Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set

Abstract

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.

Figure 1

New plant expression vectors for the high-throughput generation of fluorescent fusion constructs. (a) The pNIGEL vector series drives expression in Arabidopsis from the endogenous, intron-bearing UBQ10 promoter. Based on the pGREENII plant transformation vectors, pNIGELs carry an Amp, instead of Kan, resistance for compatibility with the pUNI open reading frame (ORF) library. The loxP site following the different fluorescence-tag sequences allows for translational fusion to the N-terminus of pUNI ORFs. (b) Schematic diagram of the recombination/selection procedure, as described in Liu et al. (1998). CRE recombinase mediates a single recombination event that leads to fusion of the pUNI and HOST vectors. Recombined products are selected on Kan/Amp double-selection plates. In addition, the origin of pUNI is incompatible with standard Escherichia coli strains. (c) The UBQ10 promoter drives detectable expression in many cell types of interest. The YFP signals from a plasma membrane-localized marker protein are presented here. The expression is homogenous, and shows no sign of patchiness or variability in expression levels, as is often observed in 35S-driven marker lines. Scale bars: early embryo, 10 μm; all other panels, 50 μm.

Figure 2

The multicolor Wave markers allow the rapid, parallel visualization of subcellular structures in planta. (a) The YFP-signals of the 20 selected Wave lines all show reproducible staining of membrane compartments, and could all be propagated as homozygotes into the T₄ generation. Root meristem cells at the onset of elongation are shown here. Most of the central membrane compartments, such as Golgi, vacuole or plasma membrane, are covered by multiple markers. In addition, a number of markers highlight compartments of pleiomorphic, dotty appearance, many of which apparently define subpopulations of endosomes. Scale bar: 10 μm. (b) Examples of signals of the same compartment marker tagged with Cerulean (blue), YFP (yellow) or mCherry (red). Note that all three fluorophores allow clear spectral separation, based on their excitation/emission characteristics, thereby circumventing the use of spectral-unmixing algorithms. Scale bars: 10 μm.

Figure 3

Functional mapping of membrane compartments by quantitative and combinatorial co-localization. (a) Co-localization of YFP-labeled compartments (green) with the FM4-64 lipidic, endocytic tracer (red) after 5–10 min of uptake. Some compartments can be clearly defined as co-localizing or non-colocalizing, whereas many show partial co-localization. (b) As in (a) but after 60–90 min of FM4-64 uptake. (c) Co-localization after co-treatment of FM4-64 with 25 lM brefeldin A (BFA) for 60 min. Partially FM4-64 co-localizing compartments can be distinguished by their different sensitivity towards the drug, compared with FM4-64. Some partially co-localizing compartments are largely resistant to BFA, whereas others completely aggregate in the BFA compartment, and strongly co-localize with FM4-64. (d) Quantifcation of co-localization based on the images shown in (a–c), measured as intensity correlation of pixels (ICQ), with a value of zero indicating no co-localization, and a value of 0.5 indicating complete overlap. Co-localization measured after 5 min (yellow) and 60 min (orange) of FM4-64 uptake. Note that some compartments show no increase in overlap, or even apparent separation from FM4-64, in response to BFA, whereas others become completely confounded. Also, note the lack of a time-dependent increase of co-localization with FM4-64. (e) Co-localization YFP-tagged Wave lines (Y) with mCherry-tagged lines (R), either with or without BFA, treated as in (d). Scale bar for panels in (a–c) (in the upper left panel in a): 10 lm. Scale bar for (d) (in the leftmost panel): 5 μm. (f) Immunogold labeling of Wave line 33Y (RabD2b) using antibodies against the additional 3 × myc epitope in the Wave Y lines. Note that most of the label appears in the trans-Golgi network (TGN) region and beyond, whereas only some of the labeling can be seen thoughout the stack. (g) Immunogold labeling of Wave line 18R (AtGot1p) using antibodies against RFP. Scale bar: 500 nm.