B.J. Vorster

Proteolysis of recombinant proteins in bioengineered plant cells

Plants are increasingly used as alternative expression hosts for the production of recombinant proteins offering many advantages including higher biomass and the ability to perform post-translational modifications on complex proteins. Key challenges for optimized accumulation of recombinant proteins in a plant system still remain, including endogenous plant proteolytic activity, which may severely compromise recombinant protein stability. Several strategies have recently been applied to improve protein stability by limiting protease action such as recombinant protein production in various sub-cellular compartments or application of protease inhibitors to limit protease action. A short update on the current strategies applied is provided here, with particular focus on sub-cellular sites previously selected for recombinant protein production and the co-expression of protease inhibitors to limit protease activity.

Transfer of genetic material between the chloroplast and nucleus: how is it related to stress in plants?

BACKGROUND The presence of chloroplast-related DNA sequences in the nuclear genome is generally regarded as a relic of the process by which genes have been transferred from the chloroplast to the nucleus. The remaining chloroplast encoded genes are not identical across the plant kingdom indicating an ongoing transfer of genes from the organelle to the nucleus. SCOPE This review focuses on the active processes by which the nuclear genome might be acquiring or removing DNA sequences from the chloroplast genome. Present knowledge of the contribution to the nuclear genome of DNA originating from the chloroplast will be reviewed. In particular, the possible effects of stressful environments on the transfer of genetic material between the chloroplast and nucleus will be considered. The significance of this research and suggestions for the future research directions to identify drivers, such as stress, of the nuclear incorporation of plastid sequences are discussed. CONCLUSIONS The transfer to the nuclear genome of most of the protein-encoding functions for chloroplast-located proteins facilitates the control of gene expression. The continual transfer of fragments, including complete functional genes, from the chloroplast to the nucleus has been observed. However, the mechanisms by which the loss of functions and physical DNA elimination from the chloroplast genome following the transfer of those functions to the nucleus remains obscure. The frequency of polymorphism across chloroplast-related DNA fragments within a species will indicate the rate at which these DNA fragments are incorporated and removed from the chromosomes.

Potential use of phytocystatins in crop improvement, with a particular focus on legumes

Phytocystatins are a well-characterized class of naturally occurring protease inhibitors that function by preventing the catalysis of papain-like cysteine proteases. The action of cystatins in biotic stress resistance has been studied intensively, but relatively little is known about their functions in plant growth and defence responses to abiotic stresses, such as drought. Extreme weather events, such as drought and flooding, will have negative impacts on the yields of crop plants, particularly grain legumes. The concepts that changes in cellular protein content and composition are required for acclimation to different abiotic stresses, and that these adjustments are achieved through regulation of proteolysis, are widely accepted. However, the nature and regulation of the protein turnover machinery that underpins essential stress-induced cellular restructuring remain poorly characterized. Cysteine proteases are intrinsic to the genetic programmes that underpin plant development and senescence, but their functions in stress-induced senescence are not well defined. Transgenic plants including soybean that have been engineered to constitutively express phytocystatins show enhanced tolerance to a range of different abiotic stresses including drought, suggesting that manipulation of cysteine protease activities by altered phytocystatin expression in crop plants might be used to improve resilience and quality in the face of climate change.

Cysteine protease and cystatin expression and activity during soybean nodule development and senescence

BackgroundNodules play an important role in fixing atmospheric nitrogen for soybean growth. Premature senescence of nodules can negatively impact on nitrogen availability for plant growth and, as such, we need a better understanding of nodule development and senescence. Cysteine proteases are known to play a role in nodule senescence, but knowledge is still fragmented regarding the function their inhibitors (cystatins) during the development and senescence of soybean nodules. This study provides the first data with regard to cystatin expression during nodule development combined with biochemical characterization of their inhibition strength.ResultsSeventy nine non-redundant cysteine protease gene sequences with homology to papain, belonging to different subfamilies, and several legumain-like cysteine proteases (vacuole processing enzymes) were identified from the soybean genome assembly with eighteen of these cysteine proteases actively transcribed during nodule development and senescence. In addition, nineteen non-redundant cystatins similar to oryzacystatin-I and belonging to cystatin subgroups A and C were identified from the soybean genome assembly with seven actively transcribed in nodules. Most cystatins had preferential affinity to cathepsin L-like cysteine proteases. Transcription of cystatins Glyma05g28250, Glyma15g12211, Glyma15g36180 particularly increased during onset of senescence, possibly regulating proteolysis when nodules senesce and undergo programmed cell death. Both actively transcribed and non-actively transcribed nodule cystatins inhibited cathepsin-L- and B-like activities in different age nodules and they also inhibited papain and cathepsin-L activity when expressed and purified from bacterial cells.ConclusionsOverlap in activities and specificities of actively and non-actively transcribed cystatins raises the question if non-transcribed cystatins provide a reservoir for response to particular environments. This data might be applicable to the development of strategies to extend the active life span of nodules or prevent environmentally induced senescence.

Drought Stress Responses in Soybean Roots and Nodules

Drought is considered to be a major threat to soybean production worldwide and yet our current understanding of the effects of drought on soybean productively is largely based on studies on above-ground traits. Although the roots and root nodules are important sensors of drought, the responses of these crucial organs and their drought tolerance features remain poorly characterized. The symbiotic interaction between soybean and rhizobia facilitates atmospheric nitrogen fixation, a process that provides essential nitrogen to support plant growth and development. Symbiotic nitrogen fixation is important for sustainable agriculture, as it sustains plant growth on nitrogen-poor soils and limits fertilizer use for crop nitrogen nutrition. Recent developments have been made in our understanding of the drought impact on soybean root architecture and nodule traits, as well as underpinning transcriptome, proteome and also emerging metabolome information, with a view to improve the selection of more drought-tolerant soybean cultivars and rhizobia in the future. We conclude that the direct screening of root and nodule traits in the field as well as identification of genes, proteins and also metabolites involved in such traits will be essential in order to gain a better understanding of the regulation of root architecture, bacteroid development and lifespan in relation to drought tolerance in soybean.

Use of transgenic oryzacystatin-I-expressing plants enhances recombinant protein production.

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter–GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein which is very likely due to the reduced activity of endogenous cysteine protease.

Use of representational difference analysis for the characterization of sequence differences between date palm varieties

Abstract. Representational difference analysis was applied to subtract the genomes of the two date palm varieties, Barhee and Medjool, for identification and characterization of unique genome differences suitable for discriminating between individual plants and the two varieties. Three different DNA difference products were isolated from Barhee representing families of dispersed, repeated variable sequences present in the genome of both varieties. Several variant members of repeated DNA were detected by sequence analysis, containing base changes from C to T and G to A and short deletions. Mutated DNA sequences could be amplified in a polymerase chain reaction-based test from a much smaller number of Barhee plants than from Medjool plants allowing the differentiation between individual plants and partial discrimination between varieties.

Review: The future of cystatin engineering

Plant cystatins are naturally occurring protease inhibitors that prevent proteolysis by papain-like cysteine proteases. Their protective action against environmental stresses has been relatively well characterised. Still, there is a need to greatly improve both potency and specificity based on the current rather poor performance of cystatins in biotechnological applications. Research in creating more potent and specific cystatins, including amino acid substitutions in either conserved cystatin motifs and/or at variable amino acid sites, is reviewed. Existing gaps for better understanding of cystatin-protease interactions are further explored. Current knowledge on multi-cystatins or hybrid protease inhibitors involving cystatins as an additional option for cystatin engineering is further outlined along with the nuances of how cystatins with rather unusual amino acid sequences might actually help in cystatin engineering. Finally, future opportunities for application of cystatins are highlighted which include applications in genetically modified transgenic plants for environmental stress protection and also as nutraceuticals, as part of more nutritious food. Further opportunities might also include the possible management of diseases and disorders, often associated with lifestyle changes, and the most immediate and promising application which is inclusion into plant-based recombinant protein production platforms.

Agroinfiltration contributes to VP1 recombinant protein degradation

ABSTRACT There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.

Potential for nitrogen fixation in the fungus-growing termite symbiosis

Termites host a gut microbiota of diverse and essential symbionts that enable specialization on dead plant material; an abundant, but nutritionally imbalanced food source. To supplement the severe shortage of dietary nitrogen (N), some termite species make use of diazotrophic bacteria to fix atmospheric nitrogen (N2). Fungus-growing termites (subfamily Macrotermitinae) host a fungal exosymbiont (genus Termitomyces) that provides digestive services and the main food source for the termites. This has been thought to obviate the need for N2-fixation by bacterial symbionts. Here, we challenge this notion by performing acetylene reduction assays of live colony material to show that N2 fixation is present in two major genera (Macrotermes and Odontotermes) of fungus-growing termites. We compare and discuss fixation rates in relation to those obtained from other termites, and suggest avenues of research that may lead to a better understanding of N2 fixation in fungus-growing and other termites.