RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Lipidic phase membrane protein serial femtosecond crystallography

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.     

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of ³H-labeled water (³H₂O) and the ¹⁴C-labeled organic acids benzoic acid ([¹⁴C]BA), salicylic acid ([¹⁴C]SA), and 2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (³H₂O/[¹⁴C]BA, ³H₂O/[¹⁴C]SA, and ³H₂O/[¹⁴C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination ³H₂O/[¹⁴C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of ³H₂O of all 12 investigated species were highly correlated with the permeances of [¹⁴C]BA (r² = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.     

Climate warming alters subsoil but not topsoil carbon dynamics in alpine grassland

Subsoil contains more than half of soil organic carbon (SOC) globally and is conventionally assumed to be relatively unresponsive to warming compared to the topsoil. Here, we show substantial changes in carbon allocation and dynamics of the subsoil but not topsoil in the Qinghai‐Tibetan alpine grasslands over 5 years of warming. Specifically, warming enhanced the accumulation of newly synthesized (¹⁴C‐enriched) carbon in the subsoil slow‐cycling pool (silt‐clay fraction) but promoted the decomposition of plant‐derived lignin in the fast‐cycling pool (macroaggregates). These changes mirrored an accumulation of lipids and sugars at the expense of lignin in the warmed bulk subsoil, likely associated with shortened soil freezing period and a deepening root system. As warming is accompanied by deepening roots in a wide range of ecosystems, root‐driven accrual of slow‐cycling pool may represent an important and overlooked mechanism for a potential long‐term carbon sink at depth. Moreover, given the contrasting sensitivity of SOC dynamics at varied depths, warming studies focusing only on surface soils may vastly misrepresent shifts in ecosystem carbon storage under climate change.     

RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.     

Apoplastic barriers to radial oxygen loss and solute penetration: a chemical and functional comparison of the exodermis of two wetland species, Phragmites australis and Glyceria maxima

Few studies have examined exodermal development in relation to the formation of barriers to both radial oxygen loss (ROL) and solute penetration along growing roots. Here, we report on the structural development, chemical composition and functional properties of the exodermis in two diverse wetland grasses, Glyceria maxima and Phragmites australis. Anatomical features, development, the biochemical composition of exodermal suberin and the penetration of apoplastic tracers and oxygen were examined. Striking interspecific differences in exodermal structure, suberin composition and quantity per unit surface area, and developmental changes along the roots were recorded. Towards the root base, ROL and periodic acid (H(5)IO(6)) penetration were virtually stopped in P. australis; in G. maxima, a tight ROL barrier restricted but did not stop H(5)IO(6) penetration and the exodermis failed to stain with lipidic dyes. Cultivation in stagnant deep hypoxia conditions or oxygenated circulating solution affected the longitudinal pattern of ROL profiles in G. maxima but statistically significant changes in exodermal suberin composition or content were not detected. Interspecific differences in barrier performance were found to be related to hypodermal structure and probably to qualitative as well as quantitative variations in suberin composition and distribution within exodermal cell walls. Implications for root system function are discussed, and it is emphasized that sufficient spatial resolution to identify the effects of developmental changes along roots is crucial for realistic evaluation of exodermal barrier properties.     

Composition of the cuticle of developing sweet cherry fruit

The composition of wax and cutin from developing sweet cherry (Prunus avium) fruit was studied by GC-MS between 22 and 85 days after full bloom (DAFB). In this and our previous study, fruit mass and surface area increased in a sigmoidal pattern with time, but mass of the cuticular membrane (CM) per unit fruit surface area decreased. On a whole fruit basis, mass of CM increased up to 36 DAFB and remained constant thereafter. At maturity, triterpenes, alkanes and alcohols accounted for 75.6%, 19.1% and 1.2% of total wax, respectively. The most abundant constituents were the triterpenes ursolic (60.0%) and oleanolic acid (7.5%), the alkanes nonacosane (13.0%) and heptacosane (3.0%), and the secondary alcohol nonacosan-10-ol (1.1%). In developing fruit triterpenes per unit area decreased, but alkanes and alcohols remained essentially constant. The cutin fraction of mature fruit consisted of mostly C16 (69.5%) and, to a lower extent, C18 monomers (19.4%) comprising alkanoic, omega-hydroxyacids, alpha,omega-dicarboxylic and midchain hydroxylated acids. The most abundant constituents were 9(10),16-dihydroxy-hexadecanoic acid (53.6%) and 9,10,18-trihydroxy-octadecanoic acid (7.8%). Amounts of C16 and C18 monomers per unit area decreased in developing fruit, but remained approximately constant on a whole fruit basis. Within both classes of monomers, opposing changes occurred. Amounts of hexadecandioic, 16-hydroxy-hexadecanoic, 9(10)-hydroxy-hexadecane-1,16-dioic and 9,10-epoxy-octadecane-1,18-dioic acids increased, but 9,10,18-trihydroxy-octadecanoic and 9,10,18-trihydroxy-octadecenoic acids decreased. There were no qualitative and minor quantitative differences in wax and cutin composition between cultivars at maturity. Our data indicate that deposition of some constituents of wax and cutin ceased during early fruit development.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Cuticular waxes from ivy leaves (Hedera helix L.): analysis of high-molecular-weight esters

Soluble waxes were extracted from the cuticle of ivy (Hedera helix L.) leaves with dichloromethane in a yield of ca. 13%. The cuticular waxes were directly analysed by GC-MS, high-temperature GC-MS and ESI-MS/MS. The GC-MS analysis showed mostly n-alkanols (45.3%), monoacids (18.8%), triterpenes (9.7%), n-aldehydes (8.7%) and n-alkanes (7.7%). The high-temperature GC-MS and the ESI-MS/MS analyses showed the presence of ester waxes, namely alkyl alkanoates and alkyl coumarates. Alkyl alkanoates comprised esters of the hexadecanoic acid with n-alkanols ranging from C16 to C34. Alkyl coumarates included esters of coumaric acid with n-alkanols ranging from C16 to C32. The cuticular waxes were hydrolysed and the resulting organic and aqueous phases analysed by GC-MS. The hydrolysate showed a major increase in the quantities of n-alkanols, hexadecanoic acid and coumaric acid derived from the alkyl and acyl moieties from the ester waxes. A content of ester waxes of 38% was estimated based on the results from the GC-MS analysis of the non-hydrolysed and hydrolysed cuticular waxes. Alkyl alkanoates were analysed by ESI-MS/MS as [M + Li]+ adduct ions and the alkyl coumarates as [M - H]- deprotonated ions. The ESI-MS/MS analysis allowed the detection of a wider range of ester waxes than high-temperature GC-MS, and was shown to be a useful technique for the qualitative analysis of ester waxes from plant cuticles.     

Suberin--a biopolyester forming apoplastic plant interfaces

Suberized cell walls form physiologically important plant-environment interfaces because they act as barriers that limit water and nutrient transport and protect plants from invasion by pathogens. Plants respond to environmental stimuli by modifying the degree of suberization in root cell walls. Salt stress or drought-induced suberization leads to a decrease in radial water transport in roots. Although reinforced, suberized cell walls never act as absolutely impermeable barriers. Deeper insights into the structure and biosynthesis of suberin are required to elucidate what determines the barrier properties. Progress has been obtained from analytical methods that enabled the structural characterization of oligomeric building blocks in suberin, and from the opening of suberin research to molecular genetic approaches by the elucidation of the chemical composition and tissue distribution of suberin in the model species Arabidopsis.