VIB - International Plant Biotechnology Outreach

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway.

Eleven day-old grass pea plants (Lathyrus sativus L.) were grown hydroponically for 96 h in the presence of 0.5 mM lead nitrate (Pb(NO(3))(2)). The survival rate was 100%. The mean lead content (measured by ICP-OES) in root tissues was 153 mg Pb g(-1) dry matter. Over three quarters of the lead was not labile. Compared with control plants, lead-exposed plants showed a six-fold, two-fold and three and a half-fold reduction in their root calcium, zinc and copper contents, respectively. Together, these results suggested that Lathyrus sativus L. was tolerant to a deficiency in essential nutrients and able to store large amounts of lead in its root tissues. Therefore, it could be used for the development of new rhizofiltration systems.

Abstract The number of studies of tropical tree species that use molecular tools is increasing, most of which collect leaf tissue for genomic DNA extraction. In tropical trees the canopy is not only frequently inaccessible, but also, once reached, the leaf tissue is often heavily defended against herbivory by high concentrations of anti-predation compounds, which may inhibit downstream applications, particularly PCR. Cambium tissue, accessed directly from the tree trunk at ground level, offers a readily accessible resource that is less hampered by the presence of defensive chemicals than leaf tissue. Here we describe a simple method for obtaining tissue from the cambial zone for DNA extraction and test the applicability of the method in a range of tropical tree species. The method was used successfully to extract DNA from 11 species in nine families. A subset of the DNA extracts was tested in more detail and proved to be highly suitable for AFLP analysis.

When we discovered that crown gall induction on plants by Agrobacterium tumefaciens is a natural event of genetic engineering, we were convinced that this was the dawn of a new era for plant science. Now, more than 30 years later, I remain overawed by how far and how rapidly we progressed with our knowledge of the molecular basis of plant growth, development, stress resistance, flowering, and ecological adaptation, thanks to the gene engineering technology. I am impressed, but also frustrated by the difficulties of applying this knowledge to improve crops and globally develop a sustainable and improved high-yielding agriculture. Now that gene engineering has become so efficient, I had hoped that thousands of teams, all over the world, would work on improving our major food crops, help domesticate new ones, and succeed in doubling or tripling biomass yields in industrial crops. We live in a world where more than a billion people are hungry or starving, while the last areas of tropical forest and wild nature are disappearing. We urgently need a better supply of raw material for our chemical industry because petroleum-based products pollute the environment and are limited in supply. Why could this new technology not bring the solutions to these challenges? Why has this not happened yet; what did we do wrong?

Validity of the taxa currently recognized in the genus Vasconcellea was analyzed by investigating morphological and molecular data from 105 specimens of this genus and six specimens of the related genus Carica. Taxon identification of these specimens was compared with clustering in two phenetic dendrograms generated with 36 morphological characters and 254 amplified fragment length polymorphic (AFLP) markers. Moreover, cytoplasmic haplotypes were assessed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of one mitochondrial and two chloroplast DNA regions. Results show that the morphological data set, containing mainly vegetative characteristics, merely reveals external resemblance between specimens, which is not directly associated with genetic relationships and taxon validity. Phenotypic plasticity and intercompatibility between several species are likely to confuse morphological delimitation of the taxa. Based on the results of our study, several specimens that could not be identified with the currently used identification key (1) could be attributed to a known taxon, which should be extended to include a higher range of morphological variability or (2) could be hypothesized to be of hybrid origin. Because of the high intraspecific variation within V. microcarpa and V. × heilbornii, revision of these taxa is recommended.

SUMMARY Annexins are calcium-binding proteins that have been associated in plants with different biological processes such as responses to abiotic stress and early nodulation stages. Until now, the implication of annexins during plant-pathogen interactions has not been reported. Here, a novel plant annexin gene induced in tobacco BY-2 cell suspension cultures infected with the phytopathogenic bacterium Rhodococcus fascians (strain D188) has been identified. Expression of this gene, called Ntann12, is also induced, but to a lower extent, by a strain (D188-5) that is unable to induce leafy gall formation. This gene was also induced in BY-2 cells infected with Pseudomonas syringae but not in cells infected with Agrobacterium tumefaciens or Escherichia coli. Ntann12 expression was also found to be stimulated by abiotic stress, including NaCl and abscissic acid, confirming a putative role in stress signal transduction pathways. In addition, promoter-GUS analyses using homozygous transgenic tobacco seedlings showed that the developmentally controlled expression of Ntann12 is altered upon R. fascians infection. Finally, up-regulation of Ntann12 during leafy gall ontogenesis was confirmed by RT-qPCR. Discussion is focused on the potential role of Ntann12 in biotic and abiotic stress responses and in plant development, both processes that may involve Ca(2+)-dependent signalling.

3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP ) is a neuroexcitatory non-protein amino acid identified in grass pea (Lathyrus sativus L.) and in ginseng (Panax spp). Its presence in the drought tolerant grass pea is thought to be responsible for the crippling disease neurolathyrism, while in the longevity promoting ginseng it is named dencichine and praised for its haemostatic properties. Some properties of -ODAP are put in perspective.

This paper draws on the importance of science-based agriculture in order to throw light on the way scientific achievements are at the basis of modern civilization. An overview of literature on plant biotechnology innovations and the need to steer agriculture towards sustainability introduces a series of perspectives on how plant biotech can contribute to the major challenge of feeding our super population with enough nutritious food without further compromise of the environment. The paper argues that science alone will not solve problems. Three major forces - science, the economy and society - shape our modern world. There is a need for a new social contract to harmonize these forces. The deployment of the technologies must be done on the basis of ethical and moral values.

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.

Abstract Neurolathyrism is a unique neurodegeneration disease caused by β-N-oxalyl-L-α, β- diaminopropionic (β-ODAP) present in grass pea seed ( Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, β-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca 2+ from mitochondria to cellular matrix. Increased Ca 2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of β1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change.

Neurolathyrism is a motor neuron disease characterized by lower limb paraparesis. It is associated with ingestion of a plant excitotoxin, beta-N-oxalyl-L-alphabeta-diaminopropionic acid (L-beta-ODAP), an agonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamatergic receptors. Previously, a limited model of neurolathyrism was reported for the rat. To improve upon the model, we stressed rat pups by separation from their mothers, followed by the subcutaneous L-beta-ODAP treatment, resulting in a 4.6-fold higher incidence (14.0-15.6%) of the paraparesis compared with the prior study. The number and size of motor neurons in these rats were decreased only in the lumbar and sacral cord segments, at approximately 13-36 weeks after treatment. Only lumbar and sacral spinal cord tissue revealed pathological insults typical of physical and ischemic spinal cord injury in the surviving motor neurons. In addition, extensive but transient hemorrhage occurred in the ventral spinal cord parenchyma of the rat, and numerous TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells were also observed. In parallel, vascular endothelial growth factor receptor (VEGFR)-2 (Flk-1) levels were significantly lowered in the lumbosacral spinal cord of the paraparetic rats compared with their controls, suggesting a failure of the VEGF system to protect neurons against L-beta-ODAP toxicity. We propose, based on these data, a novel pathological process of motor neuron death induced by peripheral L-beta-ODAP. For the first time, we present a model of the early molecular events that occur during chemically induced spinal cord injury, which can potentially be applied to other neurodegenerative disorders.

Popular advice for healthy diets that may promote health and longevity include the daily consumption of at least three servings of fruits or vegetables and the variation of foods to include items derived from different plants and those plants should belong to different botanical families Ancient civilisations in the Middle East and in America included grain legumes and cereals in well-balanced diets. In the funeral offerings found in the Egyptian pyramids various legume seeds were present, including lentils and grass pea. Apparently, legumes were a food of special consideration to be offered to kings, in contrast to the present day reputation of being the meat of the poor, with 75% of all legumes now being produced in developing countries. Excavations of ancient settlements indicate the use of both cereals and legumes A well balanced food basket promoting health and strength may have given an evolutionary advantage. The benefits of legume cultivation for soil fertility were already recognised in the 4 th century BC Legumes are important factors in the natural cycle of nitrogen, being able to fix atmospheric nitrogen in symbiosis with Rhizobium bacteria. This enables the leguminous plants to thrive on poor soil, which makes them essential partners in the maintenance of soil fertility, and to produce protein-rich seeds. However, maintenance of optimum rates of nitrogen fixation requires continued attention by plant breeders Legumes are also unusually diverse in their defence against predators by producing a large array of secondary metabolites forming their chemical armoury. Those metabolites include anti-nutrients such as inhibitors of digestion and compounds interfering with predator's metabolism reaching as far as brain function and hormonal control Interestingly, some of these metabolites are beneficial by their inhibition of human cancer cells or by antioxidant activity that can delay ageing. Although legumes have many beneficial properties, they are not a well balanced food by themselves because of deficiencies in some essential amino acids, and should not be the sole component of the food basket. In combination with cereals that are richer in those essential amino acids which are deficient in legumes such as methionine, cysteine and tryptophan, legumes are beneficial for human health and for the world's ecology. The optimum protein quality is approximated when 60-70% cereals are mixed with 30-40% cooked legumes. This would produce a combined quality of protein comparable with meat

Despite years of scientific effort to develop useful and safe biotech crops, ideologies have prevailed and genetically modified (GM-)crops have still not been fully accepted by today's society. This leads one to reflect on the role of Science in society, on what makes scientists credible, and how scientists themselves understand the world we live in. While Science remains a black box for many of the uninitiated, scientists themselves are also generally less-interested in sociology or the economy, such that the coevolution of science and daily life is often frustrated by incomprehension or even disinterest on both sides.

The excitatory amino acid L-β-N-oxalyl-α,β-diaminopropionic acid (L-β-ODAP) in Lathyrus sativus L. is proposed as the causative agent of the neurodegenerative disease neurolathyrism. We investigated the effect of L-β-ODAP on [Ca2+]i handling, redox homeostasis, and cell death in rat spinal motor neurons. L-β-ODAP and L-glutamate triggered [Ca2+]i transients, which were inhibited by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blockers; 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide and 1-naphthyl acetylspermine, the latter specifically blocking Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. In addition, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and to a lesser extent 1-naphthyl acetylspermine, protected the neurons against cell death induced by L-β-ODAP or L-glutamate. Methionine and cysteine were also protective against neuronal cell death. We conclude that deregulation of [Ca2+]i homeostasis and oxidative stress contribute to motor neuron cell death in neurolathyrism.

Abstract Within the genus Phaseolus , Phaseolus vulgaris or the common bean is the most important grain legume for direct food use. Four other Phaseolus species are also cultivated, although at a lower scale, namely P. lunatus (lima bean), P. coccinus (scarlet runner bean), P. polyanthus (year‐long bean), and P. acutifolius , the tepary bean; and the tepary bean is the focus of this chapter. The tepary bean is seen by many as one of the most drought‐tolerant annual legume in the world. It is a minor subsistence crop and, hence, is not very important from an economic point of view. Nevertheless, it has some interesting characteristics. On the one hand, its tolerance to drought, high temperatures, and common bacterial blight has inspired breeders to explore the possibilities of crosses with the common bean, P. vulgaris , for its improvement. On the other hand, the tepary bean is the only Phaseolus species that can easily be transformed with an Agrobacterium ‐based system, and for which several classes of transgenic plants have been described, and hence its coverage in this volume.

Abstract Global rice production is constrained by bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae ( Xoo ). BLB disease incidence in West Africa was between 70–85% and yield loss in farmers’ fields was in the range of 50–90% from 2005 to 2010. In the present study, African Xoo virulence gene OPP-17 2000 DNA marker was identified and purified using randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) products from 50 Xoo isolates. Genomic DNA of 50 Xoo isolates were analyzed using OPP-17 primer in RAPD-PCR during which African Xoo virulence gene OPP-17 2000 DNA marker was identified, purified, cloned, and sequenced. Cloning and DNA sequencing of African Xoo virulence gene OPP-17 2000 DNA generated a 1953 bp nucleotide sequence consequently tagged as AXaVrg -1953. BLAST homologous analysis of the AXaVrg- 1953 sequence provides comprehensive identification of the type II secretion genes and secreted proteins, type III secretion genes and secreted proteins in African Xoo virulence gene. Phylogenetic unweighted pairgroup method arithmetic (UPGMA) analysis revealed the African AXaVrg -1953 sequence was distinct from the other Xoo virulence gene sequences from China, Japan, Korea, Germany, and the United States. This information is potentially useful for effective management of BLB disease in West Africa.