Development of strategies for genetic manipulation and fine-tuning of a chloroplast retrograde signal 3'-phosphoadenosine 5'-phosphate.

Homeostasis of metabolism and regulation of stress-signaling pathways are important for plant growth. The metabolite 3'-phosphoadenosine-5'-phosphate (PAP) plays dual roles as a chloroplast retrograde signal during drought and high light stress, as well as a toxic by-product of secondary sulfur metabolism, and thus, its levels are regulated by the chloroplastic phosphatase, SAL1. Constitutive PAP accumulation in mutants improves drought tolerance but can impair growth and alter rosette morphology. Therefore, it is of interest to derive strategies to enable controlled and targeted PAP manipulation that could enhance drought tolerance while minimizing the negative effects on plant growth. We systematically tested the potential and efficiency of multiple established transgenic manipulation tools in altering PAP levels in Arabidopsis. Dexamethasone (dex)-inducible silencing of via hpRNAi [pOpOff:hpRNAi] yielded reduction in transcript and protein levels, yet failed to significantly induce PAP accumulation. Surprisingly, this was not due to insufficient silencing of the inducible system, as constitutive silencing using a strong promoter to drive hpRNAi and amiRNA targeting the transcript also failed to increase PAP content or induce a -like plant morphology despite significantly reducing the transcript levels. In contrast, using dex-inducible expression of cDNA to complement an Arabidopsis mutant successfully modulated PAP levels and restored rosette growth in a dosage-dependent manner. Results from this inducible complementation system indicate that plants with intermediate PAP levels could have improved rosette growth without compromising its drought tolerance. Additionally, preliminary evidence suggests that cDNA driven by promoters of genes expressed specifically during early developmental stages such as () could be another potential strategy for studying and optimizing PAP levels and drought tolerance while alleviating the negative impact of PAP on plant growth in . Thus, we have identified ways that can allow future dissection into multiple aspects of stress and developmental regulation mediated by this chloroplast signal.