Introduction of a terminal electron sink in chloroplasts decreases leaf cell expansion associated with higher proteasome activity and lower endoreduplication.

Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell number per leaf. Proteomic analysis showed that the biogenesis of the PETC proceeded stepwise in wild-type leaves, with accumulation of light-harvesting proteins preceding that of electron transport components, which might explain the increased energy and electron transfer to oxygen and reactive oxygen species build-up at this stage. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide formation through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation was initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.

The chloroplast redox-responsive transcriptome of solanaceous plants reveals significant nuclear gene regulatory motifs associated to stress acclimation.

KEY MESSAGE: Transcriptomes of solanaceous plants expressing a plastid-targeted antioxidant protein were analysed to identify chloroplast redox networks modulating the expression of nuclear genes associated with stress acclimation. Plastid functions depend on the coordinated expression of nuclear genes, many of them associated to developmental and stress response pathways. Plastid-generated signals mediate this coordination via retrograde signaling, which includes sensing of chloroplast redox state and levels of reactive oxygen species (ROS), although it remains a poorly understood process. Chloroplast redox poise and ROS build-up can be modified by recombinant expression of a plastid-targeted antioxidant protein, i.e., cyanobacterial flavodoxin, with the resulting plants displaying increased tolerance to multiple environmental challenges. Here we analysed the transcriptomes of these flavodoxin-expressing plants to study the coordinated transcriptional responses of the nucleus to the chloroplast redox status and ROS levels during normal growth and stress responses (drought or biotic stress) in tobacco and potato, members of the economically important Solanaceae family. We compared their transcriptomes against those from stressed and mutant plants accumulating ROS in different subcellular compartments and found distinct ROS-related imprints modulated by flavodoxin expression and/or stress. By introducing our datasets in a large-scale interaction network, we identified transcriptional factors related to ROS and stress responses potentially involved in flavodoxin-associated signaling. Finally, we discovered identical cis elements in the promoters of many genes that respond to flavodoxin in the same direction as in wild-type plants under stress, suggesting a priming effect of flavodoxin before stress manifestation. The results provide a genome-wide picture illustrating the relevance of chloroplast redox status on biotic and abiotic stress responses and suggest new cis and trans targets to generate stress-tolerant solanaceous crops.

Transcriptional and Metabolic Profiling of Potato Plants Expressing a Plastid-Targeted Electron Shuttle Reveal Modulation of Genes Associated to Drought Tolerance by Chloroplast Redox Poise.

Water limitation represents the main environmental constraint affecting crop yield worldwide. Photosynthesis is a primary drought target, resulting in over-reduction of the photosynthetic electron transport chain and increased production of reactive oxygen species in plastids. Manipulation of chloroplast electron distribution by introducing alternative electron transport sinks has been shown to increase plant tolerance to multiple environmental challenges including hydric stress, suggesting that a similar strategy could be used to improve drought tolerance in crops. We show herein that the expression of the cyanobacterial electron shuttle flavodoxin in potato chloroplasts protected photosynthetic activities even at a pre-symptomatic stage of drought. Transcriptional and metabolic profiling revealed an attenuated response to the adverse condition in flavodoxin-expressing plants, correlating with their increased stress tolerance. Interestingly, 5-6% of leaf-expressed genes were affected by flavodoxin in the absence of drought, representing pathways modulated by chloroplast redox status during normal growth. About 300 of these genes potentially contribute to stress acclimation as their modulation by flavodoxin proceeds in the same direction as their drought response in wild-type plants. Tuber yield losses under chronic water limitation were mitigated in flavodoxin-expressing plants, indicating that the flavoprotein has the potential to improve major agronomic traits in potato.

Expression of a Chloroplast-Targeted Cyanobacterial Flavodoxin in Tomato Plants Increases Harvest Index by Altering Plant Size and Productivity.

Tomato is the most important horticultural crop worldwide. Domestication has led to the selection of highly fruited genotypes, and the harvest index (HI), defined as the ratio of fruit yield over total plant biomass, is usually employed as a biomarker of agronomic value. Improvement of HI might then result from increased fruit production and/or lower vegetative growth. Reduction in vegetative biomass has been accomplished in various plant species by expression of flavodoxin, an electron shuttle flavoprotein that interacts with redox-based pathways of chloroplasts including photosynthesis. However, the effect of this genetic intervention on the development of reproductive organs has not been investigated. We show herein that expression of a plastid-targeted cyanobacterial flavodoxin in tomato resulted in significant reduction of plant size affecting stems, leaves, and fruit. Decreased size correlated with smaller cells and was accompanied by higher pigment contents and photosynthetic activities per leaf cross-section. Flavodoxin accumulated in green fruit but declined with ripening. Significant increases in HI were observed in flavodoxin-expressing lines due to the production of higher fruit number per plant in smaller plants. Therefore, overall yields can be enhanced by increasing plant density in the field. Metabolic profiling of ripe red fruit showed that levels of sugars, organic acids, and amino acids were similar or higher in transgenic plants, indicating that there was no trade-off between increased HI and fruit metabolite contents in flavodoxin-expressing plants. Taken together, our results show that flavodoxin has the potential to improve major agronomic traits when introduced in tomato.