Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world.

Genome editing in agriculture and food is leading to new, improved crops and other products. Depending on the regulatory approach taken in each country or region, commercialization of these crops and products may or may not require approval from the respective regulatory authorities. This paper describes the regulatory landscape governing genome edited agriculture and food products in a selection of countries and regions.

Plant-made therapeutics: an emerging platform in South Africa.

The field of plant-made therapeutics in South Africa is well established in the form of exploitation of the country's considerable natural plant diversity, both in the use of native plants in traditional herbal medicines over many centuries, and in the more modern extraction of pharmacologically-active compounds from plants, including those known to traditional healers. In recent years, this has been added to by the use of plants for the stable or transient expression of pharmaceutically-important compounds, largely protein-based biologics and vaccines. South Africa has a well-developed plant biotechnology community, as well as a comprehensive legislative framework for the regulation of the exploitation of local botanic resources, and of genetically-modified organisms. The review explores the investigation of both conventional and recombinant plants for pharmaceutical use in South Africa, as well as describing the relevant legislative and regulatory frameworks. Potential opportunities for national projects, as well as factors limiting biopharming in South Africa are discussed.

Downregulation of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase activity in sugarcane culms enhances sucrose accumulation due to elevated hexose-phosphate levels.

Analyses of transgenic sugarcane clones with 45-95% reduced cytosolic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypical change, but significant changes were evident in in vivo metabolite levels and fluxes during internode development. In three independent transgenic lines, sucrose concentrations increased between three- and sixfold in immature internodes, compared to the levels in the wildtype control. There was an eightfold increase in the hexose-phosphate:triose-phosphate ratio in immature internodes, a significant restriction in the triose phosphate to hexose phosphate cycle and significant increase in sucrose cycling as monitored by (13)C nuclear magnetic resonance. This suggests that an increase in the hexose-phosphate concentrations resulting from a restriction in the conversion of hexose phosphates to triose phosphates drive sucrose synthesis in the young internodes. These effects became less pronounced as the tissue matured. Decreased expression of PFP also resulted in an increase of the ATP/ADP and UTP/UDP ratios, and an increase of the total uridine nucleotide and, at a later stage, the total adenine nucleotide pool, revealing strong interactions between PPi metabolism and general energy metabolism. Finally, decreased PFP leads to a reduction of PPi levels in older internodes indicating that in these developmental stages PFP acts in the gluconeogenic direction. The lowered PPi levels might also contribute to the absence of increases in sucrose contents in the more mature tissues of transgenic sugarcane with reduced PFP activity.

The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines.

Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes.

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-beta gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.

Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation.

Suspension cultures were used as a model system to investigate sucrose metabolism in four sugarcane (Saccharum spp. interspecific hybrids) cell lines transformed with antisense neutral invertase (NI) constructs. Throughout a 14-day growth cycle two cell lines in which the antisense sequence was under the control of a tandem CaMV-35S: maize ubiquitin promoter showed a strong reduction in NI activity, as well as reduced hexose and increased sucrose concentrations in comparison to the control line. In lines where the antisense NI sequence was under the control of the weaker CaMV-35S promoter alone, changes in enzyme activity and sugar concentrations were intermediate to those of the more strongly inhibited lines and the control. In comparison to the control line, a higher sucrose to hexose ratio, i.e. increased purity, was obtained in all the lines with reduced NI activity. The in vivo rate of sucrose hydrolysis was reduced in the transgenic lines, suggesting a concomitant reduction in the flux through the 'futile cycle' of sucrose breakdown and re-synthesis. Differences between the transgenic cultures and the control were most pronounced during the early stages of the growth cycle and tapered off as the cultures matured. The transgenic cultures displayed impaired growth characteristics suggesting that the growth rate of these cells was retarded because of the reduced availability of hexoses for respiration.

Molecular and kinetic characterisation of sugarcane pyrophosphate: fructose-6-phosphate 1-phosphotransferase and its possible role in the sucrose accumulation phenotype.

The amount of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in sugarcane internodal tissue is inversely correlated with sucrose content. To help elucidate this apparent role of PFP in sucrose accumulation in sugarcane we have determined its molecular and kinetic properties. Sugarcane PFP was purified 285-fold to a final specific activity of 4.23 µmol min-1 mg-1 protein. It contained two polypeptides of 63.2 and 58.0 kDa respectively, at near equal amounts that cross-reacted with potato PFP-α and -ß antiserum. In gel filtration analyses the native enzyme eluted in three peaks of 129, 245 and 511 kDa, corresponding to dimeric, tetrameric and octameric forms, respectively and fructose 2,6-bisphosphate (Fru 2,6-P2) influenced this aggregation state. Both the glycolytic (forward) and gluconeogenic (reverse) reactions had relative broad pH optima between pH 6.7 and 8.0. The Fru 2,6-P2 saturation curves were hyperbolic with approximate Ka values of 69 and 82 nm for the forward and reverse reactions, respectively. The enzyme showed hyperbolic saturation curves for all its substrates with Km values comparable with that of other plant PFP, i.e. 150, 37, 39 and 460 µM for fructose 6-phosphate, inorganic pyrophosphate, fructose 1,6-bisphosphate and inorganic phosphate, respectively. Sugarcane PFP's molecular and kinetic characteristics differed slightly from that of other plant PFP in that: (i) Fru 2,6-P2 directly induced the octameric state from the dimeric state; (ii) Fru 2,6-P2 shifted the pH optimum for the forward reaction to a slightly more basic pH; and (iii) Fru 2,6-P2 increased the Vmax for the forward and reverse reactions by similar amounts.

Sequence analysis and transcriptional profiling of two vacuolar H+ -pyrophosphatase isoforms in Vitis vinifera.

Gene expression of grapevine vacuolar H(+)-pyrophosphatase (V-PPase EC 3.6.1.1.) during fruit ripening has previously been reported. Here we report on putative multiple V-PPase isoforms in grapevine. In this study a full-length cDNA sequence with an open reading frame of 2,295 nucleotides encoding a V-PPase gene (vpp2: acc. nr. AJ557256) was cloned. Sequence analyses of the deduced amino acid residues and RT-PCR experiments indicated that Vitis vinifera L. has at least two distinct isoforms of the V-PPase gene. Bioinformatic analyses of 13 V-PPase protein sequences revealed two highly conserved motifs associated with pyrophosphate (PPi) binding and response to stress, respectively. Both V-PPase isoforms were expressed at higher levels in the late post-véraison stage of grape berry ripening. Results also showed that the expression of grapevine V-PPase was induced by cold stress.