Protein accumulation and rumen stability of wheat γ-gliadin fusion proteins in tobacco and alfalfa.

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine-rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine-rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ-gliadin-δ-zein and γ-δ-zein, as well as δ-zein co-expressed with ß-zein, all formed protein bodies. However, the γ-gliadin-δ-zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ-gliadin-δ-zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ-gliadin-δ-zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ-gliadin-GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ-gliadin-δ-zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ-gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.

Transgene mobilization and regulatory uncertainty for non-GE fruit products of transgenic rootstocks.

Genetically engineered (GE) rootstocks may offer some advantages for biotechnology applications especially in woody perennial crops such as grape or walnut. Transgrafting combines horticultural grafting practices with modern GE methods for crop improvement. Here, a non-GE conventional scion (upper stem portion) is grafted onto a transgenic GE rootstock. Thus, the scion does not contain the genetic modification present in the rootstock genome. We examined transgene presence in walnut and tomato GE rootstocks and non-GE fruit-bearing scions. Mobilization of transgene DNA, protein, and mRNA across the graft was not detected. Though transgenic siRNA mobilization was not observed in grafted tomatoes or walnut scions, transgenic siRNA signal was detected in walnut kernels. Prospective benefits from transgrafted plants include minimized risk of GE pollen flow (Lev-Yadun and Sederoff, 2001), possible use of more than one scion per approved GE rootstock which could help curb the estimated US$136 million (CropLife International, 2011) cost to bring a GE crop to international markets, as well as potential for improved consumer and market acceptance since the consumable product is not itself GE. Thus, transgrafting provides an alternative option for agricultural industries wishing to expand their biotechnology portfolio.

Mobility of Transgenic Nucleic Acids and Proteins within Grafted Rootstocks for Agricultural Improvement.

Grafting has been used in agriculture for over 2000 years. Disease resistance and environmental tolerance are highly beneficial traits that can be provided through use of grafting, although the mechanisms, in particular for resistance, have frequently been unknown. As information emerges that describes plant disease resistance mechanisms, the proteins, and nucleic acids that play a critical role in disease management can be expressed in genetically engineered (GE) plant lines. Utilizing transgrafting, the combination of a GE rootstock with a wild-type (WT) scion, or the reverse, has the potential to provide pest and pathogen resistance, impart biotic and abiotic stress tolerance, or increase plant vigor and productivity. Of central importance to these potential benefits is the question of to what extent nucleic acids and proteins are transmitted across a graft junction and whether the movement of these molecules will affect the efficacy of the transgrafting approach. Using a variety of specific examples, this review will report on the movement of organellar DNA, RNAs, and proteins across graft unions. Attention will be specifically drawn to the use of small RNAs and gene silencing within transgrafted plants, with a particular focus on pathogen resistance. The use of GE rootstocks or scions has the potential to extend the horticultural utility of grafting by combining this ancient technique with the molecular strategies of the modern era.

An intellectual property sharing initiative in agricultural biotechnology: development of broadly accessible technologies for plant transformation.

The Public Intellectual Property Resource for Agriculture (PIPRA) was founded in 2004 by the Rockefeller Foundation in response to concerns that public investments in agricultural biotechnology benefiting developing countries were facing delays, high transaction costs and lack of access to important technologies due to intellectual property right (IPR) issues. From its inception, PIPRA has worked broadly to support a wide range of research in the public sector, in specialty and minor acreage crops as well as crops important to food security in developing countries. In this paper, we review PIPRA's work, discussing the failures, successes, and lessons learned during its years of operation. To address public sector's limited freedom-to-operate, or legal access to third-party rights, in the area of plant transformation, we describe PIPRA's patent 'pool' approach to develop open-access technologies for plant transformation which consolidate patent and tangible property rights in marker-free vector systems. The plant transformation system has been licensed and deployed for both commercial and humanitarian applications in the United States (US) and Africa, respectively.

Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato.

Vitamin C (L-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 µmol m(-2) s(-1)). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato.

In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex.

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.

The DNA-compacting protein DCP68 from soybean chloroplasts is ferredoxin:sulfite reductase and co-localizes with the organellar nucleoid.

The multiple copies of the chloroplast genome (plastome) are condensed and organized into nucleoids by a set of proteins. One of these, the DNA-binding protein DCP68 from soybean, has previously been shown to compact DNA and to inhibit DNA synthesis in vitro. N-terminal amino acid analysis and the absorption spectrum of the purified protein suggest that DCP68 is the siroheme protein sulfite reductase, a ferredoxin-dependent enzyme that participates in sulfur assimilation for cysteine and methionine biosynthesis. The in vivo association of this protein with chloroplast nucleoids was confirmed by immuno-colocalization with antibodies against sulfite reductase from Arabidopsis thaliana. These results suggest that DCP68 is a bifunctional chloroplast protein that participates in reductive sulfur assimilation and plays a role in organellar nucleoid organization. The fact that dephosphorylation by alkaline phosphatase affects the binding of purified DCP68 to DNA in vitro might be indicative of the way the interaction of the protein with the nucleoid is regulated in vivo.