Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Hair growth-promoting activity of components derived from sweet potato shochu.

Sweet potato shochu oil is one of the by-products of sweet potato shochu production. We investigated the functionality and industrial use of shochu oil as a food-derived raw material. Because of the increased incidence of self-consciousness in people owing to thinning hair, in this study, we examined the hair growth-inducing effects of shochu oil. Minoxidil, the only topical medication approved for hair growth treatment in Japan, was used as a control for the evaluation of hair growth-promoting activity of shochu oil. Human follicle dermal papilla cells treated with shochu oil showed upregulated expression of vascular endothelial growth factor in a concentration-dependent manner, indicating that shochu oil induced the activation of the hair growth cycle. In vivo, epidermal treatment with shochu oil also promoted hair growth in C3H mice. More than 35 components were detected in shochu oil via gas chromatography-mass spectrometry. The main components, accounting for 98.5% of shochu oil, were as follows, in order of decreasing concentration: ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, ethyl caprate, ethyl laurate, ethyl myristate, and ethyl α-linolenate. Among these, ethyl palmitate, ethyl linoleate, and ethyl α-linolenate promoted hair growth in C3H mice. These results indicate that shochu oil can be used as a hair restorer. To the best of our knowledge, this study is the first to demonstrate the hair growth-promoting activity of shochu oil.

Comparative transcriptome analysis implied a ZEP paralog was a key gene involved in carotenoid accumulation in yellow-fleshed sweetpotato.

The mechanisms of carotenoid accumulation in yellow-fleshed sweetpotato cultivars are unclear. In this study, we compared the transcriptome profiles of a yellow-fleshed cultivar, Beniharuka (BH) and two of its spontaneous white-fleshed mutants (WH2 and WH3) to reveal the genes involved in yellow flesh. As a result of RNA sequencing, a total of 185 differentially expressed genes (DEGs) were commonly detected in WH2 and WH3 compared to BH. Of these genes, 85 DEGs and 100 DEGs were commonly upregulated and downregulated in WH2 and WH3 compared to BH, respectively. g1103.t1, a paralog of zeaxanthin epoxidase (ZEP), was only DEG common to WH2 and WH3 among 38 genes considered to be involved in carotenoid biosynthesis in storage roots. The expression level of g1103.t1 was also considerably lower in five white-fleshed cultivars than in five yellow-fleshed cultivars. Analysis of carotenoid composition in the storage roots showed that the epoxidised carotenoids were drastically reduced in both WH2 and WH3. Therefore, we propose that the ZEP paralog, g1103.t1, may be involved in carotenoid accumulation through the epoxidation of ß-carotene and ß-cryptoxanthin in sweetpotato.

Arabidopsis R1R2R3-Myb proteins are essential for inhibiting cell division in response to DNA damage.

Inhibition of cell division is an active response to DNA damage that enables cells to maintain genome integrity. However, how DNA damage arrests the plant cell cycle is largely unknown. Here, we show that the repressor-type R1R2R3-Myb transcription factors (Rep-MYBs), which suppress G2/M-specific genes, are required to inhibit cell division in response to DNA damage. Knockout mutants are resistant to agents that cause DNA double-strand breaks and replication stress. Cyclin-dependent kinases (CDKs) can phosphorylate Rep-MYBs in vitro and are involved in their proteasomal degradation. DNA damage reduces CDK activities and causes accumulation of Rep-MYBs and cytological changes consistent with cell cycle arrest. Our results suggest that CDK suppressors such as CDK inhibitors are not sufficient to arrest the cell cycle in response to DNA damage but that Rep-MYB-dependent repression of G2/M-specific genes is crucial, indicating an essential function for Rep-MYBs in the DNA damage response.Inhibition of cell division maintains genome integrity in response to DNA damage. Here Chen et al. propose that DNA damage causes cell cycle arrest in the Arabidopsis root via Rep-MYB transcription factor-mediated repression of G2/M-specific gene expression in response to reduced cyclin-dependent kinase activity.

Effects of a sweetpotato protein digest on lipid metabolism in mice administered a high-fat diet.

Sweetpotato peptide (SPP) was prepared by enzyme digestion of sweetpotato protein from starch wastewater. Animal experiments assessed the effect of SPP on body weight, abdominal adipose tissue mass, serum lipids and adipocytokines. Body and liver weight and epididymal and mesenteric fat of mice fed a high-fat diet containing 0.5% or 5% SPP for 28 days were significantly lower than control mice. Triglyceride and cholesterol in VLDL and LDL and leptin levels were significantly lower in the serum of SPP-administered mice compared to control mice. Biomarker arrays showed that adiponectin, melanocyte-stimulating-hormone-alpha and neuromedin U were more than 1.5 times higher, while TNF-alpha was about 1.5 times lower in the livers of SPP-administered mice compared to control mice. These results suggest SPP mitigated leptin resistance in mice administered a high-fat diet, and maintained anorexigenic peptide levels. SPP administration may suppress lipogenesis by increasing adiponectin levels and decreasing TNF-alpha levels in adipocytes.

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots.

The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex), respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

Identification of putative target genes of bZIP19, a transcription factor essential for Arabidopsis adaptation to Zn deficiency in roots.

Zinc (Zn) depletion adversely affects plant growth. To avoid lethal depletion of cellular Zn, plants have evolved mechanisms to adjust the expression of genes associated with Zn homeostasis, the details of which are poorly understood. In the present study, we isolated an Arabidopsis thaliana T-DNA insertion mutant that exhibited hypersensitivity to Zn depletion. By monitoring root development under Zn-deficient conditions, we isolated a single mutant lacking the basic-region leucine-zipper transcription factor gene bZIP19. To identify proteins whose expression is affected by bZIP19, an iTRAQ-based quantitative proteomics analysis was performed using microsomal proteins from wild-type and the bzip19 mutant A. thaliana roots grown on Basal and Zn-deficient media. Of the 797 proteins identified, expression of two members of the Zrt- and Irt-related protein family, ZIP3 and ZIP9, and three defensin-like family proteins was markedly induced in wild-type but not in the bzip19 mutant under Zn-deficient conditions. Furthermore, selected reaction monitoring and quantitative real-time PCR revealed that ZIP9 expression is mediated by bZIP19 and may be partly supported by bZIP23, a homolog of bZIP19. Mutant analysis revealed that ZIP9 is involved in uptake of Zn by the roots, and the mutant lacking ZIP9 was significantly more sensitive to Zn depletion than the wild-type. These results demonstrate that bZIP19 mainly contributes to expression of genes, such as ZIP9, under Zn-deficient conditions.

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency.

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe-deficient plants. To understand cross-talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ-OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe-deficient media compared to basal MGRL solid medium. iTRAQ-OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross-talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe-deficient conditions. Plants grown on Fe-deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.

Correlation analysis of proteins responsive to Zn, Mn, or Fe deficiency in Arabidopsis roots based on iTRAQ analysis.

KEY MESSAGE: For discovering the functional correlation between the identified and quantified proteins by iTRAQ analysis, here we propose a correlation analysis method with cosine correlation coefficients as a powerful tool. iTRAQ analysis is a quantitative proteomics approach that enables identification and quantification of a large number of proteins. In order to obtain proteins responsive to Zn, Mn, or Fe mineral deficiency, we conducted iTRAQ analysis using a microsomal fraction of protein extractions from Arabidopsis root tissues. We identified and quantified 730 common proteins in three biological replicates with less than 1 % false discovery rate. To determine the role of these proteins in tolerating mineral deficiencies and their relation to each other, we calculated cosine correlation coefficients and represented the outcomes on a correlation map for visual understanding of functional relations among the identified proteins. Functionally similar proteins were gathered into the same clusters. Interestingly, a cluster of proteins (FRO2, IRT1, AHA2, PDR9/ABCG37, and GLP5) highly responsive to Fe deficiency was identified, which included both known and unknown novel proteins involved in tolerating Fe deficiency. We propose that the correlation analysis with the cosine correlation coefficients is a powerful method for finding important proteins of interest to several biological processes through comprehensive data sets.

Peptide separation methodologies for in-depth proteomics.

The integration of proteomics to other omics technologies and generation of proteome maps of a particular cell/tissue requires the identification and quantification of a maximum number of proteins. Traditional 2-D gel-based approach though provides a clear proteome map has its limitations, such as time consuming, requiring high skill, and most importantly, inability to identify low-abundance proteins. The most common drawback of 2-D gel electrophoresis is the masking of low amount proteins by the highly expressed (high abundance) proteins. Therefore, the elucidation of complete regulatory networks of a cell/tissue demands identification of low-abundance proteins. Low-abundance protein identification requires the use of usually gel-free mass spectrometry (MS)-based approaches. Using Arabidopsis thaliana as a model system, in this chapter, we describe all the steps followed for the extraction of microsomal proteins to MS analysis of separated peptides with a major focus on three different methods, namely, OFFGEL fractionation, 2D-LC, and long-column method for the identification of low-abundance proteins. Separation of such peptides will lead to in-depth proteomics-based investigations to answer biological questions.

Characteristics of a root hair-less line of Arabidopsis thaliana under physiological stresses.

The plasma membrane-associated Ca(2+)-binding protein-2 of Arabidopsis thaliana is involved in the growth of root hair tips. Several transgenic lines that overexpress the 23 residue N-terminal domain of this protein under the control of the root hair-specific EXPANSIN A7 promoter lack root hairs completely. The role of root hairs under normal and stress conditions was examined in one of these root hair-less lines (NR23). Compared with the wild type, NR23 showed a 47% reduction in water absorption, decreased drought tolerance, and a lower ability to adapt to heat. Growth of NR23 was suppressed in media deficient in phosphorus, iron, calcium, zinc, copper, or potassium. Also, the content of an individual mineral in NR23 grown in normal medium, or in medium lacking a specific mineral, was relatively low. In wild-type plants, the primary and lateral roots produce numerous root hairs that become elongated under phosphate-deficient conditions; NR23 did not produce root hairs. Although several isoforms of the plasma membrane phosphate transporters including PHT1;1-PHT1;6 were markedly induced after growth in phosphate-deficient medium, the levels induced in NR23 were less than half those observed in the wild type. In phosphate-deficient medium, the amounts of acid phosphatase, malate, and citrate secreted from NR23 roots were 38, 9, and 16% of the levels secreted from wild-type roots. The present results suggest that root hairs play significant roles in the absorption of water and several minerals, secretion of acid phosphatase(s) and organic acids, and in penetration of the primary roots into gels.

Peptide separation methodologies for in-depth proteomics in Arabidopsis.

In the post-genome era, several tools that have increased our global understanding of the molecular basis of several cell-based phenomena have been developed. However, proteomics has not been efficiently integrated with the other 'omics' (e.g. transcriptomics and metabolomics), because of the relatively low number of proteins identified by mass spectrometry (MS). Peptides from low-abundance proteins are often not detected by MS due to ionization suppression. To improve the number of peptide identifications in MS analyses, we propose three separation methodologies; namely, OFFGEL electrophoresis, 2D-liquid chromatography (LC) and the long monolithic silica-C18 capillary column method, with the common aim to decrease peptide complexity prior to MS analyses. Proteomics using the above three peptide separation methods were separately applied to protoplasts collected from the epidermal cell layer of Arabidopsis roots using fluorescence-activated cell sorting. In each method alone, 1,132, 836 and 795 proteins were specifically identified, respectively. This has allowed the identification of 1,493 proteins with no redundancy and with <1.0% false discovery rate. Moreover, approximately two-thirds of these proteins are identified here for the first time in the epidermal cell layer. These results show that use of different proteomic approaches can increase the total number of proteins identified. We propose that the integration of data from these methodologies represents a powerful tool for generation of proteome maps by enabling identification of low-abundance proteins in the various Arabidopsis root cell layers.

Unraveling the iron deficiency responsive proteome in Arabidopsis shoot by iTRAQ-OFFGEL approach.

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.

iTRAQ analysis reveals mechanisms of growth defects due to excess zinc in Arabidopsis.

The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H(+)-ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role.

AAA+ proteins RUVBL1 and RUVBL2 coordinate PIKK activity and function in nonsense-mediated mRNA decay.

Phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins play essential roles in DNA-based and RNA-based processes, such as the response to DNA damage, messenger RNA (mRNA) quality control, transcription, and translation, where they contribute to the maintenance of genome integrity and accurate gene expression. The adenosine triphosphatases associated with diverse cellular activities (AAA+) family proteins RuvB-like 1 (RUVBL1) and RUVBL2 are involved in various cellular processes, including transcription, RNA modification, DNA repair, and telomere maintenance. We show that RUVBL1 and RUVBL2 associate with each PIKK family member. We also show that RUVBL1 and RUVBL2 control PIKK abundance at least at the mRNA level. Knockdown of RUVBL1 or RUVBL2 decreased PIKK abundance and impaired PIKK-mediated signaling. Analysis of SMG-1, a PIKK family member involved in nonsense-mediated mRNA decay (NMD), revealed an essential role for RUVBL1 and RUVBL2 in NMD. RUVBL1 and RUVBL2 associated with SMG-1 and the messenger ribonucleoproteins in the cytoplasm and promoted the formation of mRNA surveillance complexes during NMD. Thus, RUVBL1 and RUVBL2 regulate PIKK functions on two different levels: They control the abundance of PIKKs, and they stimulate the formation of PIKK-containing molecular complexes, such as those involved in NMD.

SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed "SURF" that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.

Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves.

Sweetpotato leaves (Ipomoea batatas L.) contain a high content of polyphenolics that consist of caffeic acid, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. We investigated the suppression of the proliferation of selected human cancer cells by phenolic compounds isolated from sweetpotato leaf. The human cancer cells used in this research included a stomach cancer (Kato III), a colon cancer (DLD-1), and a promyelocytic leukemia cell (HL-60). Caffeic acid and di- and tricaffeoylquinic acids dose-dependently depressed cancer cell proliferation, and the difference in sensitivity between caffeoylquinic acid derivatives and each kind of cancer cell was observed. Specifically, 3,4,5-tri-O-caffeoylquinic acid effectively depressed the growth of three kinds of cancer cells, and caffeic acid had an exceptionally higher effect against HL-60 cells than other di- and tricaffeoylquinic acids. In attempting to clarify the mechanism of growth suppression with the addition of the apoptotic inhibitor N-ethylmaleimide, we observed that the nuclear granulation in 3,4,5-tri-O-caffeoylquinic acid-treated HL-60 cells suggested apoptosis induction. This effect was confirmed by DNA fragmentation, an increase of caspase-3 activity, and expression of c-Jun. Growth suppression of HL-60 cells by 3,4,5-tri-O-caffeoylquinic acid was determined to be the result of apoptotic death of the cells. These results indicate that 3,4,5-tri-O-caffeoylquinic acid may have potential for cancer prevention.

Mutation spectrum induced by dihydropyrazines in Escherichia coli.

Dihydropyrazine (DHP), which induces mutagenesis in E. coli, was investigated. From analyzing mutations in the chromosomal rpoB gene, the mutation spectrum in uvrB strain revealed the different behavior on exposure to two DHP derivatives 3-hydro-2,2,5,6-tetramethylpyrazine (HTMP), and 2,3-dihydro-5,6-dimethylpyrazine (DHDMP). A higher level of DHP-induced mutation was observed, with base substitutions at G : C pairs predominant. HTMP and DHDMP increased the frequency of G : C to T : A transversions. HTMP increased the frequency of G : C to A : T transitions, than did DHDMP. These findings suggest that DHPs prefer to attack the G : C pair and that different DHP derivatives may prefer distinct mutagenic base pairs; and further, that nucleotide excision repair may be involved in the repair of DHP-induced mutations.