In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors.

Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

Biomonitoring of heavy metals and their phytoremediation by duckweeds: Advances and prospects.

Heavy metals (HMs) contamination of water bodies severely threatens human and ecosystem health. There is growing interest in the use of duckweeds for HMs biomonitoring and phytoremediation due to their fast growth, low cultivation costs, and excellent HM uptake efficiency. In this review, we summarize the current state of knowledge on duckweeds and their suitability for HM biomonitoring and phytoremediation. Duckweeds have been used for phytotoxicity assays since the 1930s. Some toxicity tests based on duckweeds have been listed in international guidelines. Duckweeds have also been recognized for their ability to facilitate HM phytoremediation in aquatic environments. Large-scale screening of duckweed germplasm optimized for HM biomonitoring and phytoremediation is still essential. We further discuss the morphological, physiological, and molecular effects of HMs on duckweeds. However, the existing data are clearly insufficient, especially in regard to dissection of the transcriptome, metabolome, proteome responses and molecular mechanisms of duckweeds under HM stresses. We also evaluate the influence of environmental factors, exogenous substances, duckweed community composition, and HM interactions on their HM sensitivity and HM accumulation, which need to be considered in practical application scenarios. Finally, we identify challenges and propose approaches for improving the effectiveness of duckweeds for bioremediation from the aspects of selection of duckweed strain, cultivation optimization, engineered duckweeds. We foresee great promise for duckweeds as phytoremediation agents, providing environmentally safe and economically efficient means for HM removal. However, the primary limiting issue is that so few researchers have recognized the outstanding advantages of duckweeds. We hope that this review can pique the interest and attention of more researchers.

A converged ubiquitin-proteasome pathway for the degradation of TOC and TOM tail-anchored receptors.

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon on the outer chloroplast membrane) and the TOM (translocon on the outer mitochondrial membrane) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA+ adenosine triphosphatase CDC48, after which a previously uncharacterized cytosolic protein, transmembrane domain (TMD)-binding protein for tail-anchored outer membrane proteins (TTOP), binds to the exposed TMDs at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin-Benson-Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2- concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

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.

Differential RNA Editing and Intron Splicing in Soybean Mitochondria during Nodulation.

Nitrogen fixation in soybean consumes a tremendous amount of energy, leading to substantial differences in energy metabolism and mitochondrial activities between nodules and uninoculated roots. While C-to-U RNA editing and intron splicing of mitochondrial transcripts are common in plant species, their roles in relation to nodule functions are still elusive. In this study, we performed RNA-seq to compare transcript profiles and RNA editing of mitochondrial genes in soybean nodules and roots. A total of 631 RNA editing sites were identified on mitochondrial transcripts, with 12% or 74 sites differentially edited among the transcripts isolated from nodules, stripped roots, and uninoculated roots. Eight out of these 74 differentially edited sites are located on the matR transcript, of which the degrees of RNA editing were the highest in the nodule sample. The degree of mitochondrial intron splicing was also examined. The splicing efficiencies of several introns in nodules and stripped roots were higher than in uninoculated roots. These include nad1 introns 2/3/4, nad4 intron 3, nad5 introns 2/3, cox2 intron 1, and ccmFc intron 1. A greater splicing efficiency of nad4 intron 1, a higher NAD4 protein abundance, and a reduction in supercomplex I + III2 were also observed in nodules, although the causal relationship between these observations requires further investigation.

Environmental Risks of Nano Zerovalent Iron for Arsenate Remediation: Impacts on Cytosolic Levels of Inorganic Phosphate and MgATP2- in Arabidopsis thaliana.

The use of nano zerovalent iron (nZVI) for arsenate (As(V)) remediation has proven effective, but full-scale injection of nZVI into the subsurface has aroused serious concerns for associated environmental risks. This study evaluated the efficacy of nZVI treatment for arsenate remediation and its potential hazards to plants using Arabidopsis thaliana grown in a hydroponic system. Biosensors for inorganic phosphate (Pi) and MgATP2- were used to monitor in vivo Pi and MgATP2- levels in plant cells. The results showed that nZVI could remove As(V) from growth media, decrease As uptake by plants, and mitigate As(V) toxicity to plants. However, excess nZVI could cause Pi starvation in plants leading to detrimental effects on plant growth. Due to the competitive adsorption of As(V) and Pi on nZVI, removing As(V) via nZVI treatment at an upstream site could relieve downstream plants from As(V) toxicity and Pi deprivation, in which case 100 mg/L of nZVI was the optimal dosage for remediation of As(V) at a concentration around 16.13 mg/L.

Transcriptomic, proteomic and metabolic changes in Arabidopsis thaliana leaves after the onset of illumination.

BACKGROUND: Light plays an important role in plant growth and development. In this study, the impact of light on physiology of 20-d-old Arabidopsis leaves was examined through transcriptomic, proteomic and metabolomic analysis. Since the energy-generating electron transport chains in chloroplasts and mitochondria are encoded by both nuclear and organellar genomes, sequencing total RNA after removal of ribosomal RNAs provides essential information on transcription of organellar genomes. The changes in the levels of ADP, ATP, NADP(+), NADPH and 41 metabolites upon illumination were also quantified. RESULTS: Upon illumination, while the transcription of the genes encoded by the plastid genome did not change significantly, the transcription of nuclear genes encoding different functional complexes in the photosystem are differentially regulated whereas members of the same complex are co-regulated with each other. The abundance of mRNAs and proteins encoded by all three genomes are, however, not always positively correlated. One such example is the negative correlation between mRNA and protein abundances of the photosystem components, which reflects the importance of post-transcriptional regulation in plant physiology. CONCLUSION: This study provides systems-wide datasets which allow plant researchers to examine the changes in leaf transcriptomes, proteomes and key metabolites upon illumination and to determine whether there are any correlations between changes in transcript and protein abundances of a particular gene or pathway upon illumination. The integration of data of the organelles and the photosystems, Calvin-Benson cycle, carbohydrate metabolism, glycolysis, the tricarboxylic acid cycle and respiratory chain, thereby provides a more complete picture to the changes in plant physiology upon illumination than has been attained to date.

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.

Comparison of Small RNA Profiles of Glycine max and Glycine soja at Early Developmental Stages.

Small RNAs, including microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs; from PHAS loci), play key roles in plant development. Cultivated soybean, Glycine max, contributes a great deal to food production, but, compared to its wild kin, Glycine soja, it may lose some genetic information during domestication. In this work, we analyzed the sRNA profiles of different tissues in both cultivated (C08) and wild soybeans (W05) at three stages of development. A total of 443 known miRNAs and 15 novel miRNAs showed varying abundances between different samples, but the miRNA profiles were generally similar in both accessions. Based on a sliding window analysis workflow that we developed, 50 PHAS loci generating 55 21-nucleotide phasiRNAs were identified in C08, and 46 phasiRNAs from 41 PHAS loci were identified in W05. In germinated seedlings, phasiRNAs were more abundant in C08 than in W05. Disease resistant TIR-NB-LRR genes constitute a very large family of PHAS loci. PhasiRNAs were also generated from several loci that encode for NAC transcription factors, Dicer-like 2 (DCL2), Pentatricopeptide Repeat (PPR), and Auxin Signaling F-box 3 (AFB3) proteins. To investigate the possible involvement of miRNAs in initiating the PHAS-phasiRNA pathway, miRNA target predictions were performed and 17 C08 miRNAs and 15 W05 miRNAs were predicted to trigger phasiRNAs biogenesis. In summary, we provide a comprehensive description of the sRNA profiles of wild versus cultivated soybeans, and discuss the possible roles of sRNAs during soybean germination.

Global small RNA analysis in fast-growing Arabidopsis thaliana with elevated concentrations of ATP and sugars.

BACKGROUND: In higher eukaryotes, small RNAs play a role in regulating gene expression. Overexpression (OE) lines of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) were shown to grow faster and exhibit higher ATP and sugar contents. Leaf microarray studies showed that many genes involved in microRNAs (miRNAs) and trans-acting siRNAs (tasiRNAs) biogenesis were significantly changed in the fast-growing lines. In this study, the sRNA profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on sRNA expression were analyzed. RESULTS: 9-13 million reads from each library were mapped to genome. miRNAs, tasiRNAs and natural antisense transcripts-generated small interfering RNAs (natsiRNAs) were identified and compared between libraries. In the leaf of OE lines, 15 known miRNAs increased in abundance and 9 miRNAs decreased in abundance, whereas in the root of OE lines, 2 known miRNAs increased in abundance and 9 miRNAs decreased in abundance. miRNAs with increased abundance in the leaf and root samples of both OE lines (miR158b and miR172a/b) were predicted to target mRNAs coding for Dof zinc finger protein and Apetala 2 (AP2) proteins, respectively. Furthermore, a significant change in the miR173-tasiRNAs-PPR/TPR network was observed in the leaves of both OE lines. CONCLUSION: In this study, the impact of high energy content on the sRNA profiles of Arabidopsis is reported. While the abundance of many stress-induced miRNAs is unaltered, the abundance of some miRNAs related to plant growth and development (miR172 and miR319) is elevated in the fast-growing lines. An induction of miR173-tasiRNAs-PPR/TPR network was also observed in the OE lines. In contrast, only few cis- and trans-natsiRNAs are altered in the fast-growing lines.

De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing.

BACKGROUND: Biofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses. RESULTS: Approximately 27 million clean "reads" filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways. CONCLUSIONS: This is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.

Global transcriptome analysis of AtPAP2--overexpressing Arabidopsis thaliana with elevated ATP.

BACKGROUND: AtPAP2 is a purple acid phosphatase that is targeted to both chloroplasts and mitochondria. Over-expression (OE) lines of AtPAP2 grew faster, produced more seeds, and contained higher leaf sucrose and glucose contents. The present study aimed to determine how high energy status affects leaf and root transcriptomes. RESULTS: ATP and ADP levels in the OE lines are 30-50% and 20-50% higher than in the wild-type (WT) plants. Global transcriptome analyses indicated that transcriptional regulation does play a role in sucrose and starch metabolism, nitrogen, potassium and iron uptake, amino acids and secondary metabolites metabolism when there is an ample supply of energy. While the transcript abundance of genes encoding protein components of photosystem I (PS I), photosystem II (PS II) and light harvesting complex I (LHCI) were unaltered, changes in transcript abundance for genes encoding proteins of LHCII are significant. The gene expressions of most enzymes of the Calvin cycle, glycolysis and the tricarboxylic acid (TCA) cycle were unaltered, as these enzymes are known to be regulated by light/redox status or allosteric modulation by the products (e.g. citrate, ATP/ADP ratio), but not at the level of transcription. CONCLUSIONS: AtPAP2 overexpression resulted in a widespread reprogramming of the transcriptome in the transgenic plants, which is characterized by changes in the carbon, nitrogen, potassium, and iron metabolism. The fast-growing AtPAP2 OE lines provide an interesting tool for studying the regulation of energy system in plant.

Movement of Lipid Droplets in the Arabidopsis Pollen Tube Is Dependent on the Actomyosin System.

The growth of pollen tubes, which depends on actin filaments, is pivotal for plant reproduction. Pharmacological experiments showed that while oryzalin and brefeldin A treatments had no significant effect on the lipid droplets (LDs) trafficking, while 2,3-butanedione monoxime (BDM), latrunculin B, SMIFH2, and cytochalasin D treatments slowed down LDs trafficking, in such a manner that only residual wobbling was observed, suggesting that trafficking of LDs in pollen tube is related to F-actin. While the trafficking of LDs in the wild-type pollen tubes and in myo11-2, myo11b1-1, myo11c1-1, and myo11c2-1 single mutants and myo11a1-1/myo11a2-1 double mutant were normal, their trafficking slowed down in a myosin-XI double knockout (myo11c1-1/myo11c2-1) mutant. These observations suggest that Myo11C1 and Myo11C2 motors are involved in LDs movement in pollen tubes, and they share functional redundancy. Hence, LDs movement in Arabidopsis pollen tubes relies on the actomyosin system.

International Collaboration on Palliative Care Development Between ASCO and the Land of Hornbills.

PURPOSE: Palliative care in Sarawak is mainly provided by health care professionals with limited formal training in palliative care. Therefore, in 2020, collaborative work between Sarawak General Hospital, University Malaysia Sarawak, and ASCO began. This study reports on the outcome of this collaboration. METHODS: The collaboration was initiated with the first ASCO Palliative Care e-course, Train the Trainer program, International Development and Education Award-Palliative Care and translation of ASCO Palliative Care Interdisciplinary Curriculum resources. RESULTS: This collaboration has resulted in the change of practice of palliative care among the oncology team of Sarawak General Hospital. CONCLUSION: It encourages more timely palliative care referrals to ensure that patients with complex physical, psychosocial, and spiritual needs have the necessary input and support from the palliative care team throughout the course of patients' illnesses.

A dual-targeted purple acid phosphatase in Arabidopsis thaliana moderates carbon metabolism and its overexpression leads to faster plant growth and higher seed yield.

• Overexpression of AtPAP2, a purple acid phosphatase (PAP) with a unique C-terminal hydrophobic motif in Arabidopsis, resulted in earlier bolting and a higher seed yield. Metabolite analysis showed that the shoots of AtPAP2 overexpression lines contained higher levels of sugars and tricarboxylic acid (TCA) metabolites. Enzyme assays showed that sucrose phosphate synthase (SPS) activity was significantly upregulated in the overexpression lines. The higher SPS activity arose from a higher level of SPS protein, and was independent of SnRK1. • AtPAP2 was found to be targeted to both plastids and mitochondria via its C-terminal hydrophobic motif. Ectopic expression of a truncated AtPAP2 without this C-terminal motif in Arabidopsis indicated that the subcellular localization of AtPAP2 is essential for its biological actions. • Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution. AtPAP2 is the first PAP shown to modulate carbon metabolism and the first shown to be dual-targeted to both plastids and mitochondria by a C-terminal targeting signal. • One PAP-like sequence carrying a hydrophobic C-terminal motif could be identified in the genome of the smallest free-living photosynthetic eukaryote, Ostreococcus tauri. This might reflect a common ancestral function of AtPAP2-like sequences in the regulation of carbon metabolism.

Bioenergetics of pollen tube growth in Arabidopsis thaliana revealed by ratiometric genetically encoded biosensors.

Pollen tube is the fastest-growing plant cell. Its polarized growth process consumes a tremendous amount of energy, which involves coordinated energy fluxes between plastids, the cytosol, and mitochondria. However, how the pollen tube obtains energy and what the biological roles of pollen plastids are in this process remain obscure. To investigate this energy-demanding process, we developed second-generation ratiometric biosensors for pyridine nucleotides which are pH insensitive between pH 7.0 to pH 8.5. By monitoring dynamic changes in ATP and NADPH concentrations and the NADH/NAD+ ratio at the subcellular level in Arabidopsis (Arabidopsis thaliana) pollen tubes, we delineate the energy metabolism that underpins pollen tube growth and illustrate how pollen plastids obtain ATP, NADPH, NADH, and acetyl-CoA for fatty acid biosynthesis. We also show that fermentation and pyruvate dehydrogenase bypass are not essential for pollen tube growth in Arabidopsis, in contrast to other plant species like tobacco and lily.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

Biochemical and molecular characterization of PvPAP3, a novel purple acid phosphatase isolated from common bean enhancing extracellular ATP utilization.

Purple acid phosphatases (PAPs) play diverse physiological roles in plants. In this study, we purified a novel PAP, PvPAP3, from the roots of common bean (Phaseolus vulgaris) grown under phosphate (Pi) starvation. PvPAP3 was identified as a 34-kD monomer acting on the specific substrate, ATP, with a broad pH range and a high heat stability. The activity of PvPAP3 was insensitive to tartrate, indicating that PvPAP3 is a PAP-like protein. Amino acid sequence alignment and phylogenetic analysis suggest that PvPAP3 belongs to the group of plant PAPs with low molecular mass. Transient expression of 35S:PvPAP3-green fluorescent protein in onion (Allium cepa) epidermal cells verified that it might anchor on plasma membrane and be secreted into apoplast. Pi starvation led to induction of PvPAP3 expression in both leaves and roots of common bean, and expression of PvPAP3 was strictly dependent on phosphorus (P) availability and duration of Pi starvation. Furthermore, induction of PvPAP3 expression was more rapid and higher in a P-efficient genotype, G19833, than in a P-inefficient genotype, DOR364, suggesting possible roles of PvPAP3 in P efficiency in bean. In vivo analysis using a transgenic hairy root system of common bean showed that both growth and P uptake of bean hairy roots from the PvPAP3 overexpression transgenic lines were significantly enhanced when ATP was supplied as the sole external P source. Taken together, our results suggest that PvPAP3 is a novel PAP that might function in the adaptation of common bean to P deficiency, possibly through enhancing utilization of extracellular ATP as a P source.

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.