Yinghong Lu

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as "vitamin E") as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Fruit of tomato (Solanum lycopersicum), like those from many species, have been characterized to undergo a shift from partially photosynthetic to truly heterotrophic metabolism. While there is plentiful evidence for functional photosynthesis in young tomato fruit, the rates of carbon assimilation rarely exceed those of carbon dioxide release, raising the question of its role in this tissue. Here, we describe the generation and characterization of lines exhibiting a fruit-specific reduction in the expression of glutamate 1-semialdehyde aminotransferase (GSA). Despite the fact that these plants contained less GSA protein and lowered chlorophyll levels and photosynthetic activity, they were characterized by few other differences. Indeed, they displayed almost no differences in fruit size, weight, or ripening capacity and furthermore displayed few alterations in other primary or intermediary metabolites. Although GSA antisense lines were characterized by significant alterations in the expression of genes associated with photosynthesis, as well as with cell wall and amino acid metabolism, these changes were not manifested at the phenotypic level. One striking feature of the antisense plants was their seed phenotype: the transformants displayed a reduced seed set and altered morphology and metabolism at early stages of fruit development, although these differences did not affect the final seed number or fecundity. Taken together, these results suggest that fruit photosynthesis is, at least under ambient conditions, not necessary for fruit energy metabolism or development but is essential for properly timed seed development and therefore may confer an advantage under conditions of stress.

To avoid photodamage plants regulate the amount of excitation energy in the membrane at the level of the light-harvesting complexes (LHCs). It has been proposed that the energy absorbed in excess is dissipated via protein conformational changes of individual LHCs. However, the exact quenching mechanism remains unclear. Here we study the mechanism of quenching in LHCs that bind a single carotenoid species and are constitutively in a dissipative conformation. Via femtosecond spectroscopy we resolve a number of carotenoid dark states, demonstrating that the carotenoid is bound to the complex in different conformations. Some of those states act as excitation energy donors for the chlorophylls, whereas others act as quenchers. Via in silico analysis we show that structural changes of carotenoids are expected in the LHC protein domains exposed to the chloroplast lumen, where acidification triggers photoprotection in vivo. We propose that structural changes of LHCs control the conformation of the carotenoids, thus permitting access to different dark states responsible for either light harvesting or photoprotection.

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.