Genome-wide association study of resistance to anthracnose in pepper (Capsicum chinense) germplasm.

BACKGROUND: Anthracnose is a fungal disease caused by Colletotrichum spp. that has a significant impact on worldwide pepper production. Colletotrichum scovillei is the most common pathogenic anthracnose-causing species in the Republic of Korea. RESULTS: The resistances of 197 pepper (Capsicum chinense) accessions deposited in Korea's National Agrobiodiversity Center were evaluated for their response against the virulent pathogens Colletotrichum acutatum isolate 'KSCa-1' and C. scovillei isolate 'Hana') in the field and in vitro methods for three consecutive years (2018 to 2020). The severity of the disease was recorded and compared between inoculation methods. Six phenotypically resistant pepper accessions were selected based on three years of disease data. All of the selected resistant pepper accessions outperformed the control resistant pepper in terms of resistance (PI 594,137). A genome-wide association study (GWAS) was carried out to identify single nucleotide polymorphisms (SNPs) associated with anthracnose resistance. An association analysis was performed using 53,518 SNPs and the disease score of the 2020 field and in vitro experiment results. Both field and in vitro experiments revealed 25 and 32 significantly associated SNPs, respectively. These SNPs were found on all chromosomes except Ch06 and Ch07 in the field experiment, whereas in the in vitro experiment they were found on all chromosomes except Ch04 and Ch11. CONCLUSION: In this study, six resistant C. chinense accessions were selected. Additionally, in this study, significantly associated SNPs were found in a gene that codes for a protein kinase receptor, such as serine/threonine-protein kinase, and other genes that are known to be involved in disease resistance. This may strengthen the role of these genes in the development of anthracnose resistance in Capsicum spp. As a result, the SNPs discovered to be strongly linked in this study can be used to identify a potential marker for selecting pepper material resistant to anthracnose, which will assist in the development of resistant varieties.

Exploring horticultural traits and disease resistance in Capsicum baccatum through segmental introgression lines.

KEY MESSAGE: Segmental introgression and advanced backcross lines were developed and validated as important tools for improving agronomically important traits in pepper, offering improved sensitivity in detecting quantitative trait loci for breeding. Segmental introgression lines (SILs) and advanced backcross lines (ABs) can accelerate genetics and genomics research and breeding in crop plants. This study presents the development of a complete collection of SILs and ABs in pepper using Capsicum annuum cv. 'CM334' as the recipient parent and Capsicum baccatum 'PBC81', which displays various agronomically important traits including powdery mildew and anthracnose resistance, as donor parent. Using embryo rescue to overcome abortion in interspecific crosses, and marker-assisted selection with genotyping-in-thousands by sequencing (GT-seq) to develop SILs and ABs containing different segments of the C. baccatum genome, we obtained 63 SILs and 44 ABs, covering 94.8% of the C. baccatum genome. We characterized them for traits including powdery mildew resistance, anthracnose resistance, anthocyanin accumulation, trichome density, plant architecture, and fruit morphology. We validated previously known loci for these traits and discovered new sources of variation and quantitative trait loci (QTLs). A total of 15 QTLs were identified, including four for anthracnose resistance with three novel loci, seven for plant architecture, and four for fruit morphology. This is the first complete collection of pepper SILs and ABs validated for agronomic traits and will enhance QTL detection and serve as valuable breeding resources. Further, these SILs and ABs will be useful for comparative genomics and to better understand the genetic mechanisms underlying important agronomic traits in pepper, ultimately leading to improved crop productivity and sustainability.

Description of Salinimicrobium tongyeongense sp. nov., isolated from seawater.

A Gram-stain-negative, non-motile by gliding and moderately halophilic rod-shaped bacterium HN-2-9-2T was isolated from seawater in Tongyeong, Republic of Korea. The strain grew at concentrations of 0.5‒7 % (w/v) NaCl, at pH 5.5‒8.5 and in a temperature range of 18‒45 °C. HN-2-9-2T shared the highest 16S rRNA gene sequence percentage with Salinimicrobium xinjiangense BH206T (98.2 %). The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridisation (dDDH) values between HN-2-9-2T and the S. xinjiangense BH206T were 76.0 %, 81.9 % and 19.7 %, respectively. The genome comprised 3 509 958 bp with a DNA G+C content of 43.0%. HN-2-9-2T contained MK-6 as the sole menaquinone. The predominant fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 3-OH, iso-C16 : 0, iso-C15 : 1G and summed feature 9, comprising iso-C17 : 1ω6c/C16 : 1 10-methyl. The polar lipids contained phosphatidylethanolamine, one unidentified phospholipid, two unidentified aminolipids, an unidentified glycolipid and six unidentified lipids. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Salinimicrobium, for which the name Salinimicrobium tongyeongense sp. nov. is proposed. The type strain is HN-2-9-2T (=KCTC 82934T=NBRC 115920T).

Description of Brachybacterium sillae sp. nov., a thermophilic bacterium isolated from a hot spring.

The taxonomic position of strain EF45031T, isolated from the Neungam Carbonate hot spring, was examined using the polyphasic taxonomic approach. Strain EF45031T shared the highest percentage of 16S rRNA gene sequence with Brachybacterium nesterenkovii CIP 104813 T (97.7%). The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between strain EF45031T and the type strains B. nesterenkovii CIP 104813 T and B. phenoliresistens Phenol-AT were 77.0%, 69.15%, 21.9% and 75.73%, 68.81%, 20.5%, respectively. Phylogenomic analysis using an up-to-date bacterial core gene (UBCG) set revealed that strain EF45031T belonged to the genus Brachybacterium. Growth occurred between 25 and 50 ℃ at pH 6.0-9.0 and could tolerate salinity up to 5% (w/v). Strain had anteiso-C15:0 and anteiso-C17:0 as major fatty acids. Menaquinone-7 (MK-7) was the predominant respiratory menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, three aminolipids, and two unidentified glycolipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid as a diagnostic diamino acid. The genome comprised 2,663,796 bp, with a G + C content of 70.9%. Stress-responsive periplasmic chaperone/protease coding genes were identified in the genome of EF45031T and were not detected in other Brachybacterium species. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Brachybacterium, for which the name Brachybacterium sillae sp. nov. is proposed. The type strain is EF45031T (= KCTC 49702 T = NBRC 115869 T).

Description of Microbacterium neungamense sp. nov. isolated from a hot spring.

The Gram-positive, nonmotile, rod-shaped bacterium EF45044T was isolated from a hot spring in Chungju, South Korea. The strain was able to grow at concentrations of 0‒5% (w/v) NaCl, at pH 6.0‒10.0 and in the temperature range of 18‒50 °C. Strain EF45044T showed the highest 16S rRNA gene sequence similarity (98.2%) with Microbacterium ketosireducens DSM 12510T, and the digital DNA‒DNA hybridization (dDDH), average amino acid identity (AAI), and average nucleotide identity (ANI) values were all lower than the accepted species threshold. Strain EF45044T contained MK‒12 and MK‒13 as the predominant respiratory quinones and anteiso‒C17:0, anteiso‒C15:0, and iso‒C16:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, and glycolipid were detected as the major polar lipids. The cell-wall peptidoglycan contained ornithine. The DNA G + C content was 71.4 mol%. Based on the polyphasic data, strain EF45044T (= KCTC 49703T) presents a novel species of the genus Microbacterium, for which the name Microbacterium neungamense sp. nov. is proposed.

Serum phosphorus and subclinical coronary atherosclerosis in asymptomatic subjects without kidney dysfunction.

BACKGROUND AND AIMS: There is limited data on the association between serum phosphorus concentration (SPC) and subclinical coronary atherosclerosis in low-risk asymptomatic subjects without kidney dysfunction. MATERIALS AND METHODS: We retrospectively analyzed 1,636 Korean individuals (mean age 52.6 ± 7.6 years; males: 712 (43.5%)) without traditional cardiovascular risk factors (CVRFs) and kidney dysfunction who voluntarily underwent coronary computed tomography angiography (CCTA) as part of a general health examination. Traditional CVRFs were defined as follows: systolic/diastolic blood pressure ≥ 140/90 mmHg, fasting blood glucose ≥ 126 mg/dL, hemoglobin A1c ≥ 6.5%, total cholesterol ≥ 240 mg/dL, low-density lipoprotein cholesterol ≥ 160 mg/dL, high-density lipoprotein cholesterol < 40 mg/dL, body mass index ≥ 25.0 kg/m2, currently smoking, and medical history of hypertension, diabetes, and hyperlipidemia. Study participants were stratified into tertiles according to their SPC levels (≤ 3.2, 3.3 - 3.6, and ≥ 3.7 mg/dL). RESULTS: 297 (18.2%) study participants had subclinical coronary atherosclerosis, characterized by any coronary plaque on CCTA. In multivariable regression analysis, the risk of subclinical coronary atherosclerosis increased in the second (odds ratio (OR): 1.629; 95% confidence interval (CI): 1.149 - 2.308; p = 0.006) and third (OR: 1.645; 95% CI: 1.093 - 2.476; p = 0.017) SPC tertiles compared to the first SPC tertile. In addition, the risk of calcified plaque increased in the second (OR: 1.605; 95% CI: 1.124 - 2.292; p = 0.009) and third (OR 1.790; 95% CI 1.179 - 2.716; p = 0.006) SPC tertiles. CONCLUSION: In low-risk asymptomatic Korean individuals without kidney dysfunction, a higher SPC level was an independent predictor of subclinical coronary atherosclerosis.

A large-scale metagenomic study for enzyme profiles using the focused identification of the NGS-based definitive enzyme research (FINDER) strategy.

Excavating the molecular details of many diverse enzymes from metagenomes remains challenging in agriculture, food, health, and environmental fields. We present a versatile method that accelerates metabolic enzyme discovery for highly selective gene capture in metagenomes using next-generation sequencing. Culture-independent enzyme mining of environmental DNA is based on a set of short identifying degenerate sequences specific for a wide range of enzyme superfamilies, followed by multiplexed DNA barcode sequencing. A strategy of 'focused identification of next-generation sequencing-based definitive enzyme research' enabled us to generate targeted enzyme datasets from metagenomes, resulting in minimal hands-on obtention of high-throughput biological diversity and potential function profiles, without being time-consuming. This method also provided a targeted inventory of predicted proteins and molecular features of metabolic activities from several metagenomic samples. We suggest that the efficiency and sensitivity of this method will accelerate the decryption of microbial diversity and the signature of proteins and their metabolism from environmental samples.

Cost-effective production of tag-less recombinant protein in Nicotiana benthamiana.

Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost-effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost-effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose-binding domain (CBM3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin-related modifier (SUMO) and SUMO-specific protease were used to remove it. This method, together with size-exclusion chromatography, enabled purification of human interleukin-6 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium-mediated transient expression. Plant-produced hIL6 (P-hIL6) contained less than 0.2 EU/µg (0.02 ng/mL) endotoxin. P-hIL6 activated the Janus kinase-signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL-21 in activated mouse CD4+ T cells. This approach is thus a powerful method for producing recombinant proteins in plants.

Fibrillin 5 Is Essential for Plastoquinone-9 Biosynthesis by Binding to Solanesyl Diphosphate Synthases in Arabidopsis.

Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. They accumulate in chromoplasts and sequester carotenoids during the development of flowers and fruits. However, little is known about the functions of fibrillins in leaf tissues. Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis thaliana. Homozygous fbn5-1 mutations were seedling-lethal, and XVE:FBN5-B transgenic plants expressing low levels of FBN5-B had a slower growth rate and were smaller than wild-type plants. In chloroplasts, FBN5-B specifically interacted with solanesyl diphosphate synthases (SPSs) 1 and 2, which biosynthesize the solanesyl moiety of PQ-9. Plants containing defective FBN5-B accumulated less PQ-9 and its cyclized product, plastochromanol-8, but the levels of tocopherols were not affected. The reduced PQ-9 content of XVE:FBN5-B transgenic plants was consistent with their lower photosynthetic performance and higher levels of hydrogen peroxide under cold stress. These results indicate that FBN5-B is required for PQ-9 biosynthesis through its interaction with SPS. Our study adds FBN5 as a structural component involved in the biosynthesis of PQ-9. FBN5 binding to the hydrophobic solanesyl moiety, which is generated by SPS1 and SPS2, in FBN5-B/SPS homodimeric complexes stimulates the enzyme activity of SPS1 and SPS2.

Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability.

Thermophilic l-arabinose isomerase (AI), which catalyzes the interconversion of l-arabinose and l-ribulose, can be used to produce d-tagatose, a sugar substitute, from d-galactose. Unlike mesophilic AIs, thermophilic AIs are highly dependent on divalent metal ions for their catalytic activity and thermostability at elevated temperatures. However, the molecular basis underlying the substrate preferences and metal requirements of multimeric AIs remains unclear. Here we report the first crystal structure of the apo and holo forms of thermophilic Geobacillus kaustophilus AI (GKAI) in hexamer form. The structures, including those of GKAI in complex with l-arabitol, and biochemical analyses revealed not only how the substrate-binding site of GKAI is formed through displacement of residues at the intersubunit interface when it is bound to Mn(2+), but also revealed the water-mediated H-bonding networks that contribute to the structural integrity of GKAI during catalysis. These observations suggest metal-mediated isomerization reactions brought about by intersubunit interactions at elevated temperatures are responsible for the distinct active site features that promote the substrate specificity and thermostability of thermophilic AIs.

Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var. frutescens.

KEY MESSAGE: We described identification, expression, subcellular localization, and functions of genes that encode fatty acid desaturase enzymes in Perilla frutescens var. frutescens. Perilla (Perilla frutescens var. frutescens) seeds contain approximately 40 % of oil, of which α-linolenic acid (18:3) comprise more than 60 % in seed oil and 56 % of total fatty acids (FAs) in leaf, respectively. In perilla, endoplasmic reticulum (ER)-localized and chloroplast-localized ω-3 FA desaturase genes (PfrFAD3 and PfrFAD7, respectively) have already been reported, however, microsomal oleate 12-desaturase gene (PfrFAD2) has not yet. Here, four perilla FA desaturase genes, PfrFAD2-1, PfrFAD2-2, PfrFAD3-2 and PfrFAD7-2, were newly identified and characterized using random amplification of complementary DNA ends and sequence data from RNAseq analysis, respectively. According to the data of transcriptome and gene cloning, perilla expresses two PfrFAD2 and PfrFAD3 genes, respectively, coding for proteins that possess three histidine boxes, transmembrane domains, and an ER retrieval motif at its C-terminal, and two chloroplast-localized ω-3 FA desaturase genes, PfrFAD7-1 and PfrFAD7-2. Arabidopsis protoplasts transformed with perilla genes fused to green fluorescence protein gene demonstrated that PfrFAD2-1 and PfrFAD3-2 were localized in the ER, and PfrFAD7-1 and PfrFAD7-2 were localized in the chloroplasts. PfrFAD2 and perilla ω-3 FA desaturases were functional in budding yeast (Saccharomyces cerevisiae) indicated by the presence of 18:2 and 16:2 in yeast harboring the PfrFAD2 gene. 18:2 supplementation of yeast harboring ω-3 FA desaturase gene led to the production of 18:3. Therefore, perilla expresses two functional FAD2 and FAD3 genes, and two chloroplast-localized ω-3 FA desaturase genes, which support an evidence that P. frutescens cultivar is allotetraploid plant.

The immediate upstream region of the 5'-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana.

The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5'-untranslated region (5'-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5'-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions -1 to -21 of the 5'-UTRs in Arabidopsis genes. In particular, the A residue in the 5'-UTR from positions -1 to -5 was required for a high-level translational efficiency. In contrast, the T residue in the 5'-UTR from positions -1 to -5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the -1 to -21 region of the 5'-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes.

Biochemical and structural characterization of a keratin-degrading M32 carboxypeptidase from Fervidobacterium islandicum AW-1.

Comparative genomics of the keratin-degrading extremophilic eubacterium Fervidobacterium islandicum AW-1 and the closely related Fervidobacterium nodosum with no keratinolytic activity suggested that the FIAW1_1600 gene encoding a carboxypeptidase (CP) plays an important role in keratin degradation. The presumptive 489 amino acid sequence of the gene showed a conserved HEXXH motif with low levels of sequence identity (<38%) to reported thermostable M32 CPs. To identify its functional role, the FIAW1_1600 gene was overexpressed in Escherichia coli, and the recombinant enzyme was purified and characterized in detail. F. islandicum AW-1 CP (FisCP) formed a homodimer with a molecular mass of 107 kDa, and its apoenzyme exhibited maximal activity at 80 °C and pH 7.0 in the presence of Co(2+). This metalloenzyme mainly cleaved the C-termini of peptides with a basic amino acid sequence. The crystal structure of FisCP at 2.2 Å resolution showed high levels of structural similarities (root-mean-square deviations of <1.7 Å) to those of other M32 CP homologs. Remarkably, the enzyme significantly enhanced the degradation of native chicken feathers. This study suggests that FisCP, a keratinolytic member of the thermostable M32 CP family, plays an important role in keratin degradation for cellular metabolism in F. islandicum AW-1.

Abscisic acid uridine diphosphate glucosyltransferases play a crucial role in abscisic acid homeostasis in Arabidopsis.

The phytohormone abscisic acid (ABA) is crucial for plant growth and adaptive responses to various stress conditions. Plants continuously adjust the ABA level to meet physiological needs, but how ABA homeostasis occurs is not fully understood. This study provides evidence that UGT71B6, an ABA uridine diphosphate glucosyltransferase (UGT), and its two closely related homologs, UGT71B7 and UGT71B8, play crucial roles in ABA homeostasis and in adaptation to dehydration, osmotic stress, and high-salinity stresses in Arabidopsis (Arabidopsis thaliana). UGT RNA interference plants that had low levels of these three UGT transcripts displayed hypersensitivity to exogenous ABA and high-salt conditions during germination and exhibited a defect in plant growth. However, the ectopic expression of UGT71B6 in the atbg1 (for ß-glucosidase) mutant background aggravated the ABA-deficient phenotype of atbg1 mutant plants. In addition, modulation of the expression of the three UGTs affects the expression of CYP707A1 to CYP707A4, which encode ABA 8'-hydroxylases; four CYP707As were expressed at higher levels in the UGT RNA interference plants but at lower levels in the UGT71B6:GFP-overexpressing plants. Based on these data, this study proposes that UGT71B6 and its two homologs play a critical role in ABA homeostasis by converting active ABA to an inactive form (abscisic acid-glucose ester) depending on intrinsic cellular and environmental conditions in plants.

Trafficking of vacuolar proteins: the crucial role of Arabidopsis vacuolar protein sorting 29 in recycling vacuolar sorting receptor.

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.

The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.

Biohydrogen production: strategies to improve process efficiency through microbial routes.

The current fossil fuel-based generation of energy has led to large-scale industrial development. However, the reliance on fossil fuels leads to the significant depletion of natural resources of buried combustible geologic deposits and to negative effects on the global climate with emissions of greenhouse gases. Accordingly, enormous efforts are directed to transition from fossil fuels to nonpolluting and renewable energy sources. One potential alternative is biohydrogen (H2), a clean energy carrier with high-energy yields; upon the combustion of H2, H2O is the only major by-product. In recent decades, the attractive and renewable characteristics of H2 led us to develop a variety of biological routes for the production of H2. Based on the mode of H2 generation, the biological routes for H2 production are categorized into four groups: photobiological fermentation, anaerobic fermentation, enzymatic and microbial electrolysis, and a combination of these processes. Thus, this review primarily focuses on the evaluation of the biological routes for the production of H2. In particular, we assess the efficiency and feasibility of these bioprocesses with respect to the factors that affect operations, and we delineate the limitations. Additionally, alternative options such as bioaugmentation, multiple process integration, and microbial electrolysis to improve process efficiency are discussed to address industrial-level applications.

Recruitment of Arf1-GDP to Golgi by Glo3p-type ArfGAPs is crucial for golgi maintenance and plant growth.

ADP-ribosylation factor1 (Arf1), a member of the small GTP-binding proteins, plays a pivotal role in protein trafficking to multiple organelles. In its GDP-bound form, Arf1 is recruited from the cytosol to organelle membranes, where it functions in vesicle-mediated protein trafficking. However, the mechanism of Arf1-GDP recruitment remains unknown. Here, we provide evidence that two Glo3p-type Arf GTPase-activating proteins (ArfGAPs), ArfGAP domain8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis (Arabidopsis thaliana). RNA interference plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNA interference plants, Arf1 was poorly recruited to the Golgi apparatus. Conversely, high levels of AGD8 and AGD9 induced Arf1 accumulation at the Golgi and suppressed Golgi disruption and inhibition of vacuolar trafficking that was caused by overexpression of AGD7. Based on these results, we propose that the Glo3p-type ArfGAPs AGD8 and AGD9 recruit Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth.

Functional identification of sorting receptors involved in trafficking of soluble lytic vacuolar proteins in vegetative cells of Arabidopsis.

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis ßFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

Exposure of iron nanoparticles to Arabidopsis thaliana enhances root elongation by triggering cell wall loosening.

In this study, we investigated the effect of nZVI on plant root elongation in Arabidopsis thaliana and showed, for the first time, that nZVI enhanced root elongation by inducing OH radical-induced cell wall loosening. Exposure of plants to 0.5 g/L nZVI enhanced root elongation by 150-200% over that in the control, and further mechanistic studies showed that this occurred via nZVI-mediated OH radical-induced cell wall loosening. The oxidation capacity of nZVI, leading to release of H2O2, allowed it to cause OH radical-induced cell wall loosening in roots. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis clearly revealed that pectin-polysaccharides in roots were degraded; they are one of the main matrix-polysaccharide-connecting and load-bearing polymers in cell walls. Rapid root elongation led to structural changes in root cell walls: reduction of cell wall thickness and a bias on the orientation of cellulose microfibrils. Additionally, the asymmetrical distribution of tensional strength resulted from the OH radical-induced cell wall loosening enhanced endocytosis. These findings emphasize that OH radical-induced cell wall loosening is important for mechanical regulation of the cell wall and provide new insights into the cellular responses of plants exposed to reactive metal nanoparticles.