The production of human glucocerebrosidase in glyco‐engineered Nicotiana benthamiana plants

For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (NbGNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this NbGNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in NbGNTI‐RNAi plants (GC^gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC^gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI‐RNAi line, producing GC, was stable and the NbGNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.     

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus‐based vector (clbvINpr‐AtFT and clbvINpr‐CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr‐AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.     

PARylation of the forkhead‐associated domain protein DAWDLE regulates plant immunity

Protein poly(ADP‐ribosyl)ation (PARylation) primarily catalyzed by poly(ADP‐ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead‐associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non‐adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis, respectively.     

Wetland ecosystem comparison using a suite of plant assessment measures

In light of extensive human impact on wetlands it is necessary that we develop an effective way to monitor the effects of impact in order to prevent further destruction. One method is plant community assessment, specifically Floristic Quality Assessment (FQA), which is common, but can be subjective. In this case study, we implement FQA, as well as specific morphological and chemical assessment measures over a two-year period in order to compare two wetlands in the Lake George watershed in the Adirondack mountains and their response to human impact. While the wetlands studied demonstrated very different water chemistry profiles makeups, FQA did not reveal substantial differences between plant communities. However, more specific analyses of plant morphology and tissue chemistry did reveal significant differences that reflected the level of impact at these two sites. Namely, the simple plant Lemna minor had consistently shorter roots and Nuphar lutea contained higher amounts of nitrogen in above ground tissues when growing in an anthropogenically impacted wetland. We suggest that FQA and specific plant morphology and tissue chemistry measurements be performed concurrently to provide indication of both long- and short-term effects of human impact in wetland ecosystems.     

Effect of light environment on intra-specific variation in herbivory in the carnivorous plant Pinguicula moranensis (Lentibulariaceae)

Identifying the factors that affect a plant’s probability of being found and damaged by herbivores has been a central topic in the study of herbivory. Although herbivory could have important negative consequences on carnivorous plants, their interaction with herbivores remains largely unexplored. We evaluated the effect of spatial variation in light environment (sunny, shade and full-shade sites) on the pattern of leaf herbivory and florivory of the carnivorous plant Pinguicula moranensis. Plants’ overall probability of leaf damage was high (74.24%). Mean herbivory was four times higher in the sunny and shade sites than the observed in the full-shade site. Nearly 8% of plants suffered damage to reproductive structures, although the probability of florivory was similar among sites. Discussion addressed the inter-site variation in mean herbivory considering the effect of light exposure and the impact that herbivory could have on fitness components of this carnivorous plant.     

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Red mud (RM)-Induced enhancement of iron plaque formation reduces arsenic and metal accumulation in two wetland plant species

Human activities have resulted in arsenic (As) and heavy metals accumulation in paddy soils in China. Phytoremediation has been suggested as an effective and low-cost method to clean up contaminated soils. A combined soil-sand pot experiment was conducted to investigate the influence of red mud (RM) supply on iron plaque formation and As and heavy metal accumulation in two wetland plant species (Cyperus alternifolius Rottb., Echinodorus amazonicus Rataj), using As and heavy metals polluted paddy soil combined with three rates of RM application (0, 2%, 5%). The results showed that RM supply significantly decreased As and heavy metals accumulation in shoots of the two plants due to the decrease of As and heavy metal availability and the enhancement of the formation of iron plaque on the root surface and in the rhizosphere. Both wetland plants supplied with RM tended to have more Fe plaque, higher As and heavy metals on roots and in their rhizospheres, and were more tolerant of As and heavy metal toxicity. The results suggest that RM-induced enhancement of the formation of iron plaque on the root surface and in the rhizosphere of wetland plants may be significant for remediation of soils contaminated with As and heavy metals.