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).     

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3′end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (−60) minimal promoter [AG-I-35S (−60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (−60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (−60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (−60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.     

Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants

The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility but it is highly host-specific. To ascertain whether a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Analyses of transgenic plants bearing each respective promoter fused to the β-glucuronidase (GUS) reporter gene showed that all four promoters are able, like the AtAGIP, to drive very similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues. These results indicate that unlike their counterparts in rice and maize, the tobacco NtAGI-1 and NtAGI-2 enhancers share a highly conserved regulatory function. Interestingly, all four promoters display additional tissue specificity in petals, and their activity is influenced by the orientation of the incorporated enhancer, with reverse-oriented enhancers exhibiting approximately double the effectiveness of forward-oriented enhancers. These properties are novel and have not been observed with the AtAGIP promoter in Arabidopsis. As expected, these highly specific promoters can also direct the expression of the DT-A cytotoxic gene exclusively in carpels, stamens and petals, resulting in complete sterility through the precise ablation of targeted floral organs. Further analyses demonstrated that the resulting trait is mitotically stable, which is critical for the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions.     

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG'mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.     

The Arabidopsis HSP18.2 promoter/GUS gene fusion in transgenic Arabidopsis plants: a powerful tool for the isolation of regulatory mutants of the heat-shock response

A detailed study of the expression of the promoter of the HSP18.2 gene from Arabidopsis fused to the bacterial gene for β-glucuronidase (GUS) in transgenic Arabidopsis plants is described. High levels of GUS activity were induced in all organs of transformants except for seeds during heat shock. The optimum temperature for expression of GUS in Arabidopsis was 35°C regardless of the plant growth temperature. Heat shock of 40°C did not induce any detectable levels of GUS activity. Pre-incubation at 35°C was found to have a protective effect on the induction of GUS activity at 40°C. GUS activity was also increased in response to a gradual increase in temperature. Histochemical analysis revealed that basal levels of GUS activity were induced in the vascular tissue of leaves and sepals, as well as at the tips of carpels, at the normal growth temperature. Heat treatment of a limited part of the plant tissue did not appear to cause systemic induction of GUS activity. To extend the analysis of the plant heat-shock response, we attempted to screen mutations in genes involved in the regulation of the induction of heat-shock protein (HSP) genes, using the GUS gene as a selection marker in transgenic Arabidopsis plants, and the results of this analysis are described.     

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.     

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.     

Two tobacco AP1-like gene promoters drive highly specific,tightly regulated and unique expression patterns during floral transition,initiation and development

The genetic engineering of agronomic traits requires an array of highly specific and tightly regulated promoters that drive expression in floral tissues. In this study, we isolated and characterized two tobacco APETALA1-like (AP1-like) promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using the GUS reporter system, along with tissue-specific ablation analyses. Our results demonstrated that the two promoters are active in floral inflorescences but not in vegetative apical meristems or other vegetative tissues, as reflected by strong GUS staining and DT-A-mediated ablation of apical shoot tips during reproductive but not vegetative growth. We also showed that the NtAP1Lb1 promoter was more active than NtAP1La in inflorescences, as the former yielded higher frequencies and greater phenotypic evidence of tissue ablation compared to the latter. We further revealed that both promoters were uniformly expressed in the meristems of stage 1 and 2 floral buds, but were differentially expressed in floral organs later during development. While NtAP1La was found to be active in stage 4–5 carpels, later becoming confined to ovary tissue from stage 9 onwards, NtAP1Lb1 activity was apparent in all floral organs from stages 3 to 7, becoming completely absent in all floral organs from stage 11 onward. Therefore, it seems that the two tobacco promoters have acquired similar but distinct inflorescence-, floral meristem- and floral organ-specific and development-dependent regulatory features without any leaky activity in vegetative tissues. These features are novel and have rarely been observed in other flower-specific promoters characterized to date. The potential application of these promoters for engineering sterility, increasing biomass production and modifying flower architecture, as well as their putative use in flower-specific transgene excision, will be discussed.     

厚藤脱水素基因IpDHN启动子IpDHN-Pro的克隆和调控转录活性分析

为了解厚藤(Ipomoea pes-caprae)脱水素基因IpDHN (GenBank登录号:KX426069)启动子的转录活性和对非生物胁迫和植物激素ABA的响应,通过染色体步移法克隆了IpDHN的上游启动子序列IpDHN-Pro,长度为974 bp。构建IpDHN-Pro调控下GUS转基因载体,转化拟南芥(Arabidopsis thaliana)植株获得IpDHN-Pro::GUS转基因植株并进行GUS染色,验证IpDHN-Pro启动转录活性以及在氯化钠、甘露醇、ABA处理后拟南芥GUS基因表达变化。结果表明,扩增获得的IpDHN-Pro序列包含多个顺式作用元件,包括1个ABRE、3个Myb转录因子结合位点、富含TC的重复序列以及Skn-1基序等。转基因拟南芥GUS染色及qRT-PCR表明该序列可驱动GUS基因在拟南芥稳定表达,且表达受高盐、渗透压及ABA的诱导。这表明IpDHN-Pro是一个盐旱、ABA诱导的启动子序列,可应用于相关的植物抗逆遗传工程研究。     

Floral Spray Transformation Can Efficiently Generate Arabidopsis

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

Petunia <Emphasis Type="Italic">AGAMOUS</Emphasis> Enhancer-Derived Chimeric Promoters Specify a Carpel-, Stamen-, and Petal-Specific Expression Pattern Sufficient for Engineering Male and Female Sterility in Tobacco

Previous studies have shown that the AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer specifies a carpel- and stamen-specific expression in its native host species, but not in heterologous species such as tobacco, which restricts its application in the engineering of male and female sterility. These findings also imply that the AG regulatory mechanism that has evolved in Arabidopsis may, to some extent, have diverged from that of tobacco. To test whether a similar chimeric promoter created using the AG second intron/enhancer can overcome this barrier of evolutionary divergence in closely related species, we generated forward- and reverse-oriented chimeric promoters, fPtAGIP and rPtAGIP, from the petunia AG second intron/enhancer (PtAGI) fragment and tested them in tobacco, which, like petunia, belongs to the Solanaceae family. Our results demonstrate that both fPtAGIP and rPtAGIP confer similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues in tobacco as revealed by tissue-specific gene expression and tissue ablation. This pattern resembles that driven by the AtAGIP in Arabidopsis and indicates that the AG regulatory mechanism is more conserved between tobacco and petunia than between tobacco and Arabidopsis. The petunia-derived promoters also exhibited petal-specific activity, and their activities in floral organs were substantially influenced by the orientation of the PtAGI enhancer, with reverse-oriented enhancers displaying approximately double the effectiveness of forward-oriented enhancers. These two properties are novel and have not been observed previously with AtAGIP promoters. Furthermore, we found that PtAGIP promoter-driven tissue ablation is effective for engineering complete sterility in plants, and the resulting sterile trait is stable for at least three mitotic generations at various temperature regimes, which is important for the complete containment of seed-, pollen-, and fruit-mediated gene flow in field conditions.     

A Novel <Emphasis Type="Italic">PhLRR</Emphasis> Gene Promoter is Sufficient for Engineering Male Sterility in Petunia

Tissue-specific promoters can drive genes specifically expressed in the target organs and have been widely used in plant molecular breeding. In this study, a 1.2-kb promoter region of an anther-specific gene PhLRR from Petunia hybrida “Fantasy” was isolated and fused to the β-glucuronidase (GUS) gene. The pPhLRR::GUS vector was heterogeneously transformed into tobacco in which the GUS staining was only detected in the early development stage of anthers and no GUS expression in any other three floral whirls or vegetative organs was observed. It is very different from other well-studied anther-specific promoters which drive genes specifically expressed in the later development stage of anthers or only in the pollens. Furthermore, the pPhLRR::Barnase was introduced into petunia and induced complete male sterility without influencing the ornamental characteristics or the female fertility in transformed plants. These results indicate that PhLRR promoter is a new kind of petunia anther-specific promoter and could be taken as a valuable tool in ornamental plant breeding.     

Subcellular and tissue localization of NAD kinases from Arabidopsis: compartmentalization of de novo NADP biosynthesis

The de novo biosynthesis of the triphosphopyridine NADP is catalyzed solely by the ubiquitous NAD kinase family. The Arabidopsis (Arabidopsis thaliana) genome contains two genes encoding NAD⁺ kinases (NADKs), annotated as NADK1, NADK2, and one gene encoding a NADH kinase, NADK3, the latter isoform preferring NADH as a substrate. Here, we examined the tissue-specific and developmental expression patterns of the three NADKs using transgenic plants stably transformed with NADK promoter::glucuronidase (GUS) reporter gene constructs. We observed distinct spatial and temporal patterns of GUS activity among the NADK::GUS plants. All three NADK::GUS transgenes were expressed in reproductive tissue, whereas NADK1::GUS activity was found mainly in the roots, NADK2::GUS in leaves, and NADK3::GUS was restricted primarily to leaf vasculature and lateral root primordia. We also examined the subcellular distribution of the three NADK isoforms using NADK–green fluorescent protein (GFP) fusion proteins expressed transiently in Arabidopsis suspension-cultured cells. NADK1 and NADK2 were found to be localized to the cytosol and plastid stroma, respectively, consistent with previous work, whereas NADK3 localized to the peroxisomal matrix via a novel type 1 peroxisomal targeting signal. The specific subcellular and tissue distribution profiles among the three NADK isoforms and their possible non-overlapping roles in NADP(H) biosynthesis in plant cells are discussed.     

Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.     

Prevention of flower formation in dicotyledons

Prevention of flower formation is important, for example for preventing the spread of transgenes from genetically modified plants or the spread of non-native species, for increasing vegetative growth or preventing the formation of allergenic pollen. The aim of this study was to determine whether flowering of dicotyledonous plants can be prevented by genetic manipulation without harmful effects on vegetative growth. Here we describe isolation of the BpMADS1 gene (similar to SEP3, formerly AGL9) from birch and show that it is expressed only in the inflorescences. In tobacco and Arabidopsis, the expression of BpMADS1::GUS was also virtually inflorescence-specific. Transgenic tobacco and Arabidopsis containing a BpMADS1::BARNASE construct grew well. In one tobacco line the formation of the inflorescence was completely prevented; in several other lines the flowers lacked stamens and carpels and therefore were sterile. The final dry weights of the shoots of the sterile tobacco lines were 140–200% of those of controls. In Arabidopsis, some of the transgenic lines containing the BpMADS1::BARNASE construct formed inflorescences. Some of these lines formed never flowers and some others formed occasionally single fertile flowers. Some other lines did not form inflorescences, but formed up to about one hundred leaves, even in long-day conditions. These results suggest that formation of flowers or inflorescences in widely different dicotyledonous plants could be prevented using the BpMADS1::BARNASE construct and that prevention of flowering may lead to increased vegetative mass.     

Isolation and functional analysis of 4-coumarate:coenzyme A ligase gene promoters from Salvia miltiorrhiza

The enzyme 4-coumarate:coenzyme A ligase (4CL) plays an important role in phenylpropanoid metabolism. The 5′-upstream regions of two Sm4CL genes were isolated from danshen (Salvia miltiorrhiza Bunge) and their functions were characterized by promoter-directed GUS gene expression assay in transgenic Arabidopsis. Seedlings containing pSm4CL1 promoter:GUS fusions showed apparent GUS staining in hypocotyl and those harboring pSm4CL2 promoter:GUS fusions were clearly stained in cotyledon vasculars and roots. Mature Arabidopsis transformed with pSm4CL1 promoter:GUS exhibited GUS expression which was weak in the shoots and scarcely in roots and those modified with pSm4CL2 promoter:GUS displayed obvious GUS staining in roots, stigmatic papillae, stamens and sepal veins. Semi-quantitative RT-PCR revealed that Sm4CL2 was transcribed at the highest level in roots which was also shown to be the major accumulation site of salvianolic acid B. The results suggested that Sm4CL2 rather than Sm4CL1 might be responsible for the biosynthesis of salvianolic acid B in danshen roots.