Spatial pattern of long-distance symplasmic transport and communication in trees

Abstract

Symplasmic short- and long-distance communication may be regulated at different levels of plant body organization. It depends on cell-to-cell transport modulated by plasmodesmata conductivity and frequency but above all on morphogenetic fields that integrate a plant at the supracellular level. Their control of physiological and developmental processes is especially important in trees, where the continuum consists of 3-dimensional systems of: 1) stem cells in cambium, and 2) living parenchyma cells in the secondary conductive tissues. We found that long-distance symplasmic transport in trees is spatially regulated. Uneven distribution of fluorescent tracer in cambial cells along the branches examined illustrates an unknown intrinsic phenomenon that can possibly be important for plant organism integration. Here we illustrate the spatial dynamics of symplasmic transport in cambium, test and exclude the role of callose in its regulation, and discuss the mechanism that could possibly be responsible for the maintenance of this spatial pattern.

Trees are extremely large organisms built up of billions of cells that need fast and precise communication to successfully perform complex physiological and developmental processes. We still do not know how the long-distance transport of signal molecules is regulated, although it seems possible that the secondary xylem plays a crucial role in this process. This tissue is composed primarily of thick-walled dead cells water-conducting and mechanical but also of living cells of horizontal rays and axial parenchyma. The living cells are all united within the frame of a large 3-dimensional continuum within which the symplasmic transport of nutrients can be followed with application of fluorescent tracers.^1-4 Living xylem parenchyma cells are involved in numerous processes such as nutrient transportation,⁵ reserve storage accumulation, and remobilization,^6,7 as well as water conduction and embolism repair.^8-10 They also function as defense mechanisms acting against vascular pathogens¹¹ in wood structure determination^12,13 and play an important role in the biomechanics of the tree.^14,15 Therefore it is likely that they also are crucial in long-distance transport and signalization required for the maintenance of proper function and thus for the survival of the whole plant.

Experiments with fluorescent tracers, like carboxyfluorescein and HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid), loaded through the vascular system to young branches of Acer pseudoplatanus and Populus tremula x tremuloides, were harvested in the growing season, thus with active cambium (for details, see ref. 3), showed an intensive movement of applied tracers within the secondary xylem and cambium. The presence of dye in the axial and radial xylem parenchyma cells illustrated the 3-dimensional continuum of living cells in the secondary xylem region (Figs. 1A and B), but most importantly, revealed the existence of a specific spatial pattern in the cambial region (Figs. 1C-E). Along the analyzed stems, areas (domains) emerged with surprisingly uneven distribution of fluorescent symplasmic tracers in the cambial ray and fusiform cells. In one such type of domain, fluorescent signal was visible mainly in fusiform cells, whereas cambial rays were almost completely devoid of the signal (Fig. 1C). In the second type, cambial rays were almost completely filled with the tracer (Fig. 1D). One type followed another along the cambial surface in a clearly periodic manner (Fig. 1C-E). Changes in the distribution of fluorescent tracers in cambial rays along the maple (n = 7) and poplar (n = 5) branches were continuous, regular, repeatable, and statistically important (for details, see ref. 3), suggesting that the observed phenomenon presents an intrinsic process of spatial regulation of molecular transport, perhaps even of a wavy nature as implied by its regular periodicity. What kind of mechanism can regulate such peculiar spatial changes in the symplasmic transport along woody branches?

Figure 1.Acer pseudoplatanus – spatial distribution of carboxyfluorescein in the living cells of the xylem and cambial regions. (A-B) Distribution of the dye in the living parenchyma cells of the secondary xylem on radial (A) and transverse (B) hand-sections. The fluorescent signal is present in the symplast of xylem rays. (C-E) Spatial changes in dye distribution between the cambial cells along the analyzed maple branch. Tangential hand-sections localized ca. 6 cm (C), 33 cm (D), and 58 cm (E) from the site of dye application. Fluorescent signal is absent in cambial rays in the lower part of the stem (C). In the middle part of this branch, cambial rays are almost completely filled with the dye (D), while in the upper part, rays are again only partially stained (E). Abbreviations: Cr, cambial ray; F, fusiform cell; Xr, xylem rays; V, vessel. Scale bar: 50 µm.

Symplasmic tracers spread in the living plant cell systems via plasmodesmata.^16-18 It is then plausible to assume that the observed spatial differences in tracer distribution in cambium are determined by the factors that regulate plasmodesmata conductivity. One of the first possible candidates is callose, which is known, especially under stress conditions, to accumulate abundantly in the neck region of plasmodesmata.^17,19,20 We performed callose localization in the cell walls between cambial ray and fusiform cells in maple branches (n = 10) using 0.1% aqueous solution of aniline blue and β-1,3-glucan immunolocalization (5% skim milk as blocking buffer for 60 min, overnight incubation with 1:100 dilution of the primary antibody, and subsequent 2 h incubation with 1:200 dilution of the secondary antibody Alexa Fluor 488; both antibodies dissolved in PBS buffer with 1% skim milk) according to the modified protocols of refs. Seventeen and 21. Our results showed that callose deposited in the primary pit fields was evenly distributed in all the cells of the cambial region, regardless of cell type (fusiform vs. ray cell) and sample localization in the stem (Fig. 2). Equal callose distribution between all cambial cells along analyzed branches was contrary to the pattern of symplasmic dye dispersal in cambial region, where clear differences in carboxyfluorescein distribution between the cells had been found. Thus, our results prove that callose accumulation, at least at the level of its detection with the methods applied, is not responsible for spatial differences in the symplasmic tracers presence in the cambial region.

Figure 2.Acer pseudoplatanus – callose localization in cambial region. Callose was visualized using aniline blue staining (A, B) and β-1,3-glucan immunolocalization (C). (A, B) Tangential hand-sections of cambium. Equal distribution of callose is visible in the primary pit fields in all types of cambial cells about 3 cm (A) and 15cm (B) above the cut end of the branch. (C) Immunolocalization of β-1,3-glucan confirms its equal distribution between cambial cells. Abbreviations: Cr, cambial ray; F, fusiform cell. Scale bar: 25 µm (A, B) and 10 µm (C).

It is generally accepted that fast and long-distance signaling between cells in a multicellular organism must involve advanced and sophisticated mechanisms. For instance, it can be governed by morphogenetic fields acting at the supracellular level. Some of these fields were previously described in cambium, e.g., the periodic pattern of migrating, alternate domains, in which the orientation of cellular events, such as intrusive growth and anticlinal cell division, is opposite and strictly controlled^22-25 or the spatial pattern of periodically changing auxin concentration.^26-28 Moreover, it was recently shown that the regulation of the whole-sieve tube turgor has a wavy nature and is based on a rapid transfer of information on pressure/concentration waves in phloem.²⁹ These findings seem to support the hypothesis that regular, wavy patterns can indeed be significantly involved in the regulation of the different developmental processes in plants. It is likely that symplasmic transport in the xylem and cambial region also undergoes similar spatial variations, as shown in our experiments. We suggest that the uneven distribution of symplasmic tracers in the cambial region along the branches results from differences in the intensity of symplasmic transport between living cells of secondary xylem. Probably, the radial transfer of fluorescent tracer via xylem rays is intensive in these cambial regions where the dye is detected as abundant in all types of cells. Conversely, in the cambial regions where ray cells are almost completely devoid of the signal, radial dye transfer to the cambium via xylem rays is limited. According to this hypothesis, the spatial pattern observed in the cambial region suggests the precise regulation of symplasmic transport in wood, which governs axial and radial solute transfer via the xylem parenchyma cells. Thus, a deciphering of the symplasmic routes in woody species and their regulation will be our aim in future studies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.