The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression ofSKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.Facultad de Ciencias Agrarias y Forestale

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis

The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression ofSKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.Facultad de Ciencias Agrarias y Forestale

Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha

Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages

The Influence of Manga on the Graphic Novel

This material has been published in The Cambridge History of the Graphic Novel edited by Jan Baetens, Hugo Frey, Stephen E. Tabachnick. This version is free to view and download for personal use only. Not for re-distribution, re-sale or use in derivative works. © Cambridge University PressProviding a range of cogent examples, this chapter describes the influences of the Manga genre of comics strip on the Graphic Novel genre, over the last 35 years, considering the functions of domestication, foreignisation and transmedia on readers, markets and forms

AtNHX5 and AtNHX6 Are Required for the Subcellular Localization of the SNARE Complex That Mediates the Trafficking of Seed Storage Proteins in Arabidopsis.

The SNARE complex composed of VAMP727, SYP22, VTI11 and SYP51 is critical for protein trafficking and PSV biogenesis in Arabidopsis. This SNARE complex directs the fusion between the prevacuolar compartment (PVC) and the vacuole, and thus mediates protein trafficking to the vacuole. In this study, we examined the role of AtNHX5 and AtNHX6 in regulating this SNARE complex and its function in protein trafficking. We found that AtNHX5 and AtNHX6 were required for seed production, protein trafficking and PSV biogenesis. We further found that the nhx5 nhx6 syp22 triple mutant showed severe defects in seedling growth and seed development. The triple mutant had short siliques and reduced seed sets, but larger seeds. In addition, the triple mutant had numerous smaller protein storage vacuoles (PSVs) and accumulated precursors of the seed storage proteins in seeds. The PVC localization of SYP22 and VAMP727 was repressed in nhx5 nhx6, while a significant amount of SYP22 and VAMP727 was trapped in the Golgi or TGN in nhx5 nhx6. AtNHX5 and AtNHX6 were co-localized with SYP22 and VAMP727. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for the transport of the storage proteins, indicating the importance of exchange activity in protein transport. AtNHX5 or AtNHX6 did not interact physically with the SNARE complex. Taken together, AtNHX5 and AtNHX6 are required for the PVC localization of the SNARE complex and hence its function in protein transport. AtNHX5 and AtNHX6 may regulate the subcellular localization of the SNARE complex by their transport activity

Robustness of the Oxygen Uptake Efficiency Slope to Exercise Intensity in Patients with Coronary Artery Disease

Oxygen uptake efficiency slope (OUES) and ventilatory efficiency (V˙ E/V˙CO2 slope) are widely used as submaximal measurements of cardiopulmonary exercise testing as the evaluator or prognosticator of cardiac diseases. However, very few studies have compared the effects of submaximal exercise on these measurements. A total of 58 patients with coronary artery disease underwent maximal cardiopulmonary exercise testing on a treadmill. We compared the values obtained from the first 75% (V˙ E/V˙CO2 slope75 and OUES75) and 90% (V˙ E/V˙CO2 slope90 and OUES90) of the exercise period with the entire duration (V˙ E/V˙CO2 slope100 and OUES100). Although OUES100, OUES90 and OUES75 were virtually identical, submaximal calculations of V˙ E/V˙CO2 slope underestimated the measurements. The Bland-Altman method revealed that submaximal measurements of OUES agreed very well with maximal OUES (limits of agreement –5.0% to +6.0% for OUES90, and –11.5% to +12.9% for OUES75). However, the submaximal calculations of V˙ E/V˙ CO2 slope showed rather poor agreement with the maximal calculations (limit of agreement –11.8% to +3.1% for V˙ E/V˙CO2 slope90, and –20.8% to +5.3%% for V˙ E/V˙CO2 slope75). These results revealed that both the OUES and the V˙ E/V˙CO2 slopes are not overly influenced by exercise

Flowering Time Modulation by a Vacuolar SNARE via <em>FLOWERING LOCUS C</em> in <em>Arabidopsis thaliana</em>

<div><p>The transition of plant growth from vegetative to reproductive phases is one of the most important and dramatic events during the plant life cycle. In <em>Arabidopsis thaliana</em>, flowering promotion involves at least four genetically defined regulatory pathways, including the photoperiod-dependent, vernalization-dependent, gibberellin-dependent, and autonomous promotion pathways. Among these regulatory pathways, the vernalization-dependent and autonomous pathways are integrated by the expression of <em>FLOWERING LOCUS C</em> (<em>FLC</em>), a negative regulator of flowering; however, the upstream regulation of this locus has not been fully understood. The <em>SYP22</em> gene encodes a vacuolar SNARE protein that acts in vacuolar and endocytic trafficking pathways. Loss of <em>SYP22</em> function was reported to lead to late flowering in <em>A. thaliana</em> plants, but the mechanism has remained completely unknown. In this study, we demonstrated that the late flowering phenotype of <em>syp22</em> was due to elevated expression of <em>FLC</em> caused by impairment of the autonomous pathway. In addition, we investigated the DOC1/BIG pathway, which is also suggested to regulate vacuolar/endosomal trafficking. We found that elevated levels of <em>FLC</em> transcripts accumulated in the <em>doc1-1</em> mutant, and that <em>syp22</em> phenotypes were exaggerated with a double <em>syp22 doc1-1</em> mutation. We further demonstrated that the elevated expression of <em>FLC</em> was suppressed by <em>ara6-1</em>, a mutation in the gene encoding plant-unique Rab GTPase involved in endosomal trafficking. Our results indicated that vacuolar and/or endocytic trafficking is involved in the <em>FLC</em> regulation of flowering time in <em>A. thaliana</em>.</p> </div

Expression level of <i>FLC</i> was elevated in <i>syp22-1</i> mutants.

<p>The expression levels of <i>FLC, FT, LFY,</i> and <i>SOC1</i> in 14-day-old wild type (WT), <i>syp22-1</i>, and <i>fve-4</i> seedlings were examined by qRT-PCR. In <i>syp22-1</i> plants, expression levels of <i>FLC</i> were elevated, which resulted in downregulation of downstream flowering pathway integrators. <i>fve-4</i>, an autonomous pathway mutant, was used as a control. Results are presented as means ±S.D. (n = 3–12).</p