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Cryo-focused ion beam scanning electron microscopy (Cryo-FIBSEM) has become essential for preparing cryo-lamellae. Here, we present a series of improvements that speed up and enhance the efficiency of the Serial Lift-Out and SOLIST (Serialized On-grid Lift-In Sectioning for Tomography) procedures. We extend the cryo-FIBSEM session to 5 days and eliminate the need of copper or gold block intermediates and reduce curtaining effects. Finally, we report a routine to target lamellae with a precision of approximately 1 µm in X/Y/Z. We demonstrate the power of our improvements by targeting Casparian strips, suberin lamellae, as well as xylem vessels of Arabidopsis thaliana roots. This requires reaching a target of 5 micrometers in a 3 mm long and 80-120 µm thick root section. Despite ice formation in vacuoles and to some degree in the cytosol, plasma membranes and cell walls are remarkably well preserved, providing stunning insights into the native, hydrated nano-structure of plant cell walls, previously only observable with contrasting agents in a dehydrated state. This study introduces improvements to cryo-FIBSEM to enable the preparation of high-quality cryo-lamellae from complex plant samples. The 3D images of Casparian strips, suberin lamellae, and xylem vessel walls provide insights into cell wall architecture.

The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination, it protects the seedling from water loss and is, thus, critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the ABNORMAL LEAF SHAPE1 subtilase and the two GASSHO receptor-like kinases. We show that a sulfated peptide, TWISTED SEED1 (TWS1), acts as a GASSHO ligand. Cuticle surveillance depends on the action of the subtilase, which, unlike the TWS1 precursor and the GASSHO receptors, is not produced in the embryo but in the neighboring endosperm. Subtilase-mediated processing of the embryo-derived TWS1 precursor releases the active peptide, triggering GASSHO-dependent cuticle reinforcement in the embryo. Thus, a bidirectional molecular dialogue between embryo and endosperm safeguards cuticle integrity before germination.

Casparian strips are ring-like cell-wall modifications in the root endodermis of vascular plants. Their presence generates a paracellular barrier, analogous to animal tight junctions, that is thought to be crucial for selective nutrient uptake, exclusion of pathogens, and many other processes. Despite their importance, the chemical nature of Casparian strips has remained a matter of debate, confounding further molecular analysis. Suberin, lignin, lignin-like polymers, or both, have been claimed to make up Casparian strips. Here we show that, in Arabidopsis, suberin is produced much too late to take part in Casparian strip formation. In addition, we have generated plants devoid of any detectable suberin, which still establish functional Casparian strips. In contrast, manipulating lignin biosynthesis abrogates Casparian strip formation. Finally, monolignol feeding and lignin-specific chemical analysis indicates the presence of archetypal lignin in Casparian strips. Our findings establish the chemical nature of the primary root-diffusion barrier in Arabidopsis and enable a mechanistic dissection of the formation of Casparian strips, which are an independent way of generating tight junctions in eukaryotes.

Endosomes are highly dynamic membrane systems that receive endocytosed plasma membrane proteins and sort them for either degradation or recycling back to the cell surface. In addition, they receive newly synthesised proteins destined for vacuolar/lysosomal compartments. Sorting in the endosomes is necessary for the establishment and maintenance of cell polarity and it is needed to control levels and function of receptors and transporters at the cellular surface. Both processes are crucial for correct cell behaviour during tissue and organ development and for intercellular communication in general. It has therefore become an imperative to investigate structure and function of the endosomal system if we want to obtain a deeper mechanistic understanding of signal transduction and development. This review will compare our current understanding of endosomal trafficking in animals and yeast with what is known in plants, and will highlight some important breakthroughs in our understanding of the role of endosomes in signal transduction and multicellular development in Drosophila, as well as in Arabidopsis.

Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Brassinosteroids (BRs), the polyhydroxylated steroid hormones of plants, regulate the growth and differentiation of plants throughout their life cycle. Over the past several years, genetic and biochemical approaches have yielded great progress in understanding BR signaling. Unlike their animal counterparts, BRs are perceived at the plasma membrane by direct binding to the extracellular domain of the BRI1 receptor S/T kinase. BR perception initiates a signaling cascade, acting through a GSK3 kinase, BIN2, and the BSU1 phosphatase, which in turn modulates the phosphorylation state and stability of the nuclear transcription factors BES1 and BZR1. Microarray technology has been used extensively to provide a global view of BR genomic effects, as well as a specific set of target genes for BES1 and BZR1. These gene products thus provide a framework for how BRs regulate the growth of plants.

One of the mechanisms by which signalling molecules regulate cellular behaviour is modulating subcellular protein translocation. This mode of regulation is often based on specialized vesicle trafficking, termed constitutive cycling, which consists of repeated internalization and recycling of proteins to and from the plasma membrane. No such mechanism of hormone action has been shown in plants although several proteins, including the PIN auxin efflux facilitators, exhibit constitutive cycling. Here we show that a major regulator of plant development, auxin, inhibits endocytosis. This effect is specific to biologically active auxins and requires activity of the Calossin-like protein BIG. By inhibiting the internalization step of PIN constitutive cycling, auxin increases levels of PINs at the plasma membrane. Concomitantly, auxin promotes its own efflux from cells by a vesicle-trafficking-dependent mechanism. Furthermore, asymmetric auxin translocation during gravitropism is correlated with decreased PIN internalization. Our data imply a previously undescribed mode of plant hormone action: by modulating PIN protein trafficking, auxin regulates PIN abundance and activity at the cell surface, providing a mechanism for the feedback regulation of auxin transport.

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation 1 , 2 . This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown 3 . It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane 4 . The polar localization of efflux carrier candidate PIN1 supports this model 4 . Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.

The plant hormone auxin is transported in a polar manner along the shoot-root axis, which requires efflux carriers such as PIN1. Asymmetric localization of PIN1 develops from a random distribution in Arabidopsis early embryogenesis. Coordinated polar localization of PIN1 is defective in gnom embryos. GNOM is a membrane-associated guanine-nucleotide exchange factor on ADP-ribosylation factor G protein (ARF GEF). Thus, GNOM-dependent vesicle trafficking may establish cell polarity, resulting in polar auxin transport.