Processing in transgenic Arabidopsis thaliana plants of polyproteins with linker peptide variants derived from the Impatiens balsamina antimicrobial polyprotein precursor

We have previously developed a method for expression in Arabidopsis thaliana L. of transgenes encoding cleavable chimaeric polyprotein precursors. The polyprotein precursors consisted of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and a variant form of RsAFP2 originating from Raphanus sativus seeds, which were linked by an intervening linker peptide sequence originating from a natural polyprotein occurring in seeds of Impatiens balsamina. By altering the amino acid sequence of the linker peptide separating the two AMPs, we now show that it is possible to improve the accuracy of polyprotein precursor cleavage, leading to the release of both the AMPs with either no or a few additional amino acids derived from the linker peptide. Furthermore, subcellular localization indicated that both the AMPs are predominantly present in the extracellular fluid of the transgenic plants.     

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

A novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage is covalently linked to the foot‐and‐mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an ‘IntF2A’ self‐excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a ‘polyprotein’ precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C‐terminal F2A extension remains on the released POIs. We demonstrated co‐expression of as many as three proteins in plants without compromising expression levels when compared with those using single‐protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti‐His Tag antibody in N. benthamiana leaves. The IntF2A‐based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co‐expression in plants, which is particularly important for gene/trait stacking.     

The arcelin-5 Gene of Phaseolus vulgaris Directs High Seed-Specific Expression in Transgenic Phaseolus acutifolius and Arabidopsis Plants

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.     

Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene

A variety of conventional methods allow the expression of multiple foreign proteins in plants by transgene stacking or pyramiding. However, most of these approaches have significant drawbacks. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. The unique features of this polyprotein system are based on the novel activity of the 2A peptide of Foot-and-mouth disease virus (FMDV) that acts cotranslationally to effect a dissociation of the polyprotein while allowing translation to continue. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.     

Stable expression of silencing‐suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA‐guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co‐evolved viral silencing‐suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA‐regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three‐step transgenic pathway for the synthesis of arachidonic acid (AA), an ω‐6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP‐AA‐transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity

Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).     

Transgenic grain legumes obtained byin planta electroporation-mediated gene transfer

Electroporation-mediated gene transfer into intact plant tissues was demonstrated in pea, cowpea, lentil, and soybean plants. Transient expression of a chimericgus reporter gene was used to monitor the uptake and expression of the introduced DNA in electroporated nodal axillary buds in vivo. The branches that grew out of the nodal meristems were chimeric and expressed the introduced gene up to 20 d after electroporation. Transgenic R₁ pea, lentil, and cowpea plants were recovered from seeds originating on these chimeric branches as shown by Southern blot hybridization and GUS expression. Transgenic R₂ soybean and lentil plants were also obtained. Segregation ratios in these populations showed a strong bias against transgene presence or expression.     

Enhanced methionine and cysteine levels in transgenic rice seeds by the accumulation of sesame 2S albumin

A chimeric gene encoding a precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein, was expressed in transgenic rice plants under the control of the glutelin promoter with the aim of improving the nutritive value of rice. Rice grains harvested from the first generation of ten different transformed lines inherited the transgene, and the accumulated sesame 2S albumin was presumably processed correctly as its mature form in sesame seed. This transgene was specifically expressed in maturing rice seeds with its encoded sesame 2S albumin exclusively accumulated in the seeds. The crude protein content in rice grains from five putative homozygous lines was increased by 0.64-3.54%, and the methionine and cysteine contents of these transgenic rice grains were respectively elevated by 29-76% and 31-75% compared with those of wild-type rice grains.     

Co-ordinated expression of multiple enzymes in different subcellular compartments in plants

A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) NIa proteinase along with two other reporter genes (those encoding the Escherichia coli acetate kinase (ACK) and Tn9 chloramphenicol acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV NIa proteinase recognition sequence. In addition, in some instances, chloroplast targeting information (a transit peptide (TP) from a pea rbcS gene) was incorporated into the polyprotein. We found that the NIa proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP-NIa-ACK-CAT or TP-ACK-CAT-NIa failed to yield chloroplast-localized ACK and CAT proteins, although the latter did give rise to a chloroplast-localized ACK-CAT polyprotein. These results indicate that the NIa proteinase acts in cis more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK-NIa-TP-CAT and TP-ACK-NIa-TP-CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy for simultaneous engineering of multiple subcellular compartments in plants.     

Flexible control of plant architecture and yield via switchable expression of Arabidopsis gai

The growth of plants is repressed by DELLA proteins, nuclear regulators whose activities are opposed by the growth-promoting phytohormone gibberellin (GA). Mutations affecting DELLA protein function were previously used by plant breeders to create the high-yielding semidwarf wheat varieties of the green revolution. gai is an Arabidopsis mutant DELLA protein-encoding orthologue of the wheat semidwarfing genes. Here we describe the development of a transgene that confers ethanol-inducible gai expression. Transient induction of gai causes transient growth repression: growth prior to and after treatment is unaffected. Appropriate ethanol treatments result in dwarf plants that produce the same numbers of seeds as untreated controls. This new technology represents a substantial advance in the applicability of genes encoding mutant DELLA proteins to agricultural and horticultural improvement, enhancing the flexibity with which these genes can be used for the sustainable achievement of increased crop plant yields.     

Altered gene expression in plants with constitutive expression of a mitochondrial small heat shock protein suggests the involvement of retrograde regulation in the heat stress response

Heat stress can negatively affect crop productivity. One way in which plants attempt to alleviate the effects of heat stress is to induce the expression of genes encoding heat shock proteins (HSPs), including small HSPs (sHSPs). We produced transgenic lines of Arabidopsis thaliana expressing a transgene encoding a maize mitochondrial sHSP, ZmHSP22. The transgene, under the control of the cauliflower mosaic virus 35S promoter, is constitutively highly expressed in these lines. As demonstrated by confocal immunofluorescence microscopy and analyses of isolated mitochondria, ZmHSP22 is directed to the mitochondria of Arabidopsis and is processed into the mature form. These transgenic lines demonstrated altered expression of nuclear genes encoding the endogenous mitochondrial sHSP, AtHSP23.6, chloroplast localized AtHSP25.3, class I cytosolic AtHSP17.4, cytosolic AtHSP70-1 and chloroplast localized AtHSP70-6, but not cytosolic AtHSP70-15, following exposure to heat stress. This suggests that the expression of HSPs can be affected by heat-induced mitochondrial retrograde regulation. Three-week-old plants from the transgenic Arabidopsis lines expressing ZmHSP22 have increased thermotolerance, as measured by the maintenance of higher leaf mass following successive days with short periods of heat stress.     

Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.     

Ten members of the Arabidopsis gene family encoding methyl-CpG-binding domain proteins are transcriptionally active and at least one,AtMBD11, is crucial for normal development

Animal proteins that contain a methyl-CpG-binding domain (MBD) are suggested to provide a link between DNA methylation, chromatin remodelling and gene silencing. However, some MBD proteins reside in chromatin remodelling complexes, but do not have specific affinity for methylated DNA. It has recently been shown that the Arabidopsis genome contains 12 putative genes encoding proteins with domains similar to MBD, of which at least three bind symmetrically methylated DNA. Using a bioinformatics approach, we have identified additional domains in a number of these proteins and, on this basis and extended sequence similarity, divided the proteins into subgroups. Using RT-PCR we show that 10 of the AtMBD genes are active and differentially expressed in diverse tissues. To investigate the biological significance of AtMBD proteins, we have transformed Arabidopsis with a construct aimed at RNA interference with expression of the AtMBD11 gene, normally active in most tissues. The resulting 35S::AtMBD11-RNAi plants displayed a variety of phenotypic effects, including aerial rosettes, serrated leaves, abnormal position of flowers, fertility problems and late flowering. Arabidopsis lines with reduced expression of genes involved in chromatin remodelling and transgene silencing show similar phenotypes. Our results suggest an important role for AtMBD proteins in plant development.     

Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis

We demonstrate that fluorescent proteins can be used as visual selection markers for the transformation of Arabidopsis thaliana by the floral dip method. Seed-specific expression of green fluorescent protein (GFP) variants, as well as DsRed, permits the identification of mature transformed seeds in a large background of untransformed seeds by fluorescence microscopy. In planta visualization of transformed seeds in siliques shows that susceptibility to floral dip transformation is limited to a small, defined window in flower development. In the competent stage, the random transformation of up to 25% of the seeds within a single silique may occur. The use of fluorescent proteins with different spectral characteristics allows a rapid identification and genetic analysis of seeds that have received multiple genes-of-interest in co-transformation experiments. The data reveal that co-transformation does not occur at random, since the co-transformed genes are integrated at a single genetic locus in approximately 70% of the cases. This genetic linkage of the co-transformed genes greatly simplifies metabolic pathway engineering by reverse genetics in Arabidopsis. Additional advantages of using visual selection instead of antibiotic resistance include a rapid identification of the effect of the T-DNA insertion or the transgene on seed development and/or germination. This technology, of tagging and identifying transformed seeds by fluorescence provides a novel high-throughput screening system with many potential applications in plant biotechnology.     

Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds.

Acetyl-coenzyme A carboxylase (ACCase) occurs in at least two forms in rapeseed (Brassica napus): a homomeric (HO) and presumably cytosolic isozyme and a heteromeric, plastidial isozyme. We investigated whether the HO-ACCase of Arabidopsis can be targeted to plastids of B. napus seeds. A chloroplast transit peptide and the napin promoter were fused to the Arabidopsis ACC1 gene and transformed into B. napus, with the following results. (a) The small subunit transit peptide was sufficient to provide import of this very large protein into developing seed plastids. (b) HO-ACCase in isolated plastids was found to be biotinylated at a level comparable to extraplastidial HO-ACCase. (c) In vitro assays of HO-ACCase in isolated plastids from developing seeds indicate that it occurs as an enzymatically active form in the plastidial compartment. (d) ACCase activity in mature B. napus seeds is normally very low; however, plants expressing the SSU/ACC1 gene had 10- to 20-fold higher ACCase activity in mature seeds, suggesting that plastid localization prevents the turnover of HO-ACCase. (e) ACCase over-expression altered seed fatty acid composition, with the largest effect being an increase approximately 5% by the expression of HO-ACCase in plastids.