Metabolic engineering of raffinose-family oligosaccharides in the phloem reveals alterations in carbon partitioning and enhances resistance to green peach aphid

Abstract

Many plants employ energized loading strategies to accumulate osmotically-active solutes into the phloem of source organs to accentuate the hydrostatic pressure gradients that drive the flow of water, nutrients and signals from source to sinks. Proton-coupled symport of sugars from the apoplasm into the phloem symplasm is the best studied phloem-loading mechanism. As an alternative, numerous species use a polymer trapping mechanism to load through symplasm: sucrose enters the phloem through specialized plasmodesmata and is converted to raffinose-family oligosaccharides (RFOs) which accumulate because of their larger size. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which loads from the apoplasm and transports predominantly sucrose, and the fate of the sugars throughout the plant determined. Three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Three-week-old plants of both lines had RFO levels approaching 50% of total soluble sugar. RFOs were also identified in exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type (WT) leaves. Differences in starch accumulation between WT and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between WT and engineered plants, but the latter flowered slightly earlier. Finally, since the sugar composition of the translocation stream appeared altered, we tested for an impact on green peach aphid (Myzus persicae Sulzer) feeding. When given a choice between WT and transgenic plants, green peach aphids preferred settling on the WT plants. Furthermore, green peach aphid fecundity was lower on the transgenic plants compared to the WT plants. When added to an artificial diet, RFOs did not have a negative effect on aphid fecundity, suggesting that although aphid resistance in the transgenic plants is enhanced, it is not due to direct toxicity of RFO toward the insect.

Significance of galactinol and raffinose family oligosaccharide synthesis in plants

“…OE of RFO synthesizing genes and increase in RFO levels improve stress tolerance in the plant, but loss of the gene does not compromise the normal physiology. Recently, Cao et al (2013) expressed RafS/StaS/GolS directly into the ordinary companion cells of Arabidopsis to introduce RFO in the main translocation stream. Not only they observed an altered translocation stream, but they also reported lower fecundity of aphid feeding, which is not due to direct toxicity of RFO but the choice of aphids to feed on Suc-translocating plants ( Cao et al, 2013 ).…”

Expression of Sucrose Transporter cDNAs Specifically in Companion Cells Enhances Phloem Loading and Long-Distance Transport of Sucrose but Leads to an Inhibition of Growth and the Perception of a Phosphate Limitation

“…The rosettes were cut at the hypocotyl, and fresh weight was determined. The hypocotyls were then cut again under EDTA, and collection of exudates from individual plants was as described (Cao et al, 2013). Twenty-five microliters of exudate solution was mixed with 5 mL of ScintiSafe Plus 50% scintillation cocktail (Fisher Scientific), and counts per minute were determined.…”

“…To analyze 14 C transport to roots in SUT OE and wild-type plants, plants were grown on vertical plates with P supplements as described above and labeled 6 to 8 h into the 14-h light cycle as previously described (Cao et al, 2013). The distribution of 14 C in rosettes and roots of individual plants was determined by scintillation counting.…”

Metabolic Profiling of Phloem Exudates as a Tool to Improve Bread-Wheat Cultivars

“…The exudates should mainly reveal the contents of the phloem; while we cannot rule out the possibility of xylem contents and cellular leakage also occurring in the samples, such contamination would be expected to be slight [20,22]. The flag leaf and the adjacent leaf were harvested according to [21].…”