number of carpels, petals, and stamens than non-transgenic plants, and prevention of pod shatter. Significantly, the transgenic plants produced more than one silique from a single flower. The results obtained by using cDNA microarray and real-time PCR analyses in the transgenic Arabidopsis indicated that PpMADS1 might play dual roles in regulating the floral meristem development by activating or repressing different sets of genes that would determine the different fate of a floral meristem. In addition, the PpMADS1 gene promoter was further cloned, and deletion analyses were conducted by using fused GUS as a reporter gene in transgenic A. thaliana. Histochemical staining of different organs from transgenic plants revealed the region between ?197 and ?454?bp was specific for GUS expression in flower primordium, and the region between ?454 and ?678?bp was specific for GUS expression in sepals and petals. In contrast, a negative regulatory element present between ?678 and ?978?bp could suppress GUS expression in filament.     

Rice sucrose transporter1 (OsSUT1) up‐regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles

The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3‐week‐old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1‐ to 10‐day infestation time course, using a combination of gene expression and β‐glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide‐field fluorescence microscopy was used to confirm the expression of OsSUT1‐promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up‐regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up‐regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid‐related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.     

The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2

The Arabidopsis thaliana (L.) Heynh. SUC2 gene encodes a plasma-membrane sucrose-H⁺ symporter. The DNA sequence of the SUC2 promoter has been determined. Using a translational fusion of this promoter to the N-terminus of -glucuronidase (GUS) and the GUS histochemical assay, the tissue specificity of the SUC2 promoter was studied in Arabidopsis plants transformed with this fusion construct. The SUC2 promoter directed expression of GUS activity with high specificity to the phloem of all green tissues of Arabidopsis such as rosette leaves, stems, and sepals. During leaf development the expression of SUC2-GUS activity was first seen in the tips of young rosette leaves. In older leaves and during their concomitant sink/source transition, expression proceeded from the tips to the bases of the leaves, indicating that expression of the SUC2 sucrose-H⁺ symporter is tightly coupled to the source-strength of Arabidopsis leaves. Expression of SUC2-GUS activity was also seen, however, in sink tissues such as roots and developing Arabidopsis pods, suggesting that the product of the SUC2 gene might not only be important for phloem loading, but also for phloem unloading. A possible regulatory effect of carbohydrates (glucose and sucrose) on the activity of the SUC2 promoter was studied and excluded, both in excised leaves and young seedlings of transgenic Arabidopsis plants. The overall pattern of SUC2-GUS expression correlated well with that of the Arabidopsis thaliana AHA3 plasma-membrane H⁺ -ATPase which is also expressed in the phloem and most likely represents the primary pump generating the energy for secondary active transporters such as SUC2.Abbreviations GUS -glucuronidase - MS Murashige & Skocgmedium - X-Gluc 5-bromo-4-chloro-3-indolyl--d-glucuronic acid Accession number for SUC2-promoter sequences: The DNA sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence databases under the accession number X79702 (AtSUC2 promoter sequence)We want to thank Günther Peissig for growing the Arabidopsis thaliana plants. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 43/C5) and a grant to N.S. from the Bundesministerium für Forschung und Technologie.     

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H⁺ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The K_M-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The V_MAX but not the K_M-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.     

Construction of Flower-specific Chimeric Promoters and Analysis of Their Activities in Transgenic Torenia

Flower-specific promoters can enable transgenic enhancement of valuable ornamental traits, including flower shape and color. However, the identification of strong, tissue-specific promoters remains a limiting factor. To obtain enhanced flower-specific promoters, we constructed four chimeric promoters (p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω) combining the 35S or OCS enhancer fused to a 302 bp CHSA core promoter fragment from petunia (PCHS) or a 307 bp CHS core promoter fragment from lily (LCHS), and also containing an omega element (Ω). Each promoter was fused to the β-glucuronidase (GUS) reporter gene, and we examined the levels and tissue specificity of GUS expression in transgenic Torenia fournieri. p35S-PCHS-Ω and p35S-LCHS-Ω drove strong, constitutive GUS expression in all tissues, especially in colored corollas (p35S-PCHS-Ω) or in colored corollas and roots (p35S-LCHS-Ω). pOCS-PCHS-Ω drove stronger GUS expression in colored corollas than in other tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω drove GUS in colored corollas but also in roots. Among the four chimeric promoters, pOCS-PCHS-Ω exhibited stronger activity only in colored corollas, making it useful for transgenic enhancement of floral traits, such as expressing ‘blue genes’ in lily to produce new lines with blue flowers.     

A pFBP6::Barnase Construct Resulted in Stigma and Style Ablation and Floral Abscission in Transgenic Tobacco

The potential for transgene dispersal through pollen, fruit, and seed is an important argument against the release of genetically modified plants. One approach toward addressing the concerns of gene flow from transgenic crops into closely related wild species involves in the use of tissue-specific promoters to engineer male and/or female sterility. In this study, we investigated the potential of Barnase ectopic expression for engineering floral sterility. A 2.6?kb promoter region of floral binding protein 6 (FBP6) from Petunia hybrida was isolated and fused to a reporter gene encoding ??-glucuronidase (GUS). The construct was introduced into tobacco plants where GUS staining was detected ubiquitously throughout the various tissues. The expression pattern of FBP6 resembled AG promoters, i.e., weak promoter activity was found in vegetative tissues, and strong activity was found in the various floral organs including the carpels and stigma. Meanwhile,The pFBP6::Barnase construct was then cotransformed into tobacco along with the Barstar gene, encoding an enzymatic inhibitor of Barnase, which was expressed at low but ubiquitous levels. Although cotransformed tobacco plants showed near normal vegetative growth, 74% of transgenic plants exhibited stigma and style ablation, and 98% of flower buds abscised before opening. Further analyses confirmed that stigma and style ablation prevented fertilization of the flower, and abscission of the bud followed rapidly. Thus, this approach has advantages for those ornamental/landscaping species where the pollen and fruit represent pollutants of the urban environment (e.g., platanus and poplar).     

Heat shock responsiveness analysis of <Emphasis Type="Italic">Athsp70b</Emphasis> upstream region

A 1431-bp upstream fragment of Athsp70b was cloned via PCR amplification and expressed in onion epidermis by particle bombardment. Furthermore, the progressive deletions of the Athsp70b upstream fragment linked to the β-glucuronidase (GUS) coding region were performed. Then, a stable GUS expression was analyzed in tobacco BY2 cells and Arabidopsis. Our present results showed that about a 500-bp region upstream ATG of Athsp70b is suitable to confer heat inducibility to the GUS reporter gene in plants and around 116 bp contain nonperfect heat-sensitive element. This promoter responds to heat, salicylic acid, and benzyladenine. GUS staining was mainly observed in the vascular tissues and root tips, implying that Athsp70b is related to water transportation.     

Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development

Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield.