A pollen-specific calmodulin-binding protein, NPG1, interacts with putative pectate lyases.

Previous genetic studies have revealed that a pollen-specific calmodulin-binding protein, No Pollen Germination 1 (NPG1), is required for pollen germination. However, its mode of action is unknown. Here we report direct interaction of NPG1 with pectate lyase-like proteins (PLLs). A truncated form of AtNPG1 lacking the N-terminal tetratricopeptide repeat 1 (TPR1) failed to interact with PLLs, suggesting that it is essential for NPG1 interaction with PLLs. Localization studies with AtNPG1 fused to a fluorescent reporter driven by its native promoter revealed its presence in the cytosol and cell wall of the pollen grain and the growing pollen tube of plasmolyzed pollen. Together, our data suggest that the function of NPG1 in regulating pollen germination is mediated through its interaction with PLLs, which may modify the pollen cell wall and regulate pollen tube emergence and growth.

Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing.

SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS.

Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass.

When grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. Tobacco possesses potent oil biosynthesis machinery and can accumulate up to 40% of seed weight in oil. In this work, we explored two metabolic engineering approaches to enhance the oil content in tobacco green tissues for potential biofuel production. First, an Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis, was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led to up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall of about a twofold increase in extracted fatty acids (FA) up to 5.8% of dry biomass in Nicotiana tabacum cv Wisconsin, and up to 6% in high-sugar tobacco variety NC-55. Modified tobacco plants also contained elevated amounts of phospholipids. This increase in lipids was accompanied by a shift in the FA composition favourable for their utilization as biodiesel. Second, we expressed in tobacco Arabidopsis gene LEAFY COTYLEDON 2 (LEC2), a master regulator of seed maturation and seed oil storage under the control of an inducible Alc promoter. Stimulation of LEC2 expression in mature tobacco plants by acetaldehyde led to the accumulation of up to 6.8% per dry weight of total extracted FA. The obtained data reveal the potential of metabolically modified plant biomass for the production of biofuel.

Plant-produced hepatitis B core protein chimera carrying anthrax protective antigen domain-4.

The hepatitis B core antigen (HBcAg) can generate a strong immune response and is recognized as an effective carrier for foreign epitopes. The domain-4 epitope of the anthrax protective antigen (PA-D4) plays an essential role in generating protective immunity against virulent Bacillus anthracis. Here we report the successful production of a recombinant protein comprised of the antigenic PA-D4 integrated into the c/e1 loop of HBcAg in transgenic low-alkaloid Nicotiana tabacum. Sera of mice injected with the plant-derived purified HB/PA-D4 protein exhibited significant anti-PA- and anti-HBcAg-specific IgG titers; however, formation of virus-like particles (VLP) was not observed. These data support the feasibility of producing complex protein chimeras in plants.

Immunogenic properties of plant-derived recombinant smallpox vaccine candidate pB5.

The extracellular virion membrane protein B5 is a potent inducer of immune responses capable of protecting mice and primates against poxvirus infections. Here, we examined the antibody response induced in mice immunized intramuscularly (i.m.) or intranasally (i.n.) with plant-derived B5 (pB5) accompanied or not with plant total soluble protein (TSP) at various concentrations. Increasing amounts of TSP inhibited the pB5-specific response in both i.m.- and i.n.-immunized mice, with more dramatic effects in the latter. pB5 administered to mucosal surfaces induced specific IgG and IgA responses, whereas i.m. immunization produced high serum IgG titers and no IgA. A 6-fold increase in pB5 dosage administered i.n. led to an antibody response comparable to that obtained by i.m. injection. Our study addresses the quality/quantity issues of the pB5 subunit preparation and demonstrates the feasibility of mucosal administration of plant-derived smallpox subunit vaccine in obtaining a potent immune response. Overall, this work points to the practicability of needle-free mucosal administration of such vaccines in light of purity, dosage and adjuvant formulation.

Regulation of plant developmental processes by a novel splicing factor.

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

Organ-specific, developmental, hormonal and stress regulation of expression of putative pectate lyase genes in Arabidopsis.

Pectate lyases catalyse the eliminative cleavage of de-esterified homogalacturonan in pectin, a major component of the primary cell walls in higher plants. In the completed genome of Arabidopsis, there are 26 genes (AtPLLs) that encode pectate lyase-like proteins. Here, we analysed the expression pattern of all AtPLLs in different organs, at different stages of seedling development and in response to various hormones and stresses. The expression of PLLs varied considerably in different organs, with no expression of some PLLs in vegetative organs. Interestingly, all PLL genes are expressed in flowers. Several PLLs are expressed highly in pollen, suggesting a role for these in pollen development and/or function. Analysis of expression of all PLL genes in seedlings treated with hormones, abiotic stresses and elicitors of defense responses revealed significant changes in the expression of some PLLs without affecting the other PLLs. The stability of transcripts of PLLs varied considerably among different genes. Our results indicate a complex regulation of expression of PLLs and involvement of PLLs in some of the hormonal and stress responses.

Smallpox subunit vaccine produced in Planta confers protection in mice.

We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems.

Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells.

Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAbP) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAbM, the plant mAbP bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAbP and mAbM showed the similar binding to FcgammaRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAbP originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.

Severe acute respiratory syndrome (SARS) S protein production in plants: development of recombinant vaccine.

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.

Nuclear localization and in vivo dynamics of a plant-specific serine/arginine-rich protein.

Serine/arginine-rich (SR) proteins in non-plant systems are known to play important roles in both constitutive and alternative splicing of pre-messenger RNAs (pre-mRNAs). Recently, we isolated a novel SR protein (SR45), which interacts with U1 snRNP 70K protein, a key protein involved in 5' splice site recognition. SR45 is found only in plants and is unique in having two SR domains separated by an RNA recognition motif (RRM). To study the localization and dynamics of SR45, we expressed it as a fusion to green fluorescent protein (GFP) in cultured cells and transgenic Arabidopsis plants. The SR45 is localized exclusively to nuclei. In interphase nuclei, GFP-SR45 was found both in speckles and nucleoplasm. The speckles exhibited intranuclear movements and changes in morphology. Inhibition of transcription and protein phosphorylation resulted in redistribution of SR45 to bigger speckles. The change in the number and morphology of speckles caused by inhibition of transcription was blocked by an inhibitor of phosphatases. These results indicate that transcription activity of the cell and protein (de)phosphorylation regulate the intranuclear distribution of SR45.

A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination.

Calmodulin (CaM), a ubiquitous multifunctional calcium sensor in all eukaryotes, mediates calcium action by regulating the activity/function of many unrelated proteins. Although calcium and CaM are known to play a crucial role in pollen germination and pollen tube growth, the proteins that mediate their action have not been identified. We isolated three closely related CaM-binding proteins (NPG1, NPGR1, and NPGR2) from Arabidopsis. NPG1 (No Pollen Germination1) is expressed only in pollen, whereas the NPG-related proteins (NPGR1 and NPGR2) are expressed in pollen and other tissues. The bacterially expressed NPG1 bound three isoforms of Arabidopsis CaM in a calcium-dependent manner. To analyze the function of NPG1, we performed a reverse genetics screen and isolated a mutant in which NPG1 is disrupted by a T-DNA insertion. Segregation and molecular analyses of the NPG1 knockout mutant and a cross with a male sterile mutant indicate that the mutated NPG1 is not transmitted through the male gametophyte. Expression of NPG1 in the knockout mutant complemented the mutant phenotype. Analysis of pollen development in the knockout mutant by light microscopy showed normal pollen development. Pollen from NPG1 mutant in the quartet background has confirmed that NPG1 is dispensable for pollen development. However, germination studies with pollen from the mutant in the quartet background indicate that pollen carrying a mutant allele does not germinate. Our genetic, histological, and pollen germination studies with the knockout mutant line indicate that NPG1 is not necessary for microsporogenesis and gametogenesis but is essential for pollen germination.

Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals.

U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1 snRNP-specific protein, is involved in the early stages of spliceosome formation. In non-plant systems, it is involved in constitutive and alternative splicing. It has been shown that U1snRNP is dispensable for in vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K in yeast (Saccharomyces cerevisiae) is not lethal. As in yeast and humans (Homo sapiens), plant U1-70K is coded by a single gene. In this study, we blocked the expression of Arabidopsis U1-70K in petals and stamens by expressing U1-70K antisense transcript using the AP3 (APETALA3) promoter specific to these floral organs. Flowers of transgenic Arabidopsis plants expressing U1-70K antisense transcript showed partially developed stamens and petals that are arrested at different stages of development. In some transgenic lines, flowers have rudimentary petals and stamens and are male sterile. The severity of the phenotype is correlated with the level of the antisense transcript. Molecular analysis of transgenic plants has confirmed that the observed phenotype is not due to disruption of whorl-specific homeotic genes, AP3 or PISTILLATA, responsible for petal and stamen development. The AP3 transcript was not detected in transgenic flowers with severe phenotype. Flowers of Arabidopsis plants transformed with a reporter gene driven by the same promoter showed no abnormalities. These results show that U1-70K is necessary for the development of sepals and petals and is an essential gene in plants.

Function and glycosylation of plant-derived antiviral monoclonal antibody.

Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current methods of antibody production in animal systems. In this study, the heavy and light chains of human anti-rabies mAb were expressed and assembled in planta under the control of two strong constitutive promoters. An alfalfa mosaic virus untranslated leader sequence and Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal were linked at the N and C terminus of the heavy chain, respectively. mAbP was as effective at neutralizing the activity of the rabies virus as the mammalian-derived antibody (mAbM) or human rabies Ig (HRIG). The mAbP contained mainly oligomannose type N-glycans (90%) and had no potentially antigenic alpha(1,3)-linked fucose residues. mAbP had a shorter half-life than mAbM. The mAbP was as efficient as HRIG for post-exposure prophylaxis against rabies virus in hamsters, indicating that differences in N-glycosylation do not affect the efficacy of the antibody in this model.

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.