Vacuolar processing enzyme positively modulates plant resistance and cell death in response to Phytophthora parasitica infection

Oomycete, particularly Phytophthora species, causes the most devastating crop diseases, such as potato late blight,and threatens the sustainable crop production worldwide. Our previous studies identified Resistance to Phytophthora parasitica 1(RTP1) as a negative regulator of Arabidopsis resistance to multiple biotrophic pathogens and RTP1 lossof-function plants displayed rapid cell death and reactive oxygen species(ROS) production during early colonization of P. parasitica. In this study, we aim to decipher the mechanism of RTP1-mediated cell death, and identify a member of vaculoar processing enzymes(VPEs), γVPE, playing a role in rtp1-mediated resistance to P. parasitica and cell death occurrence. Our results showed up-regulation of the expression of γVPE as well as increased VPE/caspase 1-like protease activity in P. parasitica-infected rtp1 mutant plants. Besides, we found that the VPE/caspase 1-like protease activity was required for the cell death occurrence in Arabidopsis plants during the infection of P. parasitica as well as rtp1-mediated resistance to P. parasitica. Further pathogenicity assays on either Arabidopsis γvpe mutant plants or leaves of Nicotiana benthamiana with transient overexpression of γVPE demonstrated γVPE could positively affect plant resistance to P. parasitica. Together, our studies suggest that γVPE might function as an important regulator of plant defense and cell death occurrence in response to P. parasitica infection, and VPE/caspase 1-like protease activity is required for rtp1-mediated resistance to P. parasitica.

Transient vacuolar changes of the crystalline lens in patients using a dispersive ophthalmic viscosurgical device

AIM:To report the clinical prognosis and pathological findings of accidental lens vacuolar changes in eyes with intraoperative exposure to a dispersive ophthalmic viscosurgical device(OVD).METHODS:Two patients who developed transient lens vacuolar changes during uneventful persistent pupillary membrane(PPM)removal surgery were presented and followed up.This event was speculated to be associated with an intraoperative dispersive OVD DisCoVisc(hyaluronic acid 1.6%-chondroitin sulfate 4.0%)exposure.Then,to provide the pathological basis for our speculation,another four cataract patients were randomly exposed to different OVDs,and their anterior lens capsules were investigated with transmission electron microscopy(TEM).RESULTS:After months,the subcapsular vacuoles in both PPM cases were gradually disappeared without visual deterioration.For the cataract patients,similar lens changes were observed intraoperatively in those exposed to a dispersive DisCoVisc but not a cohesive OVD IVIZ(sodium hyaluronate gel 1.0%).In addition,marked ultrastructural changes,including chromatin condensation,extensive cytoplasmic vacuoles,and obvious intercellular space between lens epithelial cells in the anterior lens capsules of all eyes exposed to DisCoVisc,were observed by TEM.CONCLUSION:The lens vacuolar changes may be associated with a dispersive OVD exposure.Therefore,it is not preferable to use dispersive OVDs in patients with transparent lenses or without the intention of lens extraction.In addition,close follow-ups instead of immediate lens extraction are recommended for the occurrence of similar lens lesions.

Correlation between the expressions of metastasis-associated factor-1 in colon cancer and vacuolar ATP synthase

BACKGROUND Clinical prognosis often worsens due to high recurrence rates following radical surgery for colon cancer.The examination of high-risk recurrence factors post-surgery provides critical insights for disease evaluation and treatment planning.AIM To explore the relationship between metastasis-associated factor-1 in colon cancer(MACC1)and vacuolar ATP synthase(V-ATPase)expression in colon cancer tissues,and recurrence rate in patients undergoing radical colon cancer surgery.METHODS We selected 104 patients treated with radical colon cancer surgery at our hospital from January 2018 to June 2021.Immunohistochemical staining was utilized to assess the expression levels of MACC1 and V-ATPase in these patients.RESULTS The rates of MACC1 and V-ATPase positivity were 64.42%and 67.31%,respe-ctively,in colon cancer tissues,which were significantly higher than in paracan-cerous tissues(P<0.05).Among patients with TNM stage III,medium to low differentiation,and lymph node metastasis,the positive rates of MACC1 and V-ATPase were significantly elevated in comparison to patients with TNM stage I-II,high differentiation,and no lymph node metastasis(P<0.05).The rate of MACC1 positivity was 76.67%in patients with tumor diameters>5 cm,notably higher than in patients with tumor diameters≤5 cm(P<0.05).We observed a positive correlation between MACC1 and V-ATPase expression(rs=0.797,P<0.05).The positive rates of MACC1 and V-ATPase were significantly higher in patients with recurrence compared to those without(P<0.05).Logistic regression analysis revealed TNM stage,lymph node metastasis,MACC1 expression,and V-ATPase expression as risk factors for postoperative colon cancer recurrence(OR=6.322,3.435,2.683,and 2.421;P<0.05).CONCLUSION The upregulated expression of MACC1 and V-ATPase in colon cancer patients appears to correlate with clinicopathological features and post-radical surgery recurrence.

Identification of the Rice Vacuolar ATPase B Subunit Gene and Its Expression Pattern Analysis Under Phosphorus Deficiency

A vacuolar ATPase (V-ATPase.) B subunit gene has been cloned and characterized front a phosphorus starvation induced rice root subtractive cDNA library by suppression subtractive hybridization (SSH) method and RT-PCR amplification. This gene encodes a polypeptide of 487 amino acid residues, containing a conservative ATP binding site and with a molecular weight of 54.06 kD and an isoelectric point of 4.99, southern analysis of the. genomic DNA indicates that V-ATPase B subunit is encoded by a single gene in rice genome. The amino acid homologies of V-ATPase B subunits among different organisms range from 76% to 97% and reveals that the evolution of V-ATPase B subunit is accompanied with the biological evolution. Expression pattern analysis indicated that the maximal expression of V-ATPase B subunit gene occurred at an early stage (6 - 12 h) after phosphorus starvation in roots, and lately stage (24 - 48 It) in leaves. Under phosphorus deficiency, the up-regulated expression of V-ATPase gene was presumed to strengthen the proton transport and provide the required energy to maintain an electrochemical gradient across the tonoplast to facilitate Phosphorus transport.

Mechanisms of cadmium phytoremediation and detoxification in plants

As a consequence of industrial development,soil Cd pollution leads to crop contamination by Cd,posing a threat to food safety and human health.Excessive accumulation of Cd in plants also inhibits their growth via oxidative stress damage to their photosynthetic systems.Through evolutionary selection,plants have developed a set of efficient strategies to respond to Cd in their environments.These include the accumulation and detoxification of heavy metals.Cd is absorbed by plant roots through the apoplastic and symplastic pathways and then translocated to plant shoots via xylem loading,long-distance transport,and phloem redistribution.Simultaneously,plants initiate a series of mechanisms to reduce Cd toxicity,including cell wall adsorption,cytoplasmic chelation,and vacuolar sequestration.This review summarizes current knowledge of Cd accumulation and detoxification in plants.

Effect of High Temperature on Sucrose Content and Sucrose Cleaving Enzyme Activity in Rice Grain During the Filling Stage

Dynamic changes of sucrose, fructose, glucose contents and differences in activities of sucrose synthase, vacuolar invertase, and cell wall bound invertase in rice grain after flowering stage were studied under natural and high temperatures by using two japonica rice varieties Koshihikari and Sasanishiki. In rice grains, the sucrose synthase activity was higher than that of invertase, which was significantly correlated with starch accumulation rate, indicating that the sucrose synthase played an important role in sucrose degradation and starch synthesis. Under high temperature, the significant increase in grain sucrose content without any increase in fructose and glucose contents, suggested that the high temperature treatment enhanced sucrose accumulation, while diminished sucrose degradation in rice grains. Compared with the control plants, the decrease in activities of sucrose synthase, vacuolar invertase, and cell wall bound invertase with high temperature treated plants indicated that the deceleration of sucrose-degradation was related to the decrease in activities of sucrose synthase and invertase.

Vacuolar invertase genes SbVIN1 and SbVIN2 are differently associated with stem and grain traits in sorghum (Sorghum bicolor)

In higher plants, vacuolar invertases play essential roles in sugar metabolism, organ development, and sink strength. In sorghum(Sorghum bicolor), two vacuolar invertase genes,Sb VIN1(Sobic. 004 G004800) and Sb VIN2(Sobic. 006 G160700) have been reported, but their enzymatic properties and functional differences are largely unknown. We combined molecular, biochemical and genomic approaches to investigate their roles in sorghum stem and grain traits. Sb VIN1 and Sb VIN2 showed different expression levels in internodes,leaves, and panicles, indicating that their importance in each organ was different. In an in vitro sucrose hydrolysis assay, proteins of both genes heterologously expressed in Pichia pastoris displayed similar enzyme properties including the same optimum reaction p H(5)and similar Kmfor sucroe(49 mmol L-1 and 45 mmol L-1 for Sb VIN1 and Sb VIN2,respectively). The optimum reaction temperatures of Sb VIN1 and Sb VIN2 were 45 °C and65 °C, respectively. Sb VIN2 showed higher tolerance to high temperature than Sb VIN1. We characterized the sequence variation of these two vacuolar invertase genes in a panel of 216 diverse inbred lines of sweet and grain sorghum and performed gene-based association analysis. Sb VIN1 showed significant associations with stem traits including stem length,stem diameter, internode number, stem fresh weight, and Brix, as well as grain traits including hundred-grain weight and grain width. Significantly associated variation sites were mainly in 5′ upstream and intron regions. Sb VIN2 only associated with grain width and stem water-soluble carbohydrates(WSCs) content. We conclude that the vacuolar invertase genes Sb VIN1 and Sb VIN2 are differently associated with stem and grain traits in sorghum.

Reconstruction of liver organoid using a bioreactor

AIM： To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS： We previously reported that a radial-flow bioreactor （RFB） could provide a three-dimensional highdensity culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line （FLC-5） as hepatocytes together with mouse immortalized sinusoidal endothelial cell （SEC） line M1 and mouse immortalized hepatic stellate cell （HSC） line A7 as non parenchymal cells in the RFB. Two × 10^7 FLC-5 cells were incubated in the RFB. After 5 d, 2 × 10^7 A7 cells were added in a similar manner followed by another addition of 10^7 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy （TEM） and scanning electron microscopy （SEM）. To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS： By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles （WO） and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION： Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.

Cloning and expression analysis of GhDET3, a vacuolar H^+-ATPase subunit C gene, from cotton

Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.

Possible contribution of(pro)renin receptor to development of gestational diabetes mellitus

(Pro)renin receptor [(P)RR], a receptor for renin and prorenin, was first cloned in 2002. Since then, the pathophysiological roles of(P)RR have been growing concerns.(P)RR binds renin and prorenin, with two important consequences, nonproteolytic activation of prorenin, leading to the tissue renin-angiotensin system activation and the intracellular signalings. It is now also known to play an important role as vacuolar H+-ATPase associated protein, involving in Wnt signaling, main component of embryonic development. Extracellular domain of full-length(P)RR is cleaved in golgi-complex forming soluble(P)RR [s(P)RR]. The s(P)RR is now possible to be measured in human blood and urine. It is now measured in different pathophysiological states, and recent study showed that elevated plasma s(P)RR levels in the early stage of pregnancies are associated with higher incidence of gestational diabetes mellitus later in the pregnancies. Plasma s(P)RR levels of neonates are known to be higher than that of adults. It was also shown that, increased s(P)RR concentrations in cord blood, associated with a lower small for gestational age birth likelihood. These data suggests the involvement of(P)RR in embryo's growth. In thisreview article, we attempt to figure out the possible pathophysiological roles of the(P)RR in maternal glucose intolerance and embryo's growth, through reviewing previous studies.

Overexpression of vacuolar proton pump ATPase(V-H^+-ATPase) subunits B, C and H confers tolerance to salt and saline-alkali stresses in transgenic alfalfa(Medicago sativa L.)

The vacuolar proton pump ATPase（V-H^＋-ATPase）, which is a multi-subunit membrane protein complex, plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. In this study, three genes encoding V-H^＋-ATPase subunits B（Sc VHA-B, Gen Bank： JF826506）, C（Sc VHA-C, Gen Bank： JF826507） and H（Sc VHA-H, Gen Bank： JF826508） were isolated from the halophyte Suaeda corniculata. The transcript levels of Sc VHA-B, Sc VHA-C and Sc VHA-H were increased by salt, drought and saline-alkali treatments. V-H^＋-ATPase activity was also examined under salt, drought and saline-alkali stresses. The results showed that V-H^＋-ATPase activity was correlated with salt, drought and saline-alkali stress. Furthermore, V-H^＋-ATPase subunits B, C and H（Sc VHA-B, Sc VHA-C and Sc VHA-H） from S. corniculata were introduced separately into the alfalfa genome. The transgenic alfalfa was verified by Southern and Northern blot analysis. During salt and saline-alkali stresses, transgenic lines carrying the B, C and H subunits had higher germination rates than the wild type（WT）. More free proline, higher superoxide dismutase（SOD） activity and lower malondialdehyde（MDA） levels were detected in the transgenic plants under salt and saline-alkali treatments. Moreover, the Sc VHA-B transgenic lines showed greater tolerance to salt and saline-alkali stresses than the WT. These results suggest that overexpression of Sc VHA-B, Sc VHA-C and Sc VHA-H improves tolerance to salt and saline-alkali stresses in transgenic alfalfa.

Inhibition of HBV Replication by VPS4B and Its Dominant Negative Mutant VPS4B-K180Q In Vivo

This study examined the anti-hepatitis B virus （HBV） effect of wild-type （WT） vacuolar protein sorting 4B （VPS4B） and its dominant negative （DN） mutant VPS4B-K180Q in vivo in order to further explore the relationship between HBV and the host cellular factor VPS4. VPS4B gene was amplified from Huh7 cells by RT-PCR and cloned into the eukaryotic expression vector pXF3H. Then, the VPS4B plasmid and the VPS4B-K180Q mutation plasmid were constructed by using the overlap extension PCR site-directed mutagenesis technique. VPS4B and HBV vectors were co-delivered into mice by the hydrodynamic tail-vein injection to establish HBV vector-based models. Quantities of HBsAg and HBeAg in the mouse sera were determined by ElectroChemiLuminescence （ECL）. HBV DNA in sera was measured by real-time quantitative PCR. Southern blot analysis was used to assay the intracellular HBV nuclear capsid-related DNA, real-time quantitative PCR to detect the HBV-related mRNA and immunohistochemical staining to observe the HBcAg expression in the mouse liver tissues. Our results showed that VPS4B and its mutant VPS4B-K180Q could decrease the levels of serum HBsAg, HBeAg and HBV-DNA. In addition, the HBV DNA replication and the mRNA level of HBV in the liver tissues of treated mice could be suppressed by VPS4B and VPS4B-K180Q. It was also found that VPS4B and VPS4B-K180Q had an ability to inhibit core antigen expression in the infected mouse liver. Furthermore, the anti-HBV effect of mutant VPS4B-K180Q was more potent than that of wild-type VPS4B. Taken together, it was concluded that VPS4B and its DN mutant VPS4B-K180Q have anti-HBV effect in vivo, which helps develop molecular therapeutic strategies for HBV infection.

V-ATPase对肿瘤细胞糖酵解的影响

V-ATPase是一类特殊的膜转运蛋白,通过驱动质子转运以及参与信号转导调节细胞各种生理过程。V-ATPase在细胞中的主要功能及作用已有大量研究,而关于其激活或抑制对于细胞糖酵解的影响,以及在肿瘤细胞所起作用的相关材料比较零散。通过整理近年来关于V-ATPase以及肿瘤糖酵解等的相关文献发现,V-ATPase在肿瘤细胞发生、发展、转移等过程中起到了至关重要的作用。因此,总结V-ATPase的结构功能,以及其对细胞糖酵解的影响甚至在癌细胞中起的作用,有利于提高对于V-ATPase的进一步认识,并便于探索其在肿瘤治疗新方案,尤其靶向治疗研究中的作用。

Vacuolar phosphate transporters account for variation in phosphate accumulation in Astragalus sinicus cultivars

Astragalus sinicus is a commonly used legume green manure that fixes atmospheric N2 and accumulates mineral nutrients and organic substances that are beneficial to soils and subsequent crops.However,little is known about genotypic variation in,and molecular mechanisms of,Pi(phosphate)uptake and storage in A.sinicus.We recorded the morphological responses of six A.sinicus cultivars from four regions of China to external Pi application and measured their Pi accumulation.We identified full-length transcripts of Pi-signaling and Pi-homeostasis regulators by sequencing and measured the expression level of these genes by qRT-PCR.The major components in Pi signaling and Pi homeostasis were largely conserved between A.sinicus and the model species rice and Arabidopsis.Different A.sinicus varieties responded differently to low-phosphorus(P)stress,and their Pi accumulation was positively correlated with the expression of vacuolar Pi influx gene(SYG1/PHO81/XPR1-MAJOR FACILITATOR SUPERFAMILY(SPX-MFS)-TYPE PROTEIN)AsSPXMFS2 and negatively correlated with the expression of the vacuolar Pi efflux gene(VACUOLAR Pi EFFLUX TRANSPORTER)AsVPE1.We identified key Pi-signaling and Pihomeostasis regulators in A.sinicus.The expression of vacuolar Pi transporter genes could be used as an index to select A.sinicus accessions with high Pi accumulation.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

Effect of salt stress on the expression of NHX-type Na^+/H^+ antiporters in Populus euphratica and P.pruinosa calli

Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na＋ and K＋ contents, the expression level of vacuolar Na＋/H＋ antiporters was investigated for NaCl-induced changes in P. euphratica and t3. pru- inosa calli. Compared to R euphratica, P. pruinosa calli grew well in 200 mM NaC1 stress from 14. to 21 days. Increasing the stressed time caused an increase in Na＋ content concomitant with a decrease in K＋ content in P. euphratica calli, whereas, with the presence of 200 mM NaCI, K＋ content has a less increase in 14 and 21 days than in 7 days which was detected in R pruinosa calli. The transcript levels of six genes coding for NHX-type Na＋/H＋ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in R pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na＋ avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.

Histopathological study of the kidney after administration of methanolic extract of roots of <i>Leptadenia hastata</i>in the Albino Wistar rat

Introduction: the roots of Leptadenia hastata, (L hastata) are remedies from the Senegalese pharmacopoeia and are as widely used as the leaves. However, few researchers have devoted themselves to their toxicity, unlike the leaves. However, in the traditional pharmacopoeia, the indications for use are very different. The aim of our study was to study the effect of the administration of methanolic extracts of roots of L hastata on renal tissue, using an animal model. Materials and Methods: a cohort of 18 rats was studied with a random distribution of the animals in 3 groups (n = 6). The first group was the control group. The treated groups (Group II and III) received the methanolic extract of L. hastata with an administration of 500 mg / kg / day and 1000 mg / kg / day respectively, for 28 days. Results: The dose of 1000 mg / kg / day was lethal in group III, from the first week in females. Serum creatinine was significantly higher in rats given the root extract. There was kidney damage with vacuolar degeneration starting at 500 mg / kg / day. The lesions were more severe in group III with glomerular involvement characterized by retraction in the renal corpuscles. Conclusion: If the leaves of L hastata seem to be safe for consumption according to the majority of authors in the literature, the roots of this same plant could be harmful. In addition to the lethality observed at the doses tested, the lesions in the renal parenchyma would be dose-dependent.

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

ESCRT-Ⅲ component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice

The endosomal sorting complex required for transport(ESCRT)is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes.Although ESCRT components affect a variety of plant growth and development processes,their impact on leaf development is rarely reported.Here,we found that OsSNF7.2,an ESCRT-Ⅲ component,controls leaf rolling in rice(Oryza sativa).The Ossnf7.2 mutant rolled leaf 17(rl17)has adaxially rolled leaves due to the decreased number and size of the bulliform cells.OsSNF7.2is expressed ubiquitously in all tissues,and its protein is localized in the endosomal compartments.OsSNF7.2 homologs,including OsSNF7,OsSNF7.3,and OsSNF7.4,can physically interact with OsSNF7.2,but their single mutation did not result in leaf rolling.Other ESCRT complex subunits,namely OsVPS20,OsVPS24,and OsBRO1,also interact with OsSNF7.2.Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation.Both Osyuc8and rl17 Osyuc8 showed rolled leaves,indicating that OsYUC8 and OsSNF7.2 function in the same pathway,conferring leaf development.This study reveals a new biological function for the ESCRT-Ⅲcomponents,and provides new insights into the molecular mechanisms underlying leaf rolling.

The Formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and Implications for the Sequestration of Anthocyanin Pigments

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions （AVIs）. Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to un- derstand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside （C3G） and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-0 position （Sgt mutant） accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process （atg mutants） display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVl formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.