Cis-acting elements of the CHS1 gene from white mustard controlling promoter activity and spatial patterns of expression

Chalcone synthase (CHS) catalyses the first regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. External stimuli such as stress, light and wounding induce CHS expression that is both tissue-specific and under developmental control. In order to identify cis-acting elements involved in organ and tissue specifity, we fused varying parts of the CHS1 promoter of white mustard (Sinapis alba L.) to the GUS-coding region and analysed the expression of these constructs in stably transformed Arabidopsis plants. Two different stages of development were examined, seedlings as an early stage and flowers as the final stage of development. In seedlings, the full-length promoter showed expression in all organs except the hypocotyl; in flowers expression could be observed in all whorls. Unit 1 of the mustard CHS1 promoter, an element conserved in several CHS genes, which has been recently identified as a light responsive element, is able to mediate a tissue-specific expression pattern similar to that obtained with the full-length promoter in seedlings as well as in flowers. Other elements enhance or repress expression in combination with Unit 1, or mediate defined spatial expression independently of Unit 1. One such element, located between-907 and -655, directs expression similar to that of the full-length promoter in flowers but not in seedlings and differs therefore in function to Unit 1. Our data suggest a dominant regulation of CHS1 expression by Unit 1. Other elements within this promoter might interact with Unit 1 or confer a subset of spatial expression patterns when Unit 1 is deleted.Abbreviations ADH alcohol dehydrogenase - CaMV cauliflower mosaic virus - CHS chalcone synthase - GUS -glucuronidase     

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of -d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)₇CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.     

Construction of Stress Responsive Synthetic Promoters and Analysis of Their Activity in Transgenic Arabidopsis thaliana

To obtain strong inducible promoters to drive abiotic stress-inducible transgene expression with minimal negative effects, we constructed three artificial synthetic promoters (EKCM, EKCRM, and ECCRM) comprising multiple cis-acting stress-response elements. Each promoter was fused independently to the β-glucuronidase (GUS) reporter gene, and GUS expression was analyzed in stable expression systems in Arabidopsis thaliana. T2 transgenic progenies showed integration of the promoter-GUS construct in their genome. RT-PCR assays and histochemical staining analysis showed that GUS expression driven by each promoter increased under desiccation, cold, and high salt conditions. The activity of synthetic promoters, assessed by fluorometric quantitative analysis of GUS enzyme activity, was significantly higher than that of the rd29A promoter under various stress treatments. The most powerful promoter, EKCM, allowed about 1.29-fold in GUS activity relative to the rd29A promoter, on average, under dehydration conditions. All three synthetic promoters could drive stress-inducible GUS expression in different organs of transgenic Arabidopsis. These synthetic promoters represent valuable tools for improving the stress tolerance of crops.     

The Transition from Primary siRNAs to Amplified Secondary siRNAs That Regulate Chalcone Synthase During Development of Glycine max Seed Coats

The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat.     

Chalcone synthase and its functions in plant resistance

Chalcone synthase (CHS, EC 2.3.1.74) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway. Besides being part of the plant developmental program the CHS gene expression is induced in plants under stress conditions such as UV light, bacterial or fungal infection. CHS expression causes accumulation of flavonoid and isoflavonoid phytoalexins and is involved in the salicylic acid defense pathway. This review will discuss CHS and its function in plant resistance.     

Differential regulation of two ABA-inducible genes from Craterostigma plantagineum in transgenic Arabidopsis plants

In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the -glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter.     

Expression and regulation of theRSI-1 gene during lateral root initiation

The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots. Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process.     

大豆类黄酮生物合成关键酶CHS基因的克隆及表达分析

根据查尔酮合成酶(CHS)基因DNA序列的保守区域设计了PCR引物,通过RT-PCR扩增从大豆叶片中克隆出3个参与类黄酮合成的CHS基因,分别命名为GmCHS1、GmCHS2和GmCHS3。在大豆基因组数据库进行同源比对,发现这3个基因分别与大豆基因组上Gm08g11610、Gm05g28610和Gm08g11520相对应,DNA序列一致性达95%～98%,推导氨基酸序列一致性达98%以上。进化分析显示,大豆中3个CHS蛋白与决明、菜豆CHS蛋白亲缘关系较近。表达分析显示,这3个基因在不同品种间有表达水平的差异,这可能是不同大豆品种中类黄酮含量不同的重要原因之一。     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

Vascular-specific activity of the Arabidopsis carotenoid cleavage dioxygenase 7 gene promoter

Carotenoid cleavage dioxygenases (CCDs) are involved in the production of diverse apocarotenoids including phytohormones, the visual molecules and the aromatic volatile compounds derived from carotenoids. Here, we examined the spatial expression of four of the CCD genes (AtCcd1, 4, 7 and 8) among the nine members of this family in Arabidopsis by RT-PCR. We found that the AtCcd7 gene showed strong expression in seeds. However, the promoter activity of the 1,867-bp 5′-upstream region of this gene exhibited a vascular specificity at all developmental stages throughout the transgenic Arabidopsis plants tested. The strength of the AtCcd7 promoter was also found to be lower than that of the 35S promoter by about 60%. The whole body expression of the β-glucuronidase (GUS) reporter gene driven by the AtCcd7 promoter in Arabidopsis plants was confirmed in different organs by RT-PCR and GUS enzymatic assays. Histochemical GUS staining further revealed that the AtCcd7 promoter has utility in limiting the expression of target genes to the vascular tissues in all plant organs such as the leaf, stem, root, flower and seed.     

Characterization of the tissue-specific expression of phenylalanine ammonia-lyase gene promoter from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

We isolated the 5′ flanking region of a gene for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Pinus taeda, PtaPAL. To investigate the tissue-specific expression of the PtaPAL promoter, histochemical assay of GUS activity was performed using the transgenic tobacco expressing the PtaPAL promoter-GUS. The region of −897 to −420 in PtaPAL promoter showed high activities in the secondary xylem and response to bending stress. To characterize the cis-regulatory functions of the promoters for enzymes in phenylpropanoid biosynthesis, we examined the activity of chimeric promoters of PtaPAL and a 4-coumarate CoA ligase, Pta4CLα. The chimeric promoter showed similar activity as the Pta4CLα promoter. Electrophoretic mobility shift assays implicated −897 to –674 of PtaPAL promoter containing cis-elements of the expression in xylem of Pinus taeda. The results suggested that AC elements of PtaPAL have multiple functions in the expression under the various developmental stages and stress conditions in the transgenic tobacco. The nucleotide sequence data reported will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number AB449103 (PtaPAL promoter sequence).     

The second intron of AGAMOUS drives carpel- and stamen-specific expression sufficient to induce complete sterility in Arabidopsis

Gene containment technologies that prevent transgene dispersal through pollen, fruit and seed are in immediate demand to address concerns of gene flow from transgenic crops into wild species or close relatives. In this study, we isolated the enhancer element of Arabidopsis AGAMOUS that drives gene expression specifically in stamens and carpels. By fusing this AG enhancer to a minimal 35S promoter fragment, two tissue-specific promoters, fAGIP and rAGIP in forward and reverse orientations, respectively, were created and fused to the GUS reporter. Transgenic Arabidopsis plants harboring either fAGIP::GUS or rAGIP::GUS displayed similar GUS expression specifically in carpel and stamen tissues and their primordial cells. To test their utility for engineering sterility, the promoters were fused to the Diphtheria toxin A (DT-A) gene coding for a ribosome inactivating protein as well as the Barnase gene coding for an extracellular ribonuclease, and tested for tissue-specific ablation. Over 89% of AGIP::DT-A and 68% of AGIP::Barnase transgenic plants displayed specific and precise ablation of stamens and carpels and are completely sterile. These transgenic plants showed normal vegetative development with prolonged vegetative growth. To evaluate the stability of the sterile phenotype, 16 AGIP::DT-A lines underwent two consecutive cutback generations and showed no reversion of the floral phenotype. This study demonstrates a simple, precise and efficient approach to achieve absolute sterility through irreversible ablation of both male and female floral organs. This approach should have a practical application for transgene containment in ornamental, landscaping, and woody species, whose seeds and fruits are of no economic value.     

Tissue-specific expression of the PAL3 promoter requires the interaction of two conserved cis sequences

The bean PAL2 and PAL3 promoters confer expression in overlapping sets of tissue types in transgenic tobacco. The PAL3 promoter contains motifs that resemble two AC cis elements which are required for tissue-specific expression of the PAL2 promoter. The functions of these motifs in the PAL3 promoter were determined by analysis of mutated PAL3 promoter-GUS constructs in transgenic tobacco. This revealed that the AC motifs are necessary for tissue-specific expression of the PAL3 promoter. Therefore, a key role is indicated for AC elements, which are Myb-protein binding sites, in regulating tissue-specific expression of the bean PAL gene family.     

Isolation and Characterization of Three New Promoters from Gossypium hirsutum that Show High Activity in Reproductive Tissues

Engineering of plant protection requires well-characterized tissue-specific promoters for the targeted expression of insecticidal resistance genes. Herein, we describe the isolation of five different fragments of promoters of three distinct flower-specific cotton (Gossypium hirsutum) genes. Expression analyses of the three genes GhPME-like1, GhβGal-like1 and GhPL-like1 revealed that they are expressed highly in flowers buds ranging from 4 to 12 mm in size. Several putative regulatory cis-elements were identified in the promoter regions, including elements involved in the control of tissue-specific gene expression in pollen grains and fruits. In vivo analyses of these promoters were performed using the heterologous plant system Arabidopsis thaliana by fusing them with the gene uidA (GUS). GUS staining in Arabidopsis tissues revealed that their expression was restricted to anthers, with the majority of expression in pollen grains and in the upper portion of the carpels and siliques. A comparison between a CaMV35S::GUS constitutive promoter and the promoters isolated in this study revealed that the cotton promoters were more active and were specific to flowers and fruits, which are organs that are preferentially attacked by important pest insects such as the boll weevil (Anthonomus grandis). The activity of the promoters was also confirmed using transient expression assays in flower buds of G. hirsutum. The promoters of GhPME-like1, GhβGal-like1 and GhPL-like1 are specific to reproductive tissues and could represent important biotechnological tools for controlling insect pests, in particular the cotton boll weevil, which attacks floral and fruit tissues.