In Vivo Quantification of Cell Coupling in Plants with Different Phloem-Loading Strategies  

Abstract

Uptake of photoassimilates into the leaf phloem is the key step in carbon partitioning and phloem transport. Symplasmic and apoplasmic loading strategies have been defined in different plant taxa based on the abundance of plasmodesmata between mesophyll and phloem. For apoplasmic loading to occur, an absence of plasmodesmata is a sufficient but not a necessary criterion, as passage of molecules through plasmodesmata might well be blocked or restricted. Here, we present a noninvasive, whole-plant approach to test symplasmic coupling and quantify the intercellular flux of small molecules using photoactivation microscopy. Quantification of coupling between all cells along the prephloem pathways of the apoplasmic loader Vicia faba and Nicotiana tabacum showed, to our knowledge for the first time in vivo, that small solutes like sucrose can diffuse through plasmodesmata up to the phloem sieve element companion cell complex (SECCC). As expected, the SECCC was found to be symplasmically isolated for small solutes. In contrast, the prephloem pathway of the symplasmic loader Cucurbita maxima was found to be well coupled with the SECCC. Phloem loading in gymnosperms is not well understood, due to a profoundly different leaf anatomy and a scarcity of molecular data compared with angiosperms. A cell-coupling analysis for Pinus sylvestris showed high symplasmic coupling along the entire prephloem pathway, comprising at least seven cell border interfaces between mesophyll and sieve elements. Cell coupling together with measurements of leaf sap osmolality indicate a passive symplasmic loading type. Similarities and differences of this loading type with that of angiosperm trees are discussed.

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

“…Within each sampling time, 30 guard cells in three independent epidermal fragments were observed using a TCS-SP2 confocal laser scanning microscope (excitation at 488 nm, emission at 500-530 nm; 25°C 6 1°C). To measure the relative fluorescence intensity of guard cells, acquired images were then analyzed via region of interest analysis, provided by the Leica software (Sieberer et al, 2009;Liesche and Schulz, 2012). Data were calculated as means 6 SE of pixel intensities.…”

Leaf Hydraulic Architecture and Stomatal Conductance: A Functional Perspective

“…Thus, crossmembrane flows in the photosynthetic mesophyll set up an interesting tradeoff between water transport and carbon uptake. This tradeoff is expected to exist for the production and movement of carbohydrate from the photosynthetic mesophyll to the phloem, as gradients in sugar within the cytoplasm should be mitigated by cytoplasmic stirring, and the limiting step in transport is thought to occur across the plasmodesmata between cells (Liesche and Schulz, 2012). In this way, the high rates of gas exchange associated with a mesophyll with a large surface area-to-volume ratio may feed back on, or even drive, vein spacing.…”

Sap flow and sugar transport in plants

“…Several have investigated phloem translocation speeds by further developing this dye tracing approach (Schumacher, 1948;Froelich et al, 2011;Jensen et al, 2011;Savage, Zwieniecki, and Holbrook, 2013). Dyes have also been used to quantify the capacity of loading and unloading pathways (Oparka et al, 1994;Liesche and Schulz, 2012), as well as the impact of wounding on a sieve element function (Schulz, 1992;Knoblauch et al, 2001).…”

“…Such approaches have been used successfully in this field, e.g., by Minchin, Thorpe, and Farrar (1993), Daudet et al (2002), and Lacointe and Minchin (2008), where more complex network configurations have been modeled. Here we follow Jensen, Liesche et al (2012) in the treatment of a single tube, but with different resistivities coming from axial pressure-driven flow along the tube and lateral osmotic flows. In reality, the tubes do not necessarily have the same diameter all the way, and the tubes do not just connect sugar producing leaves (sources) to the roots, but also to other sugar consuming sinks such as young leaves as in Lacointe and Minchin (2008).…”

“…The field has developed in large part due to advances in experimental techniques. We have seen recent adaptions of x-ray computed tomography (Brodersen et al, 2010), nuclear magnetic resonance velocimetry (Windt et al, 2006), and confocal laser microscopy (Schulz, 1992;Knoblauch and Bel, 1998;Froelich et al, 2011;Liesche and Schulz, 2012) to biophysical plant research that hold the promise for further progress. Simultaneously, the development and application of biophysical models and of synthetic, biomimetic systems, which have elucidated many of the mechanisms that drive and regulate sugar and water transport in plants, have originated through collaborations between researchers in biology, physics, chemistry, engineering, informatics, and many other fields.…”