A spatial model approach for assessing windbreak growth and carbon stocks.

Agroforestry, the deliberate integration of trees into agricultural operations, sequesters carbon (C) while providing valuable services on agricultural lands. However, methods to quantify present and projected C stocks in these open-grown woody systems are limited. As an initial step to address C accounting in agroforestry systems, a spatial Markov random field model for predicting the natural logarithm (log) of the mean aboveground volume of green ash ( Marsh.) within a shelterbelt, referred to as the log of aboveground volume, was developed using data from an earlier study and web-available soil and climate information. Windbreak characteristics, site, and climate variables were used to model the large-scale trend of the log of aboveground volume. The residuals from this initial model were correlated among sites up to 24 km from a point of interest. Therefore, a spatial dependence parameter was used to incorporate information from sites within 24 km into the prediction of the log of the aboveground volume. Age is an important windbreak characteristic in the model. Thus, the log of aboveground volume can be predicted for a given windbreak age and for values of other explanatory variables associated with a site of interest. Such predictions can be exponentiated to obtain predictions of aboveground volume for windbreaks without repeated inventory. With the capability of quantifying uncertainty, the model has the potential for large regional planning efforts and C stock assessments for many deciduous tree species used in windbreaks and riparian buffers once it is calibrated.

Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

Production of biologically active human interleukin-4 in transgenic tobacco and potato.

Interleukin-4 (IL-4) is a pleiotropic cytokine that plays a key regulatory role in the immune system. Recombinant human IL-4 (rhIL-4) offers great potential for the treatment of cancer, viral and autoimmune diseases. Unfortunately, the high production cost of IL-4 associated with conventional expression systems has, until now, limited broader clinical testing, particularly with regard to the more convenient and safer oral delivery of IL-4 as opposed to parenteral injection in patients. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-4. IL-4 expression vectors with different modifications under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter were introduced into tobacco by Agrobacterium-mediated transformation. Transgenic tobaccos expressing various levels of rhIL-4 protein were generated. Higher expression was achieved through IL-4 retention in the endoplasmic reticulum (ER), with the maximal accumulation being approximately 0.1% of total soluble protein (TSP) in the leaves. No improvement in expression was further achieved by replacing the native signal peptide of IL-4 with the plant signal peptide. The best rhIL-4-expressing vector shown in tobacco was selected and further transferred into potato plants. The analysis of transgenic tubers also revealed various levels of rhIL-4, with the highest being 0.08% of TSP. Sensitive in vitro T-cell proliferation assays showed that plant-derived rhIL-4 retained full biological activity. These results suggest that plants can be used to produce biologically active rhIL-4 and probably many other mammalian proteins of medical significance. Moreover, the production of plants expressing rhIL-4 will enable the testing of plant rhIL-4 by oral delivery for the treatment of clinical diseases.

Rebaudioside F, a diterpene glycoside from Stevia rebaudiana.

The sweet diterpenoid glycoside, rebaudioside F, was isolated from leaves of a high rebaudioside C producing line of Stevia rebaudiana, and its structure was established by chemical and spectral studies.

Association of the transcriptional response of soybean plants with soybean mosaic virus systemic infection.

Compatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. To assess transcriptional changes in soybean plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation (p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean leaves with SMV infection at different time points were associated with the observed symptom development. By using stringent selection criteria (>or=2- or

pORE: a modular binary vector series suited for both monocot and dicot plant transformation.

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an 'open' set for general plant transformation, a 'reporter' set for promoter analysis and an 'expression' set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P (HPL), P (ENTCUP2) and P (TAPADH)), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).

Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4.

Induction of specific immunological unresponsiveness by feeding protein antigens is termed oral tolerance and may be a potential therapy for autoimmune diseases. Whereas oral tolerance therapy may be both simple and effective, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. We have previously demonstrated transgenic plant production and direct oral delivery of a beta cell autoantigen murine GAD67 to prevent autoimmune diabetes in nonobese diabetic mice. Mucosal adjuvants such as cholera toxin B subunit may lower the level of autoantigen required, but the development of neutralizing mucosal antibody responses may limit usefulness in enhancing long-term oral tolerance. IL-4, being an endogenous protein, would avoid this result and possibly enhance oral tolerance but has not been tested as a mucosal adjuvant. In this study, human GAD65 (hGAD65), as well as murine IL-4, was expressed in transgenic plants for feeding trials. Both IL-4 and hGAD65 plant tissue were required to protect nonobese diabetic mice from diabetes, and no benefit was found if either was used alone. Combined therapy enhanced levels of IgG1 anti-GAD antibodies, increased splenocyte IL-4/IFN-gamma cytokine responses, and produced protective regulatory T cells. These results demonstrate that orally administered plant IL-4 remains biologically active and is synergistic when given with hGAD65 in inducing robust oral immune tolerance. Using transgenic plants expressing IL-4 and GAD65 may be a novel clinical approach to the prevention of human type 1 diabetes by oral tolerance.

New non-glycosidic diterpenes from the leaves of Stevia rebaudiana.

Six new labdane-type, non-glycosidic diterpenes, sterebins I-N (1-6), were isolated from the leaves of Stevia rebaudiana. Their structures, analogous to those of the previously described sterebins A-H, were elucidated on the basis of spectroscopic and chemical studies.