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.     

Characterization of a Novel Pollen-Specific Promoter from Wheat (Triticum Aestivum L.)

PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the ??-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4?kb fragment of the promoter was enough to confer pollen-specific expression.     

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.     

PsPMEP, a pollen-specific pectin methylesterase of pea (Pisum sativum L.)

Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.     

A 286 bp upstream regulatory region of a rice anther-specific gene, OSIPP3, confers pollen-specific expression in Arabidopsis

OSIPP3 gene (coding for pectin methylesterase inhibitor protein) was isolated from a pre-pollinated inflorescence-specific cDNA library by differential screening of stage-specific libraries from Oryza sativa. OSIPP3 is present in the genome of rice as a single copy gene. OSIPP3 gene was expressed exclusively in the pre-pollinated spikelets of rice. Upstream regulatory region (URR) of OSIPP3 was isolated and a series of 5′-deletions were cloned upstream of GUS reporter gene and were used to transform Arabidopsis. OSIPP3_del1 and del2 transgenic plants showed GUS expression in root, anther and silique, while OSIPP3_del3 showed GUS activity only in anthers and siliques. Pollen-specific expression was observed in case of plants harboring OSIPP3_del4 construct. It can, therefore, be concluded that the OSIPP3 URR between ?178 and +108 bp is necessary for conferring pollen-specific expression in Arabidopsis.     

Antisense-mediated suppression of transgene expression targeted specifically to pollen

A potential problem in the field release of transgenic plants is the spread of foreign gene products via pollen. Therefore, the use of the tomato pollen-specific lat52 gene promoter was investigated as a means of targeting antisense RNA to pollen without affecting transgene expression elsewhere in the plant. A transgenic tobacco line T115, which showed GUS expression in pollen, leaves and roots were retransformed with a construct containing the pollen-specific lat52 promoter driving the GUS encoding uid A gene in antisense orientation. From 24 independent transformants obtained, 19 showed a significant reduction in pollen GUS activity. Of these lines, four showed a reproducible antisense effect in pollen in the next generation, while it was shown in one line that GUS activity in leaves and roots was also unaffected. To ascertain the effectiveness of the antisense strategy to downregulate very high levels of pollen expression, a lat52-gus antisense construct was introduced into tobacco lines containing lat52-gus, which had pollen GUS activity of up to 250 times greater than in line T115. Results showed that 30 out of 34 independent lines exhibited a significant antisense effect in pollen, confirming the effectiveness of pollen-targeted antisense strategy to reduce undesirable expression in pollen independent of expression level in pollen.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

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.     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码188个氨基酸的蛋白质,大小约20 kD,等电点为12.1。Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到。进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降。表明TaPSG719是一个花粉中晚期特异性表达基因。经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性。Southern杂交表明TaPSG719可能为一个多拷贝基因。为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控。     

Cloning and characterization of a cold inducible Pal promoter from Fagopyrum tataricum

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in biosynthesis pathway of flavonoids and plays an important role in plant stress resistance. In this study, the 5’ flanking region of phenylalanine ammonia-lyase gene was isolated from Fagopyrum tataricum by thermal asymmetric interlaced PCR method, named PFtPal (GenBank: KF463139). To investigate the functional properties of PFtPal, we constructed a series of plant expression vectors that contained different promoter fragments resulting from nest deletions and had successfully transformed them into tobacco leaves by Agrobacterium tumefaciens. Histochemical assay of GUS suggested that PFtPal could drive GUS gene expression in leaves and roots, while GUS activity was not detected in the stem. In addition, the region of ?274 bp to ?1 bp was enough to drive normal expression of GUS gene. Low temperature treatment of transgenic tobacco plants demonstrated that PFtPal conferred cold-induced expression. Taken together, our study will help to better understand the Pal promoter, and provides a candidate promoter for molecular breeding in Fagopyrum plants.     

Functional analysis of a cryptic promoter from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> reveals bidirectionality

Cryptic promoter elements play a significant role in evolution of plant gene expression patterns and are prospective tools for creating gene expression systems in plants. In a previous report, a 452 bp promoter fragment designated as cryptic root-specific promoter (AY601849) was identified immediately upstream to T-DNA insertion, in the intergenic region between divergent genes SAHH1 and SHMT4, in T-DNA tagged mutant M57 of Arabidopsis thaliana. In silico analysis of 452 bp promoter revealed typical eukaryotic promoter architecture, presence of root-specific motifs and other cis-regulatory motifs responsible for the spatial and temporal expression. GUS expression driven by 452 bp in M57 was developmentally as well as light-regulated. The AT-rich 452 bp promoter does not show homology to any known sequences. The 452 bp promoter was further proved cryptic and detailed molecular characterization of the promoter carried out through serial 5′ and 3′ deletion analysis, by cloning the promoter fragments upstream to promoter-less GUS vector. A 279 bp fragment obtained by deleting 173 bp from 5′ end of 452 bp was capable of driving root-specific expression, similar to that of full-length promoter. Further, root tip-specific, root-specific and core-regulatory motifs for root-specific expression were identified at positions 173–227, 251–323 and 408–452 bp, respectively, from the 5′ end of 452 bp. The 452 bp promoter was equally functional in inverse orientation, hence bidirectional and symmetric. In heterologous systems, such as Brassica juncea and Oryza sativa, the promoter activity was not significant since GUS was not visually detected in transient assays.     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1 172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码1 88个氨基酸的蛋白质,大小约20 kD,等电点为12.1.Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到.进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5 d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降.表明TaPSG719是一个花粉中晚期特异性表达基因.经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性.Southern杂交表明TaPSG719可能为一个多拷贝基因.为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控.     

Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems

Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accessions: OSIPA cDNA, AF220610; OSIPK cDNA, AF312920; OSIPA partial gene and upstream promoter region, AY166659; OSIPK gene-specific and upstream sequence, AY168440.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.