Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter

SBgLR (Solanum tuberosum genomic lysine-rich) is a pollen-specific gene cloned from potato (Solanum tuberosum L.). The region from −269 to −9 (The A of translation start site “ATG” as +1) of the SBgLR promoter was identified as critical for gene specific expression in pollen grains. Sequence analysis indicates a palindromic sequence “TTTCTATTATAATAGAAA” in the −227 to −209 region, in which two pollen-specific motifs TTTCT and AGAAA surround a unique putative TATA box. Moreover, nine putative pollen-specific motifs are located in the region between the TATA box and ATG. We placed the −227 to −9 region (reserving the palindrome) and the −222 to −9 region (breaking the palindrome) downstream of the CaMV35S enhancer, respectively, to construct two fusion promoters. Histochemical assays in transgenic plants demonstrated that the region from −222 to −9 is necessary and sufficient for pollen-specific expression of the uidA gene. However, the region of −227 to −9 is incapable of driving GUS expression in pollen grains and parts of vegetative tissues. A series of 5′ deletions from −269 to −9 of SBgLR promoter were constructed. A transient expression assay indicated that the region from the −227 to −9 suppressed gfp gene expression in pollen, and a positive regulatory element was present in the region of −253 to −227. The function of the palindromic sequence as a repressor inhibiting gene expression in pollen was further confirmed by the mutated promoter, breaking the palindrome by substituting its 3′-flanking five base pairs, which resumes the reporter gene expression in mature pollen.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.     

Response of transgenic rape plants bearing the <Emphasis Type="Italic">Osmyb4</Emphasis> gene from rice encoding a trans-factor to low above-zero temperature

Accumulation of soluble sugars (sucrose, fructose, and glucose), proline, phenols (total phenols and flavonoids), and antocyanins during adaptation to low-temperature stress (4°C) of two lines of spring rape (Brassica napus L., cv. Westar) characterized by weak (Bn-1) and strong (Bn-3) expression of the Osmyb4 transgene was studied. Vegetatively propagated transgenic and wild-type plants were grown in the hydroponic culture at 24°C; at the stage of 5–6 leaves, plants were exposed to 4°C for 5 days and then returned to the optimum temperature of 24°C for recovery. Transgenic plants were established to manifest improved cold and frost tolerance, which was evident from more active biomass accumulation at 4°C as compared with wild-type plants and from sustaining their viability after 2-day-long exposure to −6°C. Determination of MDA content showed that one of the reasons of their improved cold tolerance was their capability of maintaining oxidative homeostasis under low-temperature stress. This suggestion is supported by intense accumulation of phenols and antocyanins, manifesting pronounced antioxidant effects, by transgenic plants during their cold adaptation. Thus, during 2–5 days of plant exposure to 4°C, in transgenic plants the total content of phenols increased by 2.6–3.7 times, flavonoids — by 3.7–4.7 times, and antocyanins — by 3.5–5.3 times as compared with control plants growing at 24°C. Transgenic Bn-3 plants with strong expression of the Osmyb4 gene accumulated phenols and antocyanins at 4°C more actively than Bn-1 plants characterized by weak expression of this gene. Transgenic rape plants subjected to cold stress accumulated more proline, manifesting stress-protection effects, and lesser accumulation of soluble sugars. Before the beginning of experiment, the content of soluble sugars was approximately similar in wild-type plants and transgenic lines; at 4°C their level in transgenic plants was substantially lower than in control plants. As distinct from the process of cold adaptation, during recovery, the content of all tested stress-protection compounds dropped sharply. The results obtained indicate that active expression of the Osmyb4 gene from rice in the rape plants was accompanied not only by accumulation of compatible osmolytes but also by biosynthesis of antioxidants of phenolic nature.     

Multiple elements of the S 2 ?-RNase promoter from potato ( Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position −200 to bp −100, and motif II, located between bp −498 and −480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues. Received: 7 August 1997 / Accepted: 8 September 1997     

Expression of recombinant protoporphyrinogen oxidase influences growth and morphological characteristics in transgenic rice

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase (Protox), the last shared enzyme of the porphyrin pathway, in the cytoplasm (C89) or the plastids (P72) were compared with wild-type rice plants in their growth characteristics. Production of tiller buds 18 d after seeding was more profuse in transgenic plants than in wild-type plants, especially in plastid-targeted plants. Transgenic plants had 12–27% increase in tiller number and 17–33% increase in above-ground biomass compared with wild-type plants 4 and 8 weeks after transplanting of 2-week-old rice seedlings, demonstrating that tiller production and above-ground biomass correlate with each other. Cytoplasm-expressed and plastid-targeted transgenic plants also had a distinct phenotypic characteristic of narrower and more horizontal leaves than wild-type plants. Phenotypic and anatomical characteristics of the transgenic plants were clearly different from wild-type plants, indicating that regulation of porphyrin biosynthesis by expression of B. subtilis Protox in rice influences morphological characteristics of plant growth as well as biomass.     

Expression of the rice cytoplasmic cysteine synthase gene in tobacco reduces ozone-induced damage

Cysteine serves as a precursor for the synthesis of various sulfur-containing metabolites, and the cysteine synthase (CS) gene plays a central role in the sulfur cycle in nature. In the present study, rcs1, a cytosolic CS gene of rice, was introduced into the genome of tobacco (Nicotiana tabacum). The tolerance of wild-type tobacco plants as well as of the resulting transgenic tobacco plants overexpressing the rcs1 gene to toxic levels of ozone (O₃, 0.15 μ mol⁻¹) was measured after various lengths of exposure. Leaf lesions in plants exposed for 2 weeks to O₃ were more prevalent in the leaves of the wild-type plants than in those of the transgenic tobacco plants. Transgenic tobacco plants showed a higher growth rate and a higher chlorophyll content than the wild-type plants. Cysteine synthase activity and cysteine and glutathione contents were higher in transgenic plants than in wild-type plants irrespective of the length of the O₃ treatment. Our results indicate that the CS gene plays a role in the protection of the plant against toxic O₃ gas, probably through the mechanism of an over-accumulation of such sulfur-rich antioxidants as cysteine and glutathione.     

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.     

Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.     

Structural and functional characterization of a pollen-specific promoter <Emphasis Type="Italic">NTPp13</Emphasis> in tobacco

A novel 407 bp nucleotide sequence NTPp13 was isolated from tobacco (Nicotiana tabacum L.) by PCR, its structure and function were characterized. The NTPp13 sequence was highly homologous with the pollen-specific expression promoter Zm13 from maize (Zea mays L.) and contained some key motifs which controlled pollen-specific expression. The NTPp13 was fused to the β-glucuronidase (GUS) reporter gene and transferred into tobacco. Analysis of the transgenic plants revealed that this putative promoter fragment was sufficient to direct GUS expression specifically in the anther, exactly in the pollen and pollen tube, and that GUS activity reached the maximum at the stage of pollen grain began to separate. Further study showed that the expression of NTPp13 sequence at pollen was stable at the range of temperature measured. These data suggested that the NTPp13 sequence was likely the essential element of promoter region of an unknown pollen-specific gene from tobacco.     

Nuclear male sterility induced by pollen-specific expression of a ribonuclease

The promoter of a rice pollen-specific gene, PS1, has been fused to the Bacillus amyloliquefaciens Barnase gene which encodes a secreted ribonuclease. The PS1-Barnase chimeric gene has been introduced into tobacco. These transgenic tobacco plants show normal vegetative and floral development, but they display a range of reproductive properties from slightly reduced in fertility to completely sterile. Barnase mRNAs are detectable in the pollen from transgenic plants which do not show an obvious fertility-related phenotype, and in a few plants which have a mildly reduced-fertile phenotype. However, transgenic plants with a severely reduced-fertile or sterile phenotype do not accumulate detectable amounts of Barnase mRNA in their pollen, and the quality of their RNA is poor, presumably because of extensive RNA degradation. Reciprocal crosses between these transgenic plants and wild-type controls showed that the reduced-fertile phenotype is associated only with the transgenic pollen. When used as the female parent, these PS1-Barnase transgenic plants are fully fertile. Anthers in the severely sterile transgenic plants develop normally, but the majority of their pollen grains have abnormal morphology and they fail to germinate. These results indicate that expression of a pollen-specific cytotoxic gene induces lethality in pollen and may lead to severely reduced male fertility.     

Transgenic <Emphasis Type="Italic">indica</Emphasis> rice cultivar ‘Swarna’ expressing a potato chymotrypsin inhibitor <Emphasis Type="Italic">pin2</Emphasis> gene show enhanced levels of resistance to yellow stem borer

Transgenic rice was developed from ‘Swarna’, the most popular indica rice cultivar (Oryza sativa L.) in South East Asia, with a potato chymotrypsin inhibitor gene (pin2) through Agrobacterium-mediated transformation. Four out of nine primary transgenic plants had a single-copy T-DNA insertion while other five plants had two copies. Mendelian pattern of inheritance of the transgene (pin2) was observed in the T₁ generation progeny plants. Whole plant bioassays conducted at both vegetative and reproductive stages and cut stem assays showed enhanced levels of resistance of transgenic rice against yellow stem borer. The transgenic rice lines with plant derived proteinase inhibitor genes would develop into resistant cultivars to fit into resistance breeding strategies as an important component of integrated pest management in rice.     

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.     

Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrid) plants

For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based “positive” selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 μM 2,4-D, 20 g l⁻¹ sucrose and 3 g l⁻¹ mannose. Plant regeneration was obtained on MS basal medium with 2.5 μM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 μM IBA, 20 g l⁻¹ sucrose, and 1.5 g l⁻¹ mannose. An increase in mannose concentration from permissive (1.5 g l⁻¹) to selective (3 g l⁻¹) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.     

RETRACTED ARTICLE: Expression of a rice chitinase gene enhances antifungal response in transgenic litchi (cv. Bedana)

To enhance the antifungal response of litchi (Litchi chinensis Sonn.), transgenic plants were generated by transferring rice chitinase gene driven by a maize-ubiquitin promoter along with its first intron into the zygotic embryos via Agrobacterium tumefaciens-mediated transformation. After co-cultivation for 2 days, zygotic embryos were transferred onto Murashige and Skoog (MS) modified media supplemented with 25 mgl⁻¹ hygromycin and 400 mgl⁻¹ cefotaxime. Consequently embryos were selected and the antibiotic resistant transgenic plantlets were regenerated. The culture time from zygotic embryo to transgenic plants was 14 months. The integration of the transgene was confirmed by PCR, RT-PCR, Southern and western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed plants. The chitinase activity when examined using the native polyacrylamide in-gel assay, indicated that the foreign gene expression resulting in the protein of expected molecular weight that showed chitinase activity. The transgenic plants showed delayed onset of the disease and smaller lesions following in vitro inoculation of die-back, leaf spots and blight pathogen (Phomopsis sp.). The transgenic plants were adapted to the greenhouse and did not show any phenotypic alterations.