Charles Goulet

Preventing unintended proteolysis in plant protein biofactories.

Summary Numerous reports have been published over the last decade assessing the potential of plants as useful hosts for the heterologous expression of clinically useful proteins. Significant progress has been made, in particular, in optimizing transgene transcription and translation in plants, and in elucidating the complex post‐translational modifications of proteins typical of the plant cell machinery. In this article, we address the important issue of recombinant protein degradation in plant expression platforms, which directly impacts on the final yield, homogeneity and overall quality of the resulting protein product. Unlike several more stable and structurally less complex pharmaceuticals, recombinant proteins present a natural tendency to structural heterogeneity, resulting in part from the inherent instability of polypeptide chains expressed in heterologous environments. Proteolytic processing, notably, may dramatically alter the structural integrity and overall accumulation of recombinant proteins in plant expression systems, both in planta during expression and ex planta after extraction. In this article, we describe the current strategies proposed to minimize protein hydrolysis in plant protein factories, including organ‐specific transgene expression, organelle‐specific protein targeting, the grafting of stabilizing protein domains to labile proteins, protein secretion in natural fluids and the co‐expression of companion protease inhibitors.

SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato

The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C(13) cyclohexenone and C(14) mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source-sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.

The chemical interactions underlying tomato flavor preferences.

Although human perception of food flavors involves integration of multiple sensory inputs, the most salient sensations are taste and olfaction. Ortho- and retronasal olfaction are particularly crucial to flavor because they provide the qualitative diversity so important to identify safe versus dangerous foods. Historically, flavor research has prioritized aroma volatiles present at levels exceeding the orthonasally measured odor threshold, ignoring the variation in the rate at which odor intensities grow above threshold. Furthermore, the chemical composition of a food in itself tells us very little about whether or not that food will be liked. Clearly, alternative approaches are needed to elucidate flavor chemistry. Here we use targeted metabolomics and natural variation in flavor-associated sugars, acids, and aroma volatiles to evaluate the chemistry of tomato fruits, creating a predictive and testable model of liking. This nontraditional approach provides novel insights into flavor chemistry, the interactions between taste and retronasal olfaction, and a paradigm for enhancing liking of natural products. Some of the most abundant volatiles do not contribute to consumer liking, whereas other less abundant ones do. Aroma volatiles make contributions to perceived sweetness independent of sugar concentration, suggesting a novel way to increase perception of sweetness without adding sugar.

Diverse Roles of Strigolactone Signaling in Maize Architecture and the Uncoupling of a Branching-Specific Subnetwork

Strigolactones (SLs) control lateral branching in diverse species by regulating transcription factors orthologous to Teosinte branched1 (Tb1). In maize (Zea mays), however, selection for a strong central stalk during domestication is attributed primarily to the Tb1 locus, leaving the architectural roles of SLs unclear. To determine how this signaling network is altered in maize, we first examined effects of a knockout mutation in an essential SL biosynthetic gene that encodes CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8), then tested interactions between SL signaling and Tb1. Comparative genome analysis revealed that maize depends on a single CCD8 gene (ZmCCD8), unlike other panicoid grasses that have multiple CCD8 paralogs. Function of ZmCCD8 was confirmed by transgenic complementation of Arabidopsis (Arabidopsis thaliana) max4 (ccd8) and by phenotypic rescue of the maize mutant (zmccd8::Ds) using a synthetic SL (GR24). Analysis of the zmccd8 mutant revealed a modest increase in branching that contrasted with prominent pleiotropic changes that include (1) marked reduction in stem diameter, (2) reduced elongation of internodes (independent of carbon supply), and (3) a pronounced delay in development of the centrally important, nodal system of adventitious roots. Analysis of the tb1 zmccd8 double mutant revealed that Tb1 functions in an SL-independent subnetwork that is not required for the other diverse roles of SL in development. Our findings indicate that in maize, uncoupling of the Tb1 subnetwork from SL signaling has profoundly altered the balance between conserved roles of SLs in branching and diverse aspects of plant architecture.

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.

A companion protease inhibitor for the protection of cytosol‐targeted recombinant proteins in plants

We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.

2-DE proteome maps for the leaf apoplast of Nicotiana benthamiana

We provide 2‐D gel reference maps for the apoplastic proteome of Nicotiana benthamiana leaves infiltrated or not with the bacterial gene vector Agrobacterium tumefaciens. About 90 proteins were analyzed by LC‐MS/MS for identification and function assignment. We show, overall, an effective response of the plant to agroinfiltration involving a specific, cell wall maintenance‐independent up‐regulation of defense protein secretion. The proteome maps described should be a useful tool for systemic studies on plant–pathogen interactions or cell wall metabolism. They also should prove useful for the monitoring of secreted recombinant proteins and their possible pleiotropic effects along the cell secretory pathway of N. benthamiana leaves used as an expression platform for clinically useful proteins.

Role of an esterase in flavor volatile variation within the tomato clade.

Tomato flavor is dependent upon a complex mixture of volatiles including multiple acetate esters. Red-fruited species of the tomato clade accumulate a relatively low content of acetate esters in comparison with the green-fruited species. We show that the difference in volatile ester content between the red- and green-fruited species is associated with insertion of a retrotransposon adjacent to the most enzymatically active member of a family of esterases. This insertion causes higher expression of the esterase, resulting in the reduced levels of multiple esters that are negatively correlated with human preferences for tomato. The insertion was evolutionarily fixed in the red-fruited species, suggesting that high expression of the esterase and consequent low ester content may provide an adaptive advantage in the ancestor of the red-fruited species. These results illustrate at a molecular level how closely related species exhibit major differences in volatile production by altering a volatile-associated catabolic activity.

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect-resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad-spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI-expressing and control (comparator) lines, followed by a two-dimensional gel electrophoresis (2-DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI-expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2-DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2-DE showed unaltered deposition patterns in SlCDI-expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI-expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel-generation transgenic plants.

A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plants.

We describe a SELDI‐TOF MS procedure for the rapid detection and quantitation of low‐molecular‐weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens‐mediated transformation, and then used as test material for the analyses. Real‐time RT‐PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI‐TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.