Live Imaging of Companion Cells and Sieve Elements in Arabidopsis Leaves

Abstract

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Comparative Proteomics Analysis of Arabidopsis Phloem Exudates Collected During the Induction of Systemic Acquired Resistance

“…These included known phloem proteins as well as plastid-targeted proteins that are normally associated with photosynthetic, nonphloem cell types. While this may be indicative of unavoidable contamination caused by cellular leakage from nonphloem cells during exudation, companion cells and sieve elements do contain plastids that could harbor these proteins (Froelich et al, 2011;Cayla et al, 2015). In support of this idea, live imaging of Arabidopsis phloem recently determined that Rubisco-containing plastids occupy a large volume of the companion cell cytoplasm (Cayla et al, 2015).…”

“…While this may be indicative of unavoidable contamination caused by cellular leakage from nonphloem cells during exudation, companion cells and sieve elements do contain plastids that could harbor these proteins (Froelich et al, 2011;Cayla et al, 2015). In support of this idea, live imaging of Arabidopsis phloem recently determined that Rubisco-containing plastids occupy a large volume of the companion cell cytoplasm (Cayla et al, 2015). Alternatively, nucleus-encoded proteins with predicted plastid-localization peptides may localize to nonplastid subcellular sites in the phloem.…”

Are plants sentient?

“…With suitable clearing methods, the claim is up to 8 mm (De Grand & Bonfig, ). Methods for phloem imaging and in particular live imaging are available (Cayla et al, ; Furch et al, ; Truernit, ). Using green fluorescent protein (GFP) coupled proteins together with a specific sieve element promoter should greatly simplify examination and ease the assessment of anastomose numbers.…”

“…With suitable clearing methods, the claim is up to 8 mm (De Grand & Bonfig, 2015). Methods for phloem imaging and in particular live imaging are available (Cayla et al, 2015;Furch et al, 2009;Truernit, 2014).…”

Are phloem sieve tubes leaky conduits supported by numerous aquaporins?

“…Recent work has begun to elucidate the mechanisms of phloem flow (Gould et al, 2005; Knoblauch et al, 2016), wound response (Knoblauch et al, 2014), loading strategies (Rennie and Turgeon, 2009; Turgeon, 2010b; Liesche and Schulz, 2012; Comtet et al, 2017; Ross‐Elliott et al, 2017), as well as whole‐plant physiology and eco‐physiology (Woodruff, 2013; Savage et al, 2015). However, despite these advances, many questions remain unresolved as to phloem cell biology, function, and regulation (e.g., van Bel, 2003; Turgeon, 2010a; Cayla et al, 2015). This is complicated by the difficulty of studying phloem in vivo because artifacts are common when probing or making histological sections.…”