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In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1-STAP1 and dTALE2-STAP2—can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Carotenoids, such as β-carotene, accumulate in chromoplasts of various fleshy fruits, awarding them with colors, aromas, and nutrients. The Orange (CmOr) gene controls β-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressing β-carotene turnover in chromoplasts. Carotenoid isomerase (CRTISO) isomerizes yellow prolycopene into red lycopene, a prerequisite for further metabolism into β-carotene. We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants, low-β (Cmor) and yofi (Cmcrtiso). The Cmor mutation in low-β caused a major transcriptomic change in the mature fruit. In contrast, the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages. These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion, but minimally by carotenoid composition in the ripe fruit. Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts. Moreover, large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-β. CmOR family proteins were found to physically interact with light-harvesting chlorophyll a–b binding proteins, suggesting a new role of CmOR for chloroplast maintenance in melon fruit. This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.