Overlapping Functions of the Paralogous Proteins AtPAP2 and AtPAP9 in Arabidopsis thaliana.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), which is anchored to the outer membranes of chloroplasts and mitochondria, affects carbon metabolism by modulating the import of some preproteins into chloroplasts and mitochondria. AtPAP9 bears a 72% amino acid sequence identity with AtPAP2, and both proteins carry a hydrophobic motif at their C-termini. Here, we show that AtPAP9 is a tail-anchored protein targeted to the outer membrane of chloroplasts. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that both AtPAP9 and AtPAP2 bind to a small subunit of rubisco 1B (AtSSU1B) and a number of chloroplast proteins. Chloroplast import assays using [35S]-labeled AtSSU1B showed that like AtPAP2, AtPAP9 also plays a role in AtSSU1B import into chloroplasts. Based on these data, we propose that AtPAP9 and AtPAP2 perform overlapping roles in modulating the import of specific proteins into chloroplasts. Most plant genomes contain only one PAP-like sequence encoding a protein with a hydrophobic motif at the C-terminus. The presence of both AtPAP2 and AtPAP9 in the Arabidopsis genome may have arisen from genome duplication in Brassicaceae. Unlike AtPAP2 overexpression lines, the AtPAP9 overexpression lines did not exhibit early-bolting or high-seed-yield phenotypes. Their differential growth phenotypes could be due to the inability of AtPAP9 to be targeted to mitochondria, as the overexpression of AtPAP2 on mitochondria enhances the capacity of mitochondria to consume reducing equivalents.

Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis.

The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated.

Medical Resource Management in Emergency Hierarchical Diagnosis and Treatment Systems: A Research Framework.

The occurrence of major public health crises, like the COVID-19 epidemic, present significant challenges to healthcare systems and the management of emergency medical resources worldwide. This study, by examining the practices of emergency medical resource management in select countries during the COVID-19 epidemic, and reviewing the relevant literature, finds that emergency hierarchical diagnosis and treatment systems (EHDTSs) play a crucial role in managing emergency resources effectively. To address key issues of emergency resource management in EHDTSs, we examine the features of EHDTSs and develop a research framework for emergency resource management in EHDTSs, especially focusing on the management of emergency medical personnel and medical supplies during evolving epidemics. The research framework identifies key issues of emergency medical resource management in EHDTSs, including the sharing and scheduling of emergency medical supplies, the establishment and sharing of emergency medical supply warehouses, and the integrated dispatch of emergency medical personnel. The proposed framework not only offers insights for future research but also can facilitate better emergency medical resource management in EHDTSs during major public health emergencies.

A Balance between the Activities of Chloroplasts and Mitochondria Is Crucial for Optimal Plant Growth.

Energy metabolism in plant cells requires a balance between the activities of chloroplasts and mitochondria, as they are the producers and consumers of carbohydrates and reducing equivalents, respectively. Recently, we showed that the overexpression of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), a phosphatase dually anchored on the outer membranes of chloroplasts and mitochondria, can boost the plant growth and seed yield of Arabidopsis thaliana by coordinating the activities of both organelles. However, when AtPAP2 is solely overexpressed in chloroplasts, the growth-promoting effects are less optimal, indicating that active mitochondria are required for dissipating excess reducing equivalents from chloroplasts to maintain the optimal growth of plants. It is even more detrimental to plant productivity when AtPAP2 is solely overexpressed in mitochondria. Although these lines contain high level of adenosine triphosphate (ATP), they exhibit low leaf sucrose, low seed yield, and early senescence. These transgenic lines can be useful tools for studying how hyperactive chloroplasts or mitochondria affect the physiology of their counterparts and how they modify cellular metabolism and plant physiology.

Modulating the activities of chloroplasts and mitochondria promotes adenosine triphosphate production and plant growth.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin-Benson-Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

A PIF7-CONSTANS-Centered Molecular Regulatory Network Underlying Shade-Accelerated Flowering.

To compete with their neighbors for light and escape shaded environments, sun-loving plants have developed the shade-avoidance syndrome (SAS), a set of responses including alteration of plant architecture and initiation of early flowering and seed set. Previous studies on SAS mainly focused on dissecting molecular basis of hypocotyl elongation in seedlings under shade light; however, the molecular mechanisms underlying shade-accelerated flowering in adult plants remain unknown. In this study, we found that CONSTANS (CO) and PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) have an additive effect on shade-induced flowering, but that LONG HYPOCOTYL IN FAR-RED1 (HFR1) represses early flowering by binding to CO and PIF7 and preventing the binding of CO to the promoter of FLOWERING LOCUS T (FT) and the binding of PIF7 to the promoter of pri-MIR156E/F. Under shade, de-phosphorylated PIF7 and accumulated CO, balanced by HFR1, upregulate the expression of FT, TSF, SOC1, and SPLs to accelerate flowering. Moreover, we found that the function of PIF7 in flowering time is independent of phyA. Collectively, these regulatory interactions establish a crucial link between the light signal and genetic network that regulates flowering transition under shade.

Transgenic Arabidopsis thaliana containing increased levels of ATP and sucrose is more susceptible to Pseudomonas syringae.

Disease resistance exerts a fitness cost on plants, presumably due to the extra consumption of energy and carbon. In this study, we examined whether transgenic Arabidopsis thaliana with increased levels of ATP and sucrose is more resistant or susceptible to pathogen infection. Lines of A. thaliana over-expressing purple acid phosphatase 2 (AtPAP2) (OE lines) contain increased levels of ATP and sucrose, with improved growth rate and seed production. Compared to wild type (WT) and pap2 lines, the OE lines were more susceptible to several Pseudomonas syringae pv. tomato (Pst) strains carrying AvrRpm1, AvrRpt2 AvrRps4, AvrPtoB, HrcC and WT strain DC3000. The increased susceptibility of the OE lines to Pst strains cannot solely be attributed to the suppressed expression of R-genes but must also be attributed to the suppression of downstream signaling components, such as MOS2, EDS1 and EDS5. Before infection, the levels of salicylic acid (SA) and jasmonic acid (JA) precursor OPDA were similar in the leaves of OE, pap2 and WT plants, whereas the levels of JA and its derivative JA-Ile were significantly lower in the leaves of OE lines and higher in the pap2 line. The expression of JA marker defense gene PDF1.2 was up-regulated in the OE lines compared to the WT prior to Pst DC3000 infection, but its expression was lower in the OE lines after infection. In summary, high fitness Arabidopsis thaliana exhibited altered JA metabolism and broad suppression of R-genes and downstream genes as well as a higher susceptibility to Pst infections.

AtPAP2 modulates the import of the small subunit of Rubisco into chloroplasts.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.

AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria.

To date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. Using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (OM) of chloroplasts and mitochondria and exposed its N-terminal enzymatic domain to the cytosol. It was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. Mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.