Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana.

Plastids in vascular plants have various differentiated forms, among which amyloplasts are crucial for starch storage and plant productivity. Despite the vast knowledge of the binary-fission mode of chloroplast division, our understanding of the replication of non-photosynthetic plastids, including amyloplasts, remains limited. Recent studies have suggested the involvement of stromules (stroma-filled tubules) in plastid replication when the division apparatus is faulty. However, details of the underlying mechanism(s) and their relevance to normal processes have yet to be elucidated. Here, we developed a live analysis system for studying amyloplast replication using Arabidopsis (Arabidopsis thaliana) ovule integuments. We showed the full sequence of amyloplast development and demonstrated that wild-type amyloplasts adopt three modes of replication, binary fission, multiple fission, and stromule-mediated fission, via multi-way placement of the FtsZ ring. The minE mutant, with severely inhibited chloroplast division, showed marked heterogeneity in amyloplast size, caused by size-dependent but wild-type modes of plastid fission. The dynamic properties of stromules distinguish the wild-type and minE phenotypes. In minE cells, extended stromules from giant amyloplasts acquired stability, allowing FtsZ ring assembly and constriction, as well as the growth of starch grains therein. Despite hyper-stromule formation, amyloplasts did not proliferate in the ftsZ null mutant. These data clarify the differences between amyloplast and chloroplast replication and demonstrate that the structural plasticity of amyloplasts underlies the multiplicity of their replication processes. Furthermore, this study shows that stromules can generate daughter plastids via assembly of the FtsZ ring.

Lettuce-derived secretory IgA specifically neutralizes the Shiga toxin 1 activity.

MAIN CONCLUSION: An edible plant was tested as a host for the production of secretory monoclonal IgA against Shiga toxin 1 (Stx1). The lettuce-derived IgA completely protected Vero cells from Stx1. Secretory immunoglobulin A (SIgA) is thought to control mucosal infections and thus it may be applicable to oral passive immunotherapy. Edible plants are candidate hosts for producing oral formulations with SIgA against pathogenic agents. We previously established a recombinant IgA specific for the B subunit of Shiga toxin 1 (Stx1B) consisting of the Fab fragment of Stx1B-specific monoclonal IgG and the Fc region of IgA (hyIgA). Here, we developed transgenic lettuce (Lactuca sativa) that produces hyIgA in a secretory form (S-hyIgA). An Arabidopsis-derived light-harvesting complex II (LHCB) promoter was used for the expression of all four transgenes (hyIgA heavy, light and j chains, and secretory component). Agrobacterium-mediated transformation was carried out to introduce genes into lettuce leaf discs by means of a single vector harboring all four transgenes. Consistent with the tissue specificity of the LHCB promoter, the expression of hyIgA transgenes was observed in leaf and stem tissues, which contain chloroplasts, at the mRNA and protein levels. The leaves produced hyIgA in a more than tenfold higher yield as compared with stems. The lettuce-derived S-hyIgA was found to bind to Stx1B in a dose-dependent manner by means of ELISA. A leaf extract of the transgenic lettuce completely neutralized the cytotoxicity of Stx1 against Vero cells, which are highly susceptible to Stx1. In conclusion, we established a transgenic lettuce producing a secretory form of hyIgA that can bind bacterial toxin. The results indicate that edible practical plants containing S-hyIgA will provide a possible means for immunotherapy for food poisoning.

Plant-derived secretory component forms secretory IgA with shiga toxin 1-specific dimeric IgA produced by mouse cells and whole plants.

KEY MESSAGE: A key module, secretory component (SC), was efficiently expressed in Arabidopsis thaliana. The plant-based SC and immunoglobulin A of animal or plant origin formed secretory IgA that maintains antigen-binding activity. Plant expression systems are suitable for scalable and cost-effective production of biologics. Secretory immunoglobulin A (SIgA) will be useful as a therapeutic antibody against mucosal pathogens. SIgA is equipped with a secretory component (SC), which assists the performance of SIgA on the mucosal surface. Here we produced SC using a plant expression system and formed SIgA with dimeric IgAs produced by mouse cells as well as by whole plants. To increase the expression level, an endoplasmic reticulum retention signal peptide, KDEL (Lys-Asp-Glu-Leu), was added to mouse SC (SC-KDEL). The SC-KDEL cDNA was inserted into a binary vector with a translational enhancer and an efficient terminator. The SC-KDEL transgenic Arabidopsis thaliana produced SC-KDEL at the level of 2.7% of total leaf proteins. In vitro reaction of the plant-derived SC-KDEL with mouse dimeric monoclonal IgAs resulted in the formation of SIgA. When reacted with Shiga toxin 1 (Stx1)-specific ones, the antigen-binding activity was maintained. When an A. thaliana plant expressing SC-KDEL was crossed with one expressing dimeric IgA specific for Stx1, the plant-based SIgA exhibited antigen-binding activity. Leaf extracts of the crossbred transgenic plants neutralized Stx1 cytotoxicity against Stx1-sensitive cells. These results suggest that transgenic plants expressing SC-KDEL will provide a versatile means of SIgA production.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA.

Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo.

Molecular cloning and characterization of a linalool synthase from lemon myrtle.

Using a homology-based PCR strategy, we identified a cDNA with sequence similarity to linalool synthase from lemon myrtle. Functional expression of the cDNA (designated BcLS) gene in Escherichia coli yielded an active enzyme capable of catalyzing the conversion of geranyl diphosphate to (-)-linalool, i.e., an acyclic monoterpene alcohol, and to lesser amounts of cyclic monoterpenes. The kinetic parameters of BcLS were similar to those of synthases producing cyclic monoterpenes. PCR analysis revealed that the BcLS gene transcript was ubiquitously expressed in lemon myrtle and was upregulated in response to jasmonic acid treatment. Although the physiological role of neryl diphosphate (NPP) dependency of BcLS remains unclear, the cyclization activity of BcLS was enhanced when NPP was used as substrate, resulting in predominant production of cyclic monoterpenes. These findings indicate that BcLS has novel specificity and kinetic parameters, but its physiological responses to stresses such as insect damage appear to be similar to known linalool synthases.

Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells.

Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.

Transformation of Arabidopsis with plant-derived DNA sequences necessary for selecting transformants and driving an objective gene.

Most genetically modified plants are made with antibiotic-resistant genes and promoters derived from plant pathogens. To promote public acceptance, we have adapted a selectable marker and promoters all of which were derived from Arabidopsis thaliana to transformation. We have employed the gene for acetolactate synthase with its own promoter as a selectable marker, and the actin 2 gene promoter to strongly express an objective gene.

The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis.

Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In Arabidopsis thaliana, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, ARC6 and AtMinE1 are necessary for FtsZ ring formation and thus plastid division initiation, while ARC5 is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the arc5 and arc6 mutants using stroma-targeted fluorescent proteins. The arc5 PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The arc6 PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The arc5 GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The arc6 GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, arc5, arc6, and atminE1 plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.

Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation.

We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.

The Arabidopsis minD mutation causes aberrant FtsZ1 ring placement and moderate heterogeneity of chloroplasts in the leaf epidermis.

Symmetric division of leaf mesophyll chloroplasts requires MinD and MinE, which work together to suppress division other than at the mid-chloroplast. arc11 is a MinD loss-of-function mutant of Arabidopsis thaliana. In arc11 plants, asymmetric chloroplast division, as well as its delay or arrest, results in extreme size polymorphism of chloroplasts in mature mesophyll cells. The current study examined chloroplast phenotypes in the epidermis of arc11 leaves. Fluorescence microscopy analysis revealed that epidermal chloroplasts in mature leaves exhibited moderate heterogeneity in size. This probably resulted from completion of many of the previous non-equatorial or multiple division events in expanding leaves. Additionally, analyses of plastids found that epidermal chloroplasts in arc11 mutants showed several phenotypes that have not previously been described.

Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA.

Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

Development of visible markers for transgenic plants and their availability for environmental risk assessment.

Monitoring of transgenic plants in the field is important, but risk assessment has entailed laborious use of invisible marker genes. Here, we assessed three easily visible marker transgenes--green fluorescent protein (GFP), R, and Nicotiana tabacum homeobox (NTH) 15 genes--for their potential use as marker genes for monitoring genetically modified plants. Transgenic Arabidopsis thaliana plants for each of these genes were visibly distinguished from wild-type plants. We determined the germination rate, 3-week fresh weight, time to first flowering, and seed weight of the transgenic plants to evaluate whether the expression of these marker genes affected the growth of the host. Introduction of GFP gene had no effect on the evaluated parameters, and we then used the GFP gene as a marker to assess the outcrossing frequency between transgenic and two Arabidopsis species. Our results showed that the hybridization frequency between transgenic plants and Arabidopsis thaliana was 0.24%, and between transformants and Arabidopsis lyrata it was 2.6% under experimental condition. Out-crossing frequency was decreased by extending the distance between two kinds of plants. Thus, the GFP gene is a useful marker for assessing the whereabouts of transgenes/transformants in the field. We also demonstrated that the GFP gene is possibly applicable as a selection marker in the process of generation of transgenic plants.

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.

bHLH106 Integrates Functions of Multiple Genes through Their G-Box to Confer Salt Tolerance on Arabidopsis.

An activation-tagging methodology was applied to dedifferentiated calli of Arabidopsis to identify new genes involved in salt tolerance. This identified salt tolerant callus 8 (stc8) as a gene encoding the basic helix-loop-helix transcription factor bHLH106. bHLH106-knockout (KO) lines were more sensitive to NaCl, KCl, LiCl, ABA, and low temperatures than the wild-type. Back-transformation of the KO line rescued its phenotype, and over-expression (OX) of bHLH106 in differentiated plants exhibited tolerance to NaCl. Green fluorescent protein (GFP) fused with bHLH106 revealed that it was localized to the nucleus. Prepared bHLH106 protein was subjected to electrophoresis mobility shift assays against E-box sequences (5'-CANNTG-3'). The G-box sequence 5'-CACGTG-3' had the strongest interaction with bHLH106. bHLH106-OX lines were transcriptomically analyzed, and resultant up- and down-regulated genes selected on the criterion of presence of a G-box sequence. There were 198 genes positively regulated by bHLH106 and 36 genes negatively regulated; these genes possessed one or more G-box sequences in their promoter regions. Many of these genes are known to be involved in abiotic stress response. It is concluded that bHLH106 locates at a branching point in the abiotic stress response network by interacting directly to the G-box in genes conferring salt tolerance on plants.

Genome-wide screening of salt tolerant genes by activation-tagging using dedifferentiated calli of Arabidopsis and its application to finding gene for Myo-inositol-1-p-synthase.

Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis.

Production of hybrid-IgG/IgA plantibodies with neutralizing activity against Shiga toxin 1.

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.

Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase in rice: tissue-specific and light-responsive gene expression.

Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase (RNAP) were identified in Oryza sativa. The deduced amino acid sequences contain conserved regions, regions 1.2-4.2, and a novel region A/B at the N-terminus. Tissue-specific and light-responsive transcripts of OsSIG5 and OsSIG6 were observed. The N-terminal region of OsSig5 conferred import of green fluorescent protein into the chloroplast. Specific transcripts of rice psbA were synthesized in vitro by reconstituted OsSig5-RNAP holoenzymes. These results indicated that OsSig5 is a plastid sigma factor. This is the first report of the Sig5-type sigma factor in crops.