Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene

Allene oxide synthase (AOS) is encoded by a single intronless gene in Arabidopsis thaliana (L.) Heynh. The promoter region of the AOS gene exhibits, in addition to the elements of a minimal promoter and the presence of general enhancers, cis-elements that, in other promoters, are responsible for stress- and ethylene-responsiveness. Arabidopsis thaliana and Nicotiana tabacum L. were transformed with a chimaeric gene consisting of a 1.9-kb 5′-upstream sequence and the first 95 nucleotides of the AOS coding sequence translationally fused to uid A encoding β-glucuronidase (GUS). Using histochemistry, GUS activity was seen in older leaves, in the bases of petioles and in stipules, during the early stages of carpel development, in maturing pollen grains and at the base of elongated filaments, as well as in abscission-zone scars. A role for jasmonates in floral organ abscission is suggested by these findings. Furthermore, the AOS promoter was activated both locally as well as systemically upon wounding. Jasmonic acid, 12-oxophytodienoic acid and coronatine strongly induced GUS activity. This induction remained confined to the treated leaf when agonists were applied locally to a leaf, suggesting that neither jasmonic acid nor 12-oxophytodienoic acid are physiologically relevant components of the systemic wound signal complex. Rather, the data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis. Received: 21 September 1998 / Accepted: 30 December 1998     

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

 Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35 S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35 S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25  °C under a 16/8 h (day/night) photoperiod after 8 weeks of stock-culture of hairy roots and 4 weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis. Received: 28 February 2000 / Revision received: 18 August 2000 / Accepted: 22 August 2000     

Analyses in transgenic tobacco of the promoter from a Brassica napus gene highly expressed in the stigma

A genomic clone, Pis G363, containing the Brassica napus stigma-expressed gene Pis 63-2 was isolated and sequenced. The coding region of Pis G363 does not possess introns and shows 82% identity to the nucleotide sequence of a gene from Arabidopsis BAC clone T01B08. A 2-kb promoter fragment from Pis G363 was fused to the coding sequence of the marker enzyme β-glucuronidase (GUS) and introduced into tobacco via Agrobacterium-mediated transformation. The promoter fragment directed expression of the GUS gene in the stigma of transgenic tobacco. Some transformants also showed relatively low GUS activity in the pollen. Received: 25 May 1998 / Revision received: 30 July 1998 / Accepted: 21 August 1998     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

Functional Characterisation of the Oil Palm Type 3 Metallothionein-like Gene (<Emphasis Type="Italic">MT3-B</Emphasis>) Promoter

In silico analysis showed that the differentially expressed type 3 oil palm metallothionein-like genes MT3-A and MT3-B share at least 11 common putative promoter regulatory elements. The identified motifs include W-boxes, TATCCA element, binding element for cytokinin response regulators and pollen-specific elements. A high degree of conservation was observed in their genomic organisation where the coding regions are divided at two identical positions in both genes by two AT-rich introns. Promoter activity of the MT3-B gene was analysed using a transient assay by bombarding oil palm tissue slices with a β-glucuronidase (GUS) gene construct and a stable reporter assay by analysing GUS expression in transformed Arabidopsis thaliana plants. Transient expression analysis revealed MT3-B promoter activity in oil palm root tissues but not in fruit mesocarp at 12 weeks after anthesis and spear leaves. The T3 homozygous transgenic Arabidopsis plants, harbouring the MT3-B promoter/GUS construct, showed reporter activity in cotyledons and mature leaves with lower expression levels in root tissues. The expression levels in the roots of the T3 homozygous transgenic plants increased five- and 2.5-folds when treated with 80 μM of Zn²⁺ and Fe²⁺, respectively. Altogether, these results indicate that the MT3-A and MT3-B promoter activities may be regulated by a variety of abiotic factors and MT3-B promoter may potentially be manipulated for use in plant genetic engineering for induced synthesis of gene product.     

Isolation of the maize <Emphasis Type="Italic">Zpu1</Emphasis> gene promoter and its functional analysis in transgenic tobacco plants

By screening a genomic library of maize, a 2.2 kb 5′ flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including the full 5′ flanking sequence (−2267 to −1) (Z1), a 3′ deletion (−2267 to −513) (Z5) and three 5′ deletions extending to −1943 (Z2), −1143 (Z3) and −516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (−516 to −1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle.     

Structure and regulation of OPR1 and OPR2, two closely related genes encoding 12-oxophytodienoic acid-10,11-reductases from Arabidopsis thaliana

The genes of two closely related 12-oxophytodienoic acid reductases (EC 1.3.1.42), OPR1 and OPR2, were identified on a 7079-bp-long genomic fragment from Arabidopsis thaliana (L.) Heynh. The organization of these two genes was determined and putative cis elements were identified. Promoter-β-glucuronidase (GUS) fusions expressed in transgenic Arabidopsis thaliana and Nicotiana tabacum L. plants revealed differences in OPR-promoter-driven GUS expression in flowers. While the OPR1 promoter directed GUS expression in young seeds, the OPR2 promoter directed pollen-specific expression. Both OPR1 and OPR2, were predominantly expressed in roots. Stress treatments, like local and systemic wounding, UV-C illumination and coldness, resulted in transient changes in steady-state OPR mRNA levels, but no concurrent changes in polypeptide level or enzyme activity were detected. Received: 2 October 1998 / Accepted: 22 December 1998     

Production of medium-chain-length hydroxyalkanoic acids from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in pH stat

The production of β-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36–42°C for 2 h then recovery at 27°C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42°C heat shock and a recovery phase at 27°C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl β-D-glucuronic acid (MU) min⁻¹ mg⁻¹ protein at 24 h of recovery. When tissues were continuously heated at 42°C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Cloning of the promoter region of plasma membrane aquaporin<Emphasis Type="Italic">BnPIP1</Emphasis> from<Emphasis Type="Italic">Brassica napus</Emphasis> and its functional analysis

A 1.6 kb upstream regulatory sequence (GenBank accession no. AF472487) of plasma membrane aquaporinBnPIP1 gene fromBrassica napus was obtained by genomic walking based on ligation-mediated PCR method. Sequence analysis indicated that this fragment contained seed germination specific and vascular specific sequences. The 1.6 kb upstream sequence and various 5′ end deleted sequences were fused withuidA gene and constructed into plant expression vectors which were used for tobacco transformation. GUS histochemical assay showed that the 1.6 kb fragment had high levels of promoter activity and the GUS staining was mainly distributed in vascular systems and tissues with rapid expanding and proliferating cells. Promoter deletion analysis showed that the deletion of -1610 — -1030 bp resulted in a dramatic reduction in GUS activity. It was assumed that there might be cis-acting element(s) existing in this region. Whereas, the region located at -1030 — -902 bp strongly inhibited the expression ofgus and probably contained negative regulatory element(s). The fragment of -902 — -19 bp could also directgus expression at high level.     

A Phosphate Transporter Promoter from Arabidopsis thaliana AtPHT1;4 Gene Drives Preferential Gene Expression in Transgenic Maize Roots Under Phosphorus Starvation

Phosphorus (P) stress responsive genes have been identified and characterized, including the high-affinity phosphate transporter AtPHT1;4 from Arabidopsis thaliana. This gene encodes a membrane protein that is primarily expressed in roots under phosphorus deficiency. A 2.3-kb promoter region from AtPHT1;4 has been fused with the β-glucuronidase (GUS) encoding gene and introduced into maize via biolistic bombardment to evaluate its spatiotemporal activity in a heterologous system. AtPHT1;4::GUS expression is detected preferentially in transgenic maize roots under P deficiency. Further analysis of transgenic plants has also revealed that GUS activity is higher in roots than in leaves by about sixfold. These results demonstrate the ability of AtPHT1;4 promoter to direct expression of the reporter gene in a monocot root system under P stress. This property of AtPHT1;4 promoter makes it useful to engineer maize plants to modify the soil’s rhizosphere and increase efficiency of P acquisition under P stress conditions.     

A rapid<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

Isolation of the Chinese rose sHSP gene promoter and its differential regulation analysis in transgenic Arabidopsis plants

In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s response to high temperature.     

Localization and expression of serine racemase in Arabidopsis thaliana

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with β-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses.     

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the “Class II” hemoglobins. Received: 28 June 1999 / Accepted: 25 November 1999