Dissecting the role of SAL1 in metabolizing the stress signaling molecule 3′-phosphoadenosine 5′-phosphate in different cell compartments

Plants possess the most highly compartmentalized eukaryotic cells. To coordinate their intracellular functions, plastids and the mitochondria are dependent on the flow of information to and from the nuclei, known as retrograde and anterograde signals. One mobile retrograde signaling molecule is the monophosphate 3 & PRIME;-phosphoadenosine 5 & PRIME;-phosphate (PAP), which is mainly produced from 3 & PRIME;-phosphoadenosine 5 & PRIME;-phosphosulfate (PAPS) in the cytosol and regulates the expression of a set of nuclear genes that modulate plant growth in response to biotic and abiotic stresses. The adenosine bisphosphate phosphatase enzyme SAL1 dephosphorylates PAP to AMP in plastids and the mitochondria, but can also rescue sal1 Arabidopsis phenotypes (PAP accumulation, leaf morphology, growth, etc.) when expressed in the cytosol and the nucleus. To understand better the roles of the SAL1 protein in chloroplasts, the mitochondria, nuclei, and the cytosol, we have attempted to complement the sal1 mutant by specifically cargoing the transgenic SAL1 protein to these four cell compartments. Overexpression of SAL1 protein targeted to the nucleus or the mitochondria alone, or co-targeted to chloroplasts and the mitochondria, complemented most aspects of the sal1 phenotypes. Notably, targeting SAL1 to chloroplasts or the cytosol did not effectively rescue the sal1 phenotypes as these transgenic lines accumulated very low levels of SAL1 protein despite overexpressing SAL1 mRNA, suggesting a possibly lower stability of the SAL1 protein in these compartments. The diverse transgenic SAL1 lines exhibited a range of PAP levels. The latter needs to reach certain thresholds in the cell for its impacts on different processes such as leaf growth, regulation of rosette morphology, sulfate homeostasis, and glucosinolate biosynthesis. Collectively, these findings provide an initial platform for further dissection of the role of the SAL1-PAP pathway in different cellular processes under stress conditions

PAPST2 Plays Critical Roles in Removing the Stress Signaling Molecule 3 '-Phosphoadenosine 5 '-Phosphate from the Cytosol and Its Subsequent Degradation in Plastids and Mitochondria

The compartmentalization of PAPS (the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate) synthesis (mainly in plastids), PAPS consumption (in the cytosol), and PAP (the stress signaling molecule 3'-phosphoadenosine 5'-phosphate) degradation (in plastids and mitochondria) requires organellar transport systems for both PAPS and PAP. The plastidial transporter PAPST1 (PAPS TRANSPORTER1) delivers newly synthesized PAPS from the stroma to the cytosol. We investigated the activity of PAPST2, the closest homolog of PAPST1, which unlike PAPST1 is targeted to both the plastids and mitochondria. Biochemical characterization in Arabidopsis thaliana revealed that PAPST2 mediates the antiport of PAP, PAPS, ATP, and ADP. Strongly increased cellular PAP levels negatively affect plant growth, as observed in the fry1 papst2 mutant, which lacks the PAP-catabolizing enzyme SALT TOLERANCE 1 and PAPST2. PAP levels were specifically elevated in the cytosol of papst2 and fiery1 papst2, but not in papst1 or fry1 papst1. PAPST1 failed to complement the papst2 mutant phenotype in mitochondria, because it likely removes PAPS from the cell, as demonstrated by the increased expression of phytosulfokine genes. Overexpression of SAL1 in mitochondria rescued the phenotype of fry1 but not fry1 papst2. Therefore, PAPST2 represents an important organellar importer of PAP, providing a piece of the puzzle in our understanding of the organelle-to-nucleus PAP retrograde signaling pathway

The Arabidopsis Thylakoid ADP/ATP Carrier TAAC Has an Additional Role in Supplying Plastidic Phosphoadenosine 5 '-Phosphosulfate to the Cytosol

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide- sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines