No abstract

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.

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.

Transgenic sugarcane plants (Saccharum officinarum L. interspecific hybrids) were regenerated from previously described cell lines with reduced neutral invertase (NI) activity. The effects that were observed in the differentiated culm tissues at different stages of maturity paralleled those observed across the growth cycle of the suspension cultures. Reduced NI activity correlated with an increase in sucrose and a decrease in hexose levels. However, the magnitude of the reduction in enzyme activity and the accompanying changes in carbohydrate metabolism were not as pronounced as in the suspension cultures. Feeding experiments with radio-labelled fructose provided evidence that the cycling of sucrose as well as the total respiration rate correlated directly with NI activity. Sucrose synthase activity was upregulated in the transgenic plants, possibly to compensate for the reduction in invertase activity. Despite this partial compensation, the respiratory rates of the transgenic lines were still significantly lower than those of the untransformed control lines. This study clearly demonstrates the importance of NI in directing carbon towards respiratory processes in the sugarcane culm. In addition, this is the first report in which data obtained from genetically modified sugarcane suspension cell cultures and their regenerated, whole-plant counterparts are compared. The observed correlations support the use of cell cultures as a model system for sugarcane internodes, which could significantly accelerate reverse genetic studies on sugarcane carbohydrate metabolism in the future.

No abstract

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.

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.

No abstract