A mutation in Arabidopsis SAL1 alters its in vitro activity against IP3 and delays developmental leaf senescence in association with lower ROS levels.

KEY MESSAGE: Our manuscript is the first to find a link between activity of SAL1/OLD101 against IP3 and plant leaf senescence regulation and ROS levels assigning a potential biological role for IP3. Leaf senescence is a genetically programmed process that limits the longevity of a leaf. We identified and analyzed the recessive Arabidopsis stay-green mutation onset of leaf death 101 (old101). Developmental leaf longevity is extended in old101 plants, which coincided with higher peroxidase activity and decreased H2O2 levels in young 10-day-old, but not 25-day-old plants. The old101 phenotype is caused by a point mutation in SAL1, which encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity. SAL1 activity is highly specific for its substrates 3-polyadenosine 5-phosphate (PAP) and inositol 1, 4, 5-trisphosphate (IP3), where it removes the 1-phosphate group from the IP3 second messenger. The in vitro activity of recombinant old101 protein against its substrate IP3 was 2.5-fold lower than that of wild type SAL1 protein. However, the in vitro activity of recombinant old101 mutant protein against PAP remained the same as that of the wild type SAL1 protein. The results open the possibility that the activity of SAL1 against IP3 may affect the redox balance of young seedlings and that this delays the onset of leaf senescence.

The PAP/SAL1 retrograde signaling pathway is involved in iron homeostasis.

KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.

Secondary sulfur metabolism in cellular signalling and oxidative stress responses.

The sulfur metabolism pathway in plants produces a variety of compounds that are central to the acclimation response to oxidative stresses such as drought and high light. Primary sulfur assimilation provides the amino acid cysteine, which is utilized in protein synthesis and as a precursor for the cellular redox buffer glutathione. In contrast, the secondary sulfur metabolism pathway produces sulfated compounds such as glucosinolates and sulfated peptides, as well as a corresponding by-product 3'-phosphoadenosine 5'-phosphate (PAP). Emerging evidence over the past decade has shown that secondary sulfur metabolism also has a crucial engagement during oxidative stress. This occurs across various cellular, tissue, and organismal levels including chloroplast-to-nucleus retrograde signalling events mediated by PAP, modulation of hormonal signalling by sulfated compounds and PAP, control of physiological responses such as stomatal closure, and potential regulation of plant growth. In this review, we examine the contribution of the different components of plant secondary metabolism to oxidative stress homeostasis, and how this pathway is metabolically regulated. We further outline the key outstanding questions in the field that are necessary to understand how and why this 'specialized' metabolic pathway plays significant roles in plant oxidative stress tolerance.

A SAL1 Loss-of-Function Arabidopsis Mutant Exhibits Enhanced Cadmium Tolerance in Association with Alleviation of Endoplasmic Reticulum Stress.

SAL1, as a negative regulator of stress response signaling, has been studied extensively for its role in plant response to environmental stresses. However, the role of SAL1 in cadmium (Cd) stress response and the underlying mechanism is still unclear. Using an Arabidopsis thaliana loss-of-function mutant of SAL1, we assessed Cd resistance and further explored the Cd toxicity mechanism through analysis of the endoplasmic reticulum (ER) stress response. The loss of SAL1 function greatly improved Cd tolerance and significantly attenuated ER stress in Arabidopsis. Exposure to Cd induced an ER stress response in Arabidopsis as evidenced by unconventional splicing of AtbZIP60 and up-regulation of ER stress-responsive genes. Damage caused by Cd was markedly reduced in the ER stress response double mutant bzip28 bzip60 or by application of the ER stress-alleviating chemical agents, tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (4-PBA), in wild-type plants. The Cd-induced ER stress in Arabidopsis was also alleviated by loss of function of SAL1. These results identified SAL1 as a new component mediating Cd toxicity and established the role of the ER stress response in Cd toxicity. Additionally, the attenuated ER stress in the sal1 mutant might also shed new light on the mechanism of diverse abiotic stress resistance in the SAL1 loss-of-function mutants.

Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.

Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

CRISPR-Cas9 Gene Editing of the Sal1 Gene Family in Wheat.

The highly conserved Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted. Precise gene editing techniques were used to generate Sal1 mutants in hexaploid bread wheat. The CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) Cas9 system with three guide RNAs (gRNAs) was used to inactivate six Sal1 homologous genes within the Bobwhite wheat genome. The resulting mutant wheat plants with all their Sal1 genes disabled had slimmer stems, had a modest reduction in biomass and senesced more slowly in water limiting conditions, but did not exhibit improved yield under drought conditions. Our results show that multiplexed gRNAs enabled effective targeted gene editing of the Sal1 gene family in hexaploid wheat. These Sal1 mutant wheat plants will be a resource for further research studying the function of this gene family in wheat.

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'-phosphoadenosine 5'-phosphate (PAP), which is mainly produced from 3'-phosphoadenosine 5'-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.

PAP/SAL1 retrograde signaling pathway modulates iron deficiency response in alkaline soils.

Iron (Fe) is an essential micronutrient for plants and is present abundantly in the Earth's crust. However, Fe bioavailability in alkaline soils is low due to the decreased solubility of the ferric ions. Previously, we have demonstrated the relationship between the PAP/SAL1 retrograde signaling pathway, the activity of Strategy I Fe uptake genes (FIT, FRO2, IRT1), and ethylene signaling. In this work, we have characterized mutant lines that are deficient in this retrograde signaling pathway and their ability to grow in alkaline soils. This adverse growth condition caused less impact on mutant plants, which showed less reduced rosette area, and higher carotenoid, chlorophyll and Fe content than wild-type plants. Several genes involved in the biosynthesis and excretion of secondary metabolites derived from the phenylpropanoid pathway, which improve Fe uptake, were elevated in mutant plants. Finally, we observed an increase in excreted fluorescent phenolic compounds in mutant lines compared to wild-type plants. In this way, PAP/SAL1 mutants showed alterations in the biosynthesis of metabolites that mobilize Fe, which ultimately improved these plants ability to grow in alkaline soils. Results agree with the existence of a link between the PAP/SAL1 retrograde signaling pathway and the regulation of Fe deficiency responses in Arabidopsis.

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 sal1 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 SAL1 via hpRNAi [pOpOff:SAL1hpRNAi] yielded reduction in SAL1 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 SAL1 transcript also failed to increase PAP content or induce a sal1-like plant morphology despite significantly reducing the SAL1 transcript levels. In contrast, using dex-inducible expression of SAL1 cDNA to complement an Arabidopsis sal1 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 SAL1 cDNA driven by promoters of genes expressed specifically during early developmental stages such as ABA-Insensitive 3 (ABI3) 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 sal1. Thus, we have identified ways that can allow future dissection into multiple aspects of stress and developmental regulation mediated by this chloroplast signal.