Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles.

BACKGROUND: In common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles. RESULTS: By integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7 OE, Glu-1Bx13, Glu-1Bx14 (-) , Glu-1Bx14 (+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14 (+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14 (+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14 (-), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14 (+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis. CONCLUSIONS: Seven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat.

Generation of Wheat Near-Isogenic Lines Overexpressing 1Bx7 Glutenin with Increased Protein Contents and SDS-Sedimentation Values.

Overexpression of Glu-1Bx7 via allele 1Bx7OE significantly contributes to high dough strength in some wheat varieties and is useful for improving wheat quality. However, the proportion of wheat varieties containing Bx7OE is quite low. In this study, four cultivars containing 1Bx7OE were selected, and among the selected varieties, Chisholm (1Ax2*, 1Bx7OE + 1By8*, and 1Dx5 + 1Dx10) was crossed with Keumkang, a wheat variety that contains 1Bx7 (1Ax2*, 1Bx7 + 1By8, and 1Dx5 + 1Dx10). SDS-PAGE and UPLC analyses showed that the expression of the high-molecular-weight glutenin subunit (HMW-GS) 1Bx7 was significantly higher in NILs (1Ax2*, 1Bx7OE + 1By8*, and 1Dx5 + 1Dx10) compared with that in Keumkang. Wheat quality was analyzed with near infrared reflectance spectroscopy by measuring the protein content and SDS-sedimentation of NILs. The protein content of NILs (12.94%) was 21.65% higher than that of Chisholm (10.63%) and 4.54% higher than that of Keumkang (12.37%). In addition, the SDS-sedimentation value of NILs (44.29 mL) was 14.97% and 16.44% higher than that of Keumkang (38.52 mL) and Chisholm (38.03 mL), respectively. This study predicts that the quality of domestic wheat can be improved by crossbreeding with 1Bx7OE-containing cultivars.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Genome-Wide Association Study of Arabinoxylan Content from a 562 Hexaploid Wheat Collection.

The selection of wheat varieties with high arabinoxylan (AX) levels could effectively improve the daily consumption of dietary fiber. However, studies on the selection of markers for AX levels are scarce. This study analyzed AX levels in 562 wheat genotypes collected from 46 countries using a GWAS with the BLINK model in the GAPIT3. Wheat genotypes were classified into eight subpopulations that exhibited high genetic differentiation based on 31,926 SNP loci. Eight candidate genes were identified, among which those encoding F-box domain-containing proteins, disease resistance protein RPM1, and bZIP transcription factor 29 highly correlated with AX levels. The AX level was higher in the adenine allele than in the guanine alleles of these genes in the wheat collection. In addition, the AX level was approximately 10% higher in 3 adenine combinations than 2 guanine, 1 adenine, and 3 guanine combinations in genotypes of three genes (F-box domain-containing proteins, RPM1, and bZIP transcription factor 29). The adenine allele, present in 97.46% of AX-95086356 SNP, exhibited a high correlation with AX levels following classification by country. Notably, the East Asian wheat genotypes contain high adenine alleles in three genes. These results highlight the potential of these three SNPs to serve as selectable markers for high AX content.

Comparison of Gene Expression Changes in Three Wheat Varieties with Different Susceptibilities to Heat Stress Using RNA-Seq Analysis.

Wheat is highly susceptible to heat stress, which significantly reduces grain yield. In this study, we used RNA-seq technology to analyze the transcript expression at three different time-points after heat treatment in three cultivars differing in their susceptibility to heat stress: Jopum, Keumkang, and Olgeuru. A total of 11,751, 8850, and 14,711; 10,959, 7946, and 14,205; and 22,895, 13,060, and 19,408 differentially-expressed genes (log2 fold-change > 1 and FDR (padj) < 0.05) were identified in Jopum, Keumkang, and Olgeuru in the control vs. 6-h, in the control vs. 12-h, and in the 6-h vs. 12-h heat treatment, respectively. Functional enrichment analysis showed that the biological processes for DEGs, such as the cellular response to heat and oxidative stress­and including the removal of superoxide radicals and the positive regulation of superoxide dismutase activity­were significantly enriched among the three comparisons in all three cultivars. Furthermore, we investigated the differential expression patterns of reactive oxygen species (ROS)-scavenging enzymes, heat shock proteins, and heat-stress transcription factors using qRT-PCR to confirm the differences in gene expression among the three varieties under heat stress. This study contributes to a better understanding of the wheat heat-stress response at the early growth stage and the varietal differences in heat tolerance.

Plant Growth Promotion by Two Volatile Organic Compounds Emitted From the Fungus Cladosporium halotolerans NGPF1.

Microbial volatiles have beneficial roles in the agricultural ecological system, enhancing plant growth and inducing systemic resistance against plant pathogens without being hazardous to the environment. The interactions of plant and fungal volatiles have been extensively studied, but there is limited research specifically elucidating the effects of distinct volatile organic compounds (VOCs) on plant growth promotion. The current study was conducted to investigate the impact of VOCs from Cladosporium halotolerans NGPF1 on plant growth, and to elucidate the mechanisms for the plant growth-promoting (PGP) activity of these VOCs. The VOCs from C. halotolerans NGPF1 significantly promoted plant growth compared with the control, and this PGP activity of the VOCs was culture medium-dependent. Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) identified two VOC structures with profiles that differed depending on the culture medium. The two compounds that were only produced in potato dextrose (PD) medium were identified as 2-methyl-butanal and 3-methyl-butanal, and both modulated plant growth promotion and root system development. The PGP effects of the identified synthetic compounds were analyzed individually and in blends using N. benthamiana plants. A blend of the two VOCs enhanced growth promotion and root system development compared with the individual compounds. Furthermore, real-time PCR revealed markedly increased expression of genes involved in auxin, expansin, and gibberellin biosynthesis and metabolism in plant leaves exposed to the two volatile blends, while cytokinin and ethylene expression levels were decreased or similar in comparison with the control. These findings demonstrate that naturally occurring fungal VOCs can induce plant growth promotion and provide new insights into the mechanism of PGP activity. The application of stimulatory volatiles for growth enhancement could be used in the agricultural industry to increase crop yield.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types.

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.

Temporal and Spatial Expression Analysis of Shoot-Regeneration Regulatory Genes during the Adventitious Shoot Formation in Hypocotyl and Cotyledon Explants of Tomato (CV. Micro-Tom).

Enhancing the competence for plant regeneration in tissue culture studies is an important issue not only for efficient genetic transformation of commercial crops but also for the reproducibility of scientific reports. In this study, we investigated optimization of several tissue culture conditions including plant growth regulators, types and ages of explants, culture densities, and plant position in order to improve the competence of adventitious shoot formation of the tomato (Solanum lycopersicum cv. Micro-Tom). In addition, we examined the differential expression of D-type cyclin (CYCD3-1) and several shoot regeneration regulatory genes from hypocotyl and cotyledon explants of tomato during shoot organogenesis. A treatment of 1 mg L-1 Zeatin and 0.1 mg L-1 Indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium containing 3% sucrose was optimal for adventitious shoot formation from hypocotyl and cotyledon explants. The younger explants exhibited more shoot formation regardless of explant types. Additionally, those closest to the shoot apical meristem produced more shoots compared to the other regions in the hypocotyl and the cotyledon explants. Gene expression of CYCD3-1, SHOOT MERISTEMLESS (STM), and cytokinin dependent WUSCHEL (WUS) was significantly higher in younger explants than in older ones. Furthermore, an increase in CYCD3-1, STM, and WUS expression was evident at the distal part of hypocotyls and the proximal part of cotyledons compared to other regions. These differential gene expression profiles exhibited good agreement with the results of shoot formation obtained from diverse explants of tomato. These results suggest that temporal and spatial gene expression of shoot regeneration regulatory genes plays an important role in enhancing the competence and the reproducibility of adventitious shoot formation from tomato explants.

Potential of Pantoea dispersa as an effective biocontrol agent for black rot in sweet potato.

Biocontrol offers a promising alternative to synthetic fungicides for the control of a variety of pre- and post-harvest diseases of crops. Black rot, which is caused by the pathogenic fungus Ceratocytis fimbriata, is the most destructive post-harvest disease of sweet potato, but little is currently known about potential biocontrol agents for this fungus. Here, we isolated several microorganisms from the tuberous roots and shoots of field-grown sweet potato plants, and analyzed their ribosomal RNA gene sequences. The microorganisms belonging to the genus Pantoea made up a major portion of the microbes residing within the sweet potato plants, and fluorescence microscopy showed these microbes colonized the intercellular spaces of the vascular tissue in the sweet potato stems. Four P. dispersa strains strongly inhibited C. fimbriata mycelium growth and spore germination, and altered the morphology of the fungal hyphae. The detection of dead C. fimbriata cells using Evans blue staining suggested that these P. dispersa strains have fungicidal rather than fungistatic activity. Furthermore, P. dispersa strains significantly inhibited C. fimbriata growth on the leaves and tuberous roots of a susceptible sweet potato cultivar ("Yulmi"). These findings suggest that P. dispersa strains could inhibit black rot in sweet potato plants, highlighting their potential as biocontrol agents.

Development of fast and sensitive protocols for the detection of viral pathogens using a small portable convection PCR platform.

One of the most crucial steps for preventing viral pandemics is the early detection of the causative virus on site. Various molecular and immunological approaches have been developed for virus detection. In this study, we investigated the utility of the recently introduced convection polymerase chain reaction (cPCR) platform for the rapid and sensitive detection of various animal viruses in the field, including the foot-and-mouth disease virus (FMDV) and avian influenza viruses (AIVs). Primer sets were designed to simultaneously detect two highly conserved regions of the FMDV, including the 5' untranslated region (5'-UTR) and 3D gene, and to specifically amplify the NP and hemagglutinin (HA) genes of H5 and H9 subtypes of AIVs. The portable cPCR system was able to amplify from as low as 1 to 10 copies of viral cDNAs in the singleplex mode and 10 to 100 copies of viral cDNAs in the duplex mode within 21 min. Thus, our data suggest that the cPCR protocols developed in this study are highly sensitive and enable quick detection of animal viruses in biological samples.

Neobacillus endophyticus sp. nov., an endophytic bacterium isolated from Selaginella involvens roots.

A Gram-stain-positive, facultatively anaerobic, rod-shaped, endospore-forming, oxidase-positive, and catalase-negative strain designated as BRMEA1T was isolated from the surface-sterilized Selaginella involvens roots. Growth of strain BRMEA1T was found to occur at pH 6.0-8.0 (optimum, pH 7.0), 15-50 °C (optimum, 25-30 °C) and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BRMEA1T formed a lineage within the genus Neobacillus (family Bacillaceae) and showed the highest sequence similarity to Neobacillus drentensis DSM 15600T (98.3 %) and Neobacillus fumarioli KCTC 13885T (98.2 %), and less than 98.2 % 16S rRNA gene sequence similarity to the other members of the genus Neobacillus. Whole-genome analysis of strain BRMEA1T comprised a circular chromosome (5 632 809 bp in size) with 38.5 mol% G+C content. Digital DNA-DNA hybridization analyses revealed that strain BRMEA1T showed 20.5 and 22.0% genomic DNA relatedness with the closest species, N. drentensis DSM 15600T and N. fumarioli KCTC 13885T, respectively. The whole-genome sequence of strain BRMEA1T showed the presence of 11 specific conserved signature indels for the genus Neobacillus. The major cellular fatty acids (>10 %) of strain BRMEA1T were found to be iso-C15 : 0 and anteiso-C15 : 0, while the major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Polyphasic analysis results revealed that BRMEA1T represents a novel species of the genus Neobacillus, with the proposed name Neobacillus endophyticus sp. nov. The type strain is BRMEA1T (=KCTC 43208T=CCTCC AB 2020071T).

AtCAP2 is crucial for lytic vacuole biogenesis during germination by positively regulating vacuolar protein trafficking.

Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the prevacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.

The A/ENTH Domain-Containing Protein AtECA4 Is an Adaptor Protein Involved in Cargo Recycling from the trans-Golgi Network/Early Endosome to the Plasma Membrane.

Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane.

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis (Arabidopsis thaliana) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4, atpra1.f4, was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na+/K+-ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA:AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus.

Interactions between Transmembrane Helices within Monomers of the Aquaporin AtPIP2;1 Play a Crucial Role in Tetramer Formation.

Aquaporin (AQP) is a water channel protein found in various subcellular membranes of both prokaryotic and eukaryotic cells. The physiological functions of AQPs have been elucidated in many organisms. However, understanding their biogenesis remains elusive, particularly regarding how they assemble into tetramers. Here, we investigated the amino acid residues involved in the tetramer formation of the Arabidopsis plasma membrane AQP AtPIP2;1 using extensive amino acid substitution mutagenesis. The mutant proteins V41A/E44A, F51A/L52A, F87A/I91A, F92A/I93A, V95A/Y96A, and H216A/L217A, harboring alanine substitutions in the transmembrane (TM) helices of AtPIP2;1 polymerized into multiple oligomeric complexes with a variable number of subunits greater than four. Moreover, these mutant proteins failed to traffic to the plasma membrane, instead of accumulating in the endoplasmic reticulum (ER). Structure-based modeling revealed that these residues are largely involved in interactions between TM helices within monomers. These results suggest that inter-TM interactions occurring both within and between monomers play crucial roles in tetramer formation in the AtPIP2;1 complex. Moreover, the assembly of AtPIP2;1 tetramers is critical for their trafficking from the ER to the plasma membrane, as well as water permeability.

In vivo localization in Arabidopsis protoplasts and root tissue.

In eukaryotic cells, a large number of proteins are transported to their final destination after translation by a process called intracellular trafficking. Transient gene expression, either in plant protoplasts or in specific plant tissues, is a fast, flexible, and reproducible approach to study the cellular function of proteins, protein subcellular localizations, and protein-protein interactions. Here we describe the general method of protoplast isolation, polyethylene glycol-mediated protoplast transformation and immunostaining of protoplast or intact root tissues for studying the localization of protein in Arabidopsis.

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.

An Arabidopsis prenylated Rab acceptor 1 isoform, AtPRA1.B6, displays differential inhibitory effects on anterograde trafficking of proteins at the endoplasmic reticulum.

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aid the targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana) PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to the endoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In this study, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to various endomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat protein complex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescent protein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFP was inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(ΔC):mRFP was not affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency of sporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteins revealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels were controlled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER.