Advances in plant molecular farming

Plant molecular farming (PMF) is a new branch of plant biotechnology, where plants are engineered to produce recombinant pharmaceutical and industrial proteins in large quantities. As an emerging subdivision of the biopharmaceutical industry, PMF is still trying to gain comparable social acceptance as the already established production systems that produce these high valued proteins in microbial, yeast, or mammalian expression systems. This article reviews the various cost-effective technologies and strategies, which are being developed to improve yield and quality of the plant-derived pharmaceuticals, thereby making plant-based production system suitable alternatives to the existing systems. It also attempts to overview the different novel plant-derived pharmaceuticals and non-pharmaceutical protein products that are at various stages of clinical development or commercialization. It then discusses the biosafety and regulatory issues, which are crucial (if strictly adhered to) to eliminating potential health and environmental risks, which in turn is necessary to earning favorable public perception, thus ensuring the success of the industry.     

Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate

The aromatic amino acids L-phenylalanine and L-tyrosine and their plant-derived natural products are essential in human and plant metabolism and physiology. Here we identified Petunia hybrida and Arabidopsis thaliana genes encoding prephenate aminotransferases (PPA-ATs), thus completing the identification of the genes involved in phenylalanine and tyrosine biosyntheses. Biochemical and genetic characterization of enzymes showed that PPA-AT directs carbon flux from prephenate toward arogenate, making the arogenate pathway predominant in plant phenylalanine biosynthesis.     

Foreign protein production in plant tissue cultures

Foreign proteins synthesised by plants are now in the marketplace, and clinical trials for plant-derived therapeutic proteins are underway. Economic analysis of plant production systems has helped identify the types of protein that would be most suitable for manufacture using tissue culture methods. The major advantages associated with in vitro plant systems include the ability to manipulate environmental conditions for better control over protein levels and quality, the rapidity of production compared with agriculture, and the use of simpler and cheaper downstream processing schemes for product recovery from the culture medium.     

Field assessment of plant derivative compounds for managing fungal soybean diseases

Natural plant-derived compounds are currently being explored as alternatives for pest control in sustainable agriculture. This study explored the use of two compounds, sesamol and carbenoxolone, in the management of the fungal soybean disease charcoal rot (Macrophomina phaseolina). Previous studies have determined that sesamol and carbenoxolone compounds significantly inhibited fungal pathogen growth and plant disease in vitro. In order to assess the field efficacy of these compounds for fungal disease control, 2 years of field testing of these compounds have been conducted in southeast Kansas. Field treatments of the compounds sesamol and carbenoxolone at three concentrations, 0, 500 and 1000 microg/ml, were applied foliarly at four distinct plant developmental stages. Treatments were applied to plots in random triplicate array and the experiment was repeated during the 1998 and 1999 growing seasons. Disease assessments were based on visual disease ratings, plant mortality and soybean yield analysis. Data were recorded weekly for each treatment plot and statistically analysed using analysis of variance. Results indicate that sesamol and carbenoxolone treatments significantly decreased disease symptoms (11-12%) and plant mortality (24-28%) while significantly increasing soybean yields (18-38%). These results support that plant-derived compounds can have a significant impact on soybean disease management and yield under field conditions.     

The antiparasitic actions of plant jasmonates

Jasmonates are a group of small lipids produced in plants, which function as plant stress hormones. We have previously shown that jasmonates can exert significant cytotoxic effects upon human cancer cells. The purpose of the present study was to determine the effects of jasmonates on parasites. To that end, we chose 2 major human blood parasites, Plasmodium falciparum, a unicellular parasite, and Schistosoma mansoni, a multicellular helminth parasite, and studied the effects of jasmonates on these parasites in vitro. We found that jasmonates are cytotoxic toward both parasites, with P. falciparum being the more susceptible. Jasmonates did not cause any damage to control human erythrocytes at the maximum concentration used in the experiments. This is the first study demonstrating the antiparasitic potential of plant-derived jasmonates.     

Functional cloning and characterization of a plant efflux carrier for multidrug and heavy metal detoxification.

We have identified a detoxifying efflux carrier from Arabidopsis using a functional cloning strategy. A bacterial mutant, KAM3, is deficient in multidrug resistance and does not survive on medium containing norfloxacin. After transformation of KAM3 cells with an Arabidopsis cDNA library, transformants were selected for restored growth on the toxic medium. One cDNA clone that complemented KAM3 encodes a novel protein with twelve putative transmembrane domains and contains limited sequence homology to a multidrug and toxin efflux carrier from bacteria. We named this Arabidopsis protein AtDTX1 (for Arabidopsis thaliana Detoxification 1). A large gene family of at least 56 members encoding related proteins was identified from the Arabidopsis genome. Further functional analysis of AtDTX1 protein in KAM3 mutant demonstrated that AtDTX1 serves as an efflux carrier for plant-derived alkaloids, antibiotics, and other toxic compounds. Interestingly, AtDTX1 was also capable of detoxifying Cd(2+), a heavy metal. Further experiments suggest that AtDTX1 is localized in the plasma membrane in plant cells thereby mediating the efflux of plant-derived or exogenous toxic compounds from the cytoplasm.     

Impact of plant derivatives on the growth of foodborne pathogens and the functionality of probiotics

Numerous studies have been published on the antimicrobial and antioxidant properties of various plant components. However, there is relatively little information on the impact of such components on the enhancement of probiotics and production of antimicrobial compounds from these probiotics. Hence, this paper focuses on the influence of plant-derived components against pathogens, enhancement of cell viability and functionality of probiotics, and potential applications of such components in food safety and human health.     

Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals

The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.     

Analytical aspects of plant metabolite profiling platforms: current standings and future aims

Over the past years, metabolic profiling has been established as a comprehensive systems biology tool. Mass spectrometry or NMR spectroscopy-based technology platforms combined with unsupervised or supervised multivariate statistical methodologies allow a deep insight into the complex metabolite patterns of plant-derived samples. Within this review, we provide a thorough introduction to the analytical hard- and software requirements of metabolic profiling platforms. Methodological limitations are addressed, and the metabolic profiling workflow is exemplified by summarizing recent applications ranging from model systems to more applied topics.     

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

Concerns for our environment and unease with our dependence on foreign oil have renewed interest in converting plant biomass into fuels and 'green' chemicals. The volume of plant matter available makes lignocellulose conversion desirable, although no single isolated organism has been shown to depolymerize lignocellulose and efficiently metabolize the resulting sugars into a specific product. This work reviews selected chemicals and fuels that can be produced from microbial fermentation of plant-derived cell-wall sugars and directed engineering for improvement of microbial biocatalysts. Lactic acid and ethanol production are highlighted, with a focus on engineered Escherichia coli .     

In vitro processing of tomato proteinase inhibitor I by barley microsomal membranes: a system for analysis of cotranslational processing of plant endomembrane proteins

A plant-derived in vitro system for the study of cotranslational processing of plant endomembrane proteins has been developed and used to investigate cotranslational proteolytic processing of tomato proteinase inhibitor I. Translation of the inhibitor I precursor in wheat germ lysate supplemented with barley aleurone microsomal membranes resulted in cotranslational import of the protein into microsomal vesicles and cleavage of the signal sequence. NH₂-terminal sequence analysis of the translocated inhibitor I processing intermediate showed that the signal sequence was cleaved between Ala₂₃ and Arg₂₄ of the precursor protein. Parallel experiments using dog pancreas microsomal membranes indicated an identical site of cleavage, suggesting that the substrate determinants for signal sequence processing are conserved across kingdoms. The plant-derived processing system used for this study may be valuable for analysis of cotranslational processing of other plant preproteins and for characterizing the components of the cotranslational import machinery in plants.     

Hyphae of waxcap fungi colonise plant roots

The trophic strategy of the globally distributed waxcaps (Hygrophoraceae) is uncertain. Some clues point to a biotrophic mode, particularly the ¹³C and ¹⁵N (stable isotopes) signatures. The observation of dense basal hyphae of Hygrocybe fruit bodies being tightly attached to live fine roots may be indicative of a plant-derived nutritional habit. To further scrutinize this fungus–plant association, stipe base samples and attached plant fragments were examined histologically. Waxcap hyphae were found growing inside live fine roots of associated vegetation. Amplification and sequencing of waxcap DNA from living root tissues using species-specific PCR primers also confirmed their presence in live plant roots. We therefore conclude that this group of fungi has a biotrophic lifestyle with plants.     

The production of biopharmaceuticals in plant systems

Biopharmaceuticals present the fastest growing segment in the pharmaceutical industry, with an ever widening scope of applications. Whole plants as well as contained plant cell culture systems are being explored for their potential as cheap, safe, and scalable production hosts. The first plant-derived biopharmaceuticals have now reached the clinic. Many biopharmaceuticals are glycoproteins; as the Golgi N-glycosylation machinery of plants differs from the mammalian machinery, the N-glycoforms introduced on plant-produced proteins need to be taken into consideration. Potent systems have been developed to change the plant N-glycoforms to a desired or even superior form compared to the native mammalian N-glycoforms. This review describes the current status of biopharmaceutical production in plants for industrial applications. The recent advances and tools which have been utilized to generate glycoengineered plants are also summarized and compared with the relevant mammalian systems whenever applicable.     

蚜虫雄蚜与雌性母对性信息素及植物挥发物的田间反应

田间观察了桃蚜Myzus persicae (Sulzer)、绣线菊蚜Aphis spiraecola Patch 、山楂圆疣蚜Ovatus crataegarius (Walker)等3种蚜虫对性信息素[(4aS,7S,7aR)-荆芥内酯和 (1R,4aS,7S,7aR)-荆芥醇]的反应,并且调查了性信息素与植物挥发物对桃蚜的田间引诱活性的相互作用.在有冬寄主或夏寄主植物的田中,性信息素诱捕器诱捕到桃蚜雄蚜与雌性母的数量显著多于对照诱捕器的诱捕数;但在非寄主植物的田中,却引诱不到桃蚜.苯甲醛(冬寄主植物桃树Prunus persica的主要挥发物组分之一)能够增强桃蚜雄蚜的引诱作用.绣线菊蚜雄蚜和雌性母对植物中提取的荆芥内酯有反应,而山楂圆疣蚜雄蚜和雌性母对植物中提取的和人工合成的荆芥内酯都没有反应,但对荆芥醇有反应.并且在荆芥醇中添加荆芥内酯之后对山楂圆疣蚜雄蚜引诱活性显著提高.还讨论了雌性蚜产生化合物被雄蚜作为性信息素、被雌性母作为聚集信息素以及性信息素与寄主植物挥发物的相互作用.     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

Proteomic Study to Understand Promotive Effects of Plant-Derived Smoke on Soybean (Glycine max L.) Root Growth Under Flooding Stress

Plant-derived smoke plays a key role in plant growth. Proteomic technique was used for underlying mechanisms of plant-derived smoke on the growth of soybean (Glycine max L.) under flooding stress. The length and weight of soybean root increased with 2000 parts per million plant-derived smoke under flooding stress within 4 days. Altered proteins by plant-derived smoke treatment under flooding stress were mainly related to protein metabolism, stress, and redox. Furthermore, proteins related to mitochondrial electron transport chain decreased by flooding stress; however, they increased by addition of plant-derived smoke under flooding stress. Based on the results of proteomic analysis, confirmation experiments were performed. ATPase abundance and ATP content increased with the treatment of plant-derived smoke under flooding stress. Furthermore, the ascorbate/glutathione cycle was activated with the treatment of plant-derived smoke under flooding stress. These results suggest that plant-derived smoke improves the root growth of soybean with energy production and reactive oxygen scavenging even if it is under flooding stress, which might positively regulate soybean tolerance towards flooding stress.

     

植物源二萜类化合物微生物合成研究进展

植物源二萜类天然产物结构复杂且功能多样,具有抗癌、抗炎和抗菌等多种药理活性,在药品、化妆品和食品添加剂等方面广泛应用。近年来,基于植物源二萜类化合物(diterpenoids)生物合成途径中功能基因的逐步揭示和合成生物技术的发展,科研人员采用代谢工程技术构建了多种二萜类化合物的微生物细胞工厂,且多个化合物达到克级产量。本文对植物源二萜类化合物微生物细胞工厂的构建情况进行综述,介绍并探讨植物源二萜类化合物微生物合成的研究进展和改造策略,为高产二萜类化合物细胞工厂构建和工业化生产提供参考。     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

Interaction of the breast cancer resistance protein with plant polyphenols

Multidrug transporters influence drug distribution in vivo and are often associated with tumour drug resistance. Here we show that plant-derived polyphenols that interact with P-glycoprotein can also modulate the activity of the recently discovered ABC transporter, breast cancer resistance protein (BCRP/ABCG2). In two separate BCRP-overexpressing cell lines, accumulation of the established BCRP substrates mitoxantrone and bodipy-FL-prazosin was significantly increased by the flavonoids silymarin, hesperetin, quercetin, and daidzein, and the stilbene resveratrol (each at 30 microM) as measured by flow cytometry, though there was no corresponding increase in the respective wild-type cell lines. These compounds also stimulated the vanadate-inhibitable ATPase activity in membranes prepared from bacteria (Lactococcus lactis) expressing BCRP. Given the high dietary intake of polyphenols, such interactions with BCRP, particularly in the intestines, may have important consequences in vivo for the distribution of these compounds as well as other BCRP substrates.     

The fate of nitrogen (15N) released from different plant materials during decomposition under field conditions

Release of N, retention in soil, availability to a subsequent crop and total recovery of N derived from different¹⁵N-labelled plant materials decomposing in soil was investigated in two field experiments. In the first experiment five different plant species (white clover, red clover, subterranean clover, field bean and timothy) and in the second subterranean clover of different maturity (2,3 and 4 months old) were buried in mesh bags in the soil and allowed to decompose for 10 and 4 months, respectively. Most of the N released from the decaying plant materials was retained in the soil (27–46% of input). The subsequent crop (barley) took up 6–25% of input. The uptake correlated with the amount of N released from the decomposing material (r=0.936^*, I experiment). Similar amounts of subterranean clover N were taken up by barley regardless to whether the material was buried in soil in the previous autumn or just before sowing of the crop. At the end of the experiments, the total recovery of the introduced plant-derived N varied between 89 and 102%. The results present evidence that the ability of the soil to retain plant-derived N is strong in comparison with the ability of the subsequent crop and different loss mechanisms to remove it.