Early development of epiphytic roots: perspectives based on the composition of the velamen cell wall

ABSTRACT

The velamen, a root structure of some epiphytic species for water uptake, usually is stratified epidermis consisting of dead cells. In general, its cell walls exhibit variation during development, including in thickness and amount and type of impregnated substances. These changes result in diverse physical and chemical properties that can serve in water and nutrient uptake, as well as in mechanical support and protection. On this basis, the main objective of the current study was to describe the composition of the cell walls of the velamen during the development of the roots of four species of Cattleya. Anatomical, histochemical and immunocytochemical analyses were performed with samples (n=3 individuals per species) of meristematic, developing, and mature regions of the root. The development of the primary wall led to the deposition of pectins as highly methylesterified homogalacturonans, which were demetihylesterified with maturation of the velamen. The deposition of lipids (and subsequently lignins) in velamen cells marked a transition stage to the formation of the secondary wall, which gives rigidity to the tissue. For the first time, we showed that the deposition of lipids and lignins began close to the exodermis in the direction of the epivelamen.

Keywords:
Cattleya; Orchidaceae; pectins; root meristem; velamen development

Introduction

The morphological and anatomical features of the roots of epiphytic orchids have evolved in order to improve water and nutrient uptake, since these plants grow under severe fluctuations of these resources (Krauss 1948; Proença & Sajo 2008; Macedo 2013). One of these remarkable features is the velamen, considered to be an adaptive trait responsible for the efficient absorption of water and nutrients (Engard 1944; Silva & Milaneze-Gutierre 2004; Zotz & Winkler 2013). The velamen is the most external root tissue of many epiphytic groups and of other related terrestrial plants, mainly monocots (Zotz & Winkler 2013). Usually, the velamen consists of a stratified epidermis resulting from periclinal divisions of the protodermis, dead at maturity, and composed of compactly arranged cells with different secondary thickenings in the cell walls (Engard 1944; Pridgeon 1987; Joca et al. 2020). The composition of cell walls during plant organ development, including secondary thickenings, seems to be impacted by abiotic factors that change the physical, chemical and physiological properties of the cell walls (Albersheim et al. 2010; Le Gall et al. 2015).

The dynamic and complex arrangement of the cell wall components, associated with diverse physicochemical properties, triggers many signaling events that are responsible for cellular functions, such as patterns of organ and tissue development and growth (Lorenzo et al. 2019). This differential arrangement occurs early during cell wall development (Burton et al. 2010), leading to primary and/or secondary wall formation (Kraus et al. 2012; Canteri et al. 2019; Kang et al. 2019). The primary cell walls (PCW) are formed during cell division and early expansion, and are mainly composed of cellulose, hemicelluloses and pectins. The cellulose microfibrils are embedded in a hydrated matrix of polysaccharides which forms a three-dimensional network responsible for the physical, chemical and physiological properties of cell walls (Cosgrove 2005; Amar et al. 2010; Albersheim et al. 2010). The formation of the secondary cell wall occurs by lignin deposition, especially after cell expansion (Albersheim et al. 1996; Cosgrove & Jarvis 2012; Fahey et al. 2017). Although formed by dead cells at maturity, with expressive secondary thickenings, the cell walls of the velamen retain a pectic matrix. These pectins with different degrees of methylesterification seem to be essential for stability, mechanical support of the velamen cells, and even the dynamics of nutrient absorption (Joca et al. 2020). The main domains of pectins are the homogalacturonans (HGs) (Ridley et al. 2001; Willats et al. 2001), which play an important role in gel formation that in turn increases water retention and, with the presence of Ca^{2 +}, may lead to cell wall stiffening (Liu et al. 2013; Albersheim et al. 2010). They are also related to water absorption by apoplastic flow and may be involved in mechanical protection and reduction of water loss (Albersheim et al. 1996; Willats et al. 2000). The role of each component of the cell wall in the roots of orchids and the degree of pectin methylesterification may be involved in the capacity of water absorption, retention and transport (Willats et al. 2001; Joca et al. 2017; 2020). In addition, the pectic matrix can act as a moderator of cationic exchanges since naturally negatively charged carboxyl groups (COO-) can attract positively charged compounds (Zotz & Winkler 2013; Joca et al. 2020).

The deposition of suberin and lignins occurs in the final stages of development of the primary wall and during the formation of the secondary wall of velamen cells. Both are first placed mainly in the middle lamella of the cell, related to adaptations and specialized physiological functions of tissue (Monties 1989; Campbell & Sederoff 1996; Zeier & Schreiber 1997). Lignins provide mechanical stability, whereas suberin, a highly hydrophobic substance, impermeabilizes the cell walls, thus increasing the apoplastic flow in the velamen and obstructing this flow in the exodermis and endodermis (North & Nobel 1994; Enstone & Peterson 2005; Franke & Schreiber 2007; Joca et al. 2017). The compactation and chemical composition (including suberin and often the addition of lignin) of the basic cells of the exodermis and endodermis force the passage of water and nutrients through the passage cells as a preselection of solutes before reaching the vascular cylinder (Zimmermann et al. 2000; Hose et al. 2001; Franke & Schreiber 2007). Lignins also play a role in apoplastic flow throught the cortex (sometimes as a barrier or as structures leading towards the stele) (Joca et al. 2017) and, together with lignified cells of the exodermis, provide mechanical protection and decrease the loss of water from the cortex into the environment (Sanford & Adanlawo 1973; Benzing et al. 1982; Ma & Peterson 2003).

The adventitious roots in orchids have root apical meristems (RAM) with their epidermis associated with cortex and not with rootcap production (Heimsch et al. 2008). In orchids, RAM are closed, with cortical-epidermal initial cells or cortical initials separated from the epidermal initials. In addition, RAM show a bulge of the outer cortical initials towards the lateral rootcap (Heimsch et al. 2008). The early development of the roots from the RAM is decisive in order to determine the functions assigned to the roots. The periclinal divisions will determine the number of cell layers of the velamen (Engard 1944), while cellular development will end with the formation of a secondary cell wall which maximizes water and nutrient uptake (Zotz & Winkler 2013; Joca et al. 2017; 2020). Thus, we believe that the time of velamen differentiation may be related to the composition of the cell walls (including suberin impregnation and/or secondary wall development). In the present study, we describe the composition of the velamen cell wall during early development and up to root maturation of four species of Cattleya in an attempt to understand the patterns of pectin, lipid and lignin deposition. Cattleya is a native genus from Brazil, composed by epiphytic species, and with a velamen usually described with many cell layers.

Materials and methods

Plant material

In the current study we used four species of Cattleya: C. nobiliorRchb.f., C. schillerianaRchb.f., C. velutina Rchb.f. and C. walkerianaGardner, kept in the greenhouse of Universidade Federal de Uberlândia, Uberlândia, Brazil. The greenhouse was maintained in natural conditions. The temperature ranged from 12 °C to 33 °C (annual average of 22 °C), and the maximum photosynthetic active radiation was 250 µmol m^-2 s^-1. The plants received free water supply until percolation in the pots. Fragments of the root apices and mature roots (3 cm from the apex) were collected and fixed in 70 % FAA (formaldehyde: acetic acid: 70 % ethanol, 1:1:18, v:v:v) for 48 hours, and subsequently stored in 70 % ethanol (Kraus & Arduin 1997).

Histological analyses

Samples of Cattleya roots (three individuals per species) were free-hand sectioned at 3 cm from the apex to confirm the mature condition of the velamen. The sections were cleared with 50 % sodium hypochlorite (Kraus & Arduin 1997) and stained with 1 % safranin alcoholic solution and 0.5 % Astra blue (1:9 v/v) (Bukatsch 1972). Samples were glued on slides, assembled with glycerinated gelatin of Kaiser (Johansen 1940) and the material was photographed with a Leica® DM500 photomicroscope coupled to a Leica® ICC50 HD digital camera. The transverse area of ​​the roots, the number of cell layers and the thickness of the velamen, as well as the area of ​​the root occupied by the velamen were determined in ten roots with free growth (not adhered to a substrate) per species selected from the three individuals of each species. The Image J software was used (National Institute of Health, EUA) to measure three different points of each section and the mean value was calculated and used as a valid value. Data showed normal distribution and were analysed using ANOVA with posterior Tukey test to compare root area and velamen thickness thickness/area. We assumed differences below 5 % of probability.

RAM structure was analyzed on 4 cm long fragments from the apices, which were divided into three morphological regions; (a) meristematic region (purplish/brown region of the root closest to the RAM), (b) developing region (green, but with many white patches) and (c) mature region (completely white) (Fig. 1A). All samples were dehydrated in ethanol series, pre-infiltrated with 95 % ethanol and base resin 1:1 (v/v) for approximately 36 hours and embedded in base resin according to Historesin® manufacturer instructions (Leica® Biosystem). Sections (~5 µm thick) were cut with a microtome (YD315, ANCAP, Brazil) using disposable high quality stainless-steel razors, stained with 0.05 % toluidine blue in 100 ml of 0.1 M phosphate buffer, pH 4.0 (O’Brien et al. 1965) and analyzed with a Leica® DM500 photomicroscope coupled to a Leica® ICC50 HD digital camera.

For scanning electron microscopy (SEM) analyses, root fragments of the orchid species (0.5 cm - meristematic region; and 3 cm from the apex - mature region) were fixed in Karnovsky (4 % paraformaldehyde, 0.01 M glutaraldehyde and 0.2 M phosphate buffer, pH 7.2) (5:3:2, v:v:v) (Karnovsky 1965, modified by Kraus & Ardiun 1997) for at least 48 h, washed and stored in 70 % ethanol and phosphate buffer. The samples were immersed in osmium tetroxide for 1 minute, dehydrated in ethyl series and submitted to CO₂ critical point (EMCPD 300, Leica, Vienna, Austria). The fragments were metallized with gold (20 to 30 nm thick) using a Sputter Coater SCD O50, BalTec (Quorum, New York) and analyzed by SEM (VEGA 3, TESCAN, Czech Republic) calibrated at an acceleration of 10 kV voltages (as used by Pridgeon et al. 1983; Porembski & Barthlott 1988).

Histochemical analyses

For lipid detection in the velamen, samples were incubated in a 0.5 % ethanolic solution (80 % ethanol) of Sudan III for 20 to 30 minutes (Sass 1951). For histolocalization of lignins, the sections were incubated with a 1 % phloroglucinol ehtanolic solution (95 % ethanol) and 25 % hydrochloric acid solution for five minutes (Johansen 1940). We also used a DAPI filter (excitation spectrum: 385-400 nm) in an epifluorescence system (Chomicki et al. 2014; Joca et al. 2017) of a Leica® DM4000B microscope coupled to a Leica® DFC3000 G camera. For all analyses, blank sections were examined in order to confirm false-positive results, as recommended by the cited authors. All sections were mounted in distilled water and photographed using a Leica® DM500 microscope coupled to a Leica® ICC50 HD digital camera.

Immunocytochemical analyses

Samples of the apices and mature roots were embedded in Leica^® historesin, sectioned (10 μm) with a rotatory microtome (YD315, ANCAP, Brazil) and incubated in block solution (3 % powdered milk protein, Molico^®, in phosphate buffered saline (PBS), pH 7.1) for 30 minutes to prevent crosslinking. Samples were then incubated with the following primary monoclonal antibodies: JIM 5 - for HG epitopes with up to 40 % methylesterification and JIM7 - for HGs epitopes with up to 80 % methylesterification (Centre for Plant Sciences, University of Leeds, UK), for one hour. Next, the samples were washed in PBS and incubated with IgG anti-rat FITC secundary antibody (Sigma-Aldrich®) (1:100 in 3 % milk protein/PBS) for two hours in the dark. As a control, the primary antibody was suppressed. After a new washing in PBS, the sections were mounted in glycerin/distilled water (1:1) and examined under a Leica® DM500 microscope with an epifluorescence system coupled to a Leica® ICC50 HD digital camera, using an FITC filter (Blake et al. 2006; Hervé et al. 2011; Leroux et al. 2018 - modified).

Results

In the apices of the four Cattleya roots, the velamen contained living cells close to the RAM, but development began very early to lead to maturity, when some dead cells could be noted. The complexity of the velamen’s cell wall increased with maturity, including the deposition of lipids and lignins. During primary development, we observed the presence of high-methyesterified pectins predominantly in younger tissues. These pectins were also detected in the mature velamen of C. velutina and C. walqueriana. Low methylesterified HGs predominated in mature velamen of C. velutina, C. walqueriana and C. nobilior.

Structural analysis of Cattleya roots

The roots of Cattleya nobilior had the smallest transverse sectional area (5.21 ± 1.6 mm²), while C. velutina had the most calibrous roots (11.80 ± 2.4 mm²), and C. schilleriana and C. walquerian had similar areas (~ 7.8 ± 1.9 mm²) (Tab. 1). The roots were cylindrical in shape, while the roots in contact with a substrate were slightly flattened. The meristematic and developing regions (characterized by still green or sometimes brownish coloration) at the root apex were about 11.4 ± 0.14 mm long in all species, becoming white at maturity (Fig. 1A). A slight purplish/brown coloration was observed (up to 1.0 cm of the RAM) in all species due to anthocyanins in the vacuoles of the ground meristem and/or cortical parenchyma cells (Fig. 1B-C).

Table 1
Root area and the investment in velamen by Cattleya nobilior, C. schilleriana, C. velutina and C walqueriana. All species were kept in a greenhouse for approximately one year.

	Root area (mm²) in transverse section	Area of velamen in transverse section (mm²)	Velamen thickness (µm)
Cattleya nobilior	5.21 ± 1.6c	2.47 ± 1.0b	380.2 ± 83.8ª
Cattleya schilleriana	7.87 ± 2.0ab	2.64 ± 0.9b	333.5 ± 93.3ª
Cattleya velutina	11.80 ± 2.4ª	4.00 ± 1.0ª	407.2 ± 93.2ª
Cattleya walkeriana	7.88 ± 1.7b	3.41 ± 0.9ab	436.9 ± 81.1ª
P	<0.0001	<0.005	>0.05

• The data compared species using ANOVA with posterior Tukey test considering differences below 5 %.

Figure 1
Root apex of three distinct species of Cattleya. The roots were divided into three morphological regions: (1) meristematic region (RAM), (2) developing region and (3) mature region. A. The three morphological regions of C. walqueriana root. B. Longitudinal section of C. nobilior root showing the presence of anthocyanins in the meristematic region (ground meristem). C. A detail of the cells of the ground meristem in C. walqueriana roots with anthocyanin-filled vacuoles.

The four species showed the typical root structure of other epiphytic orchids, composed of a vascular cylinder, cortical parenchyma limited by endodermis and exodermis, and a multilayered velamen (Fig. 2). The vascular cylinder had 13 to 17 strands of xylem and phloem in C. nobilior, 12 to 18 in C. schilleriana (Fig. 2A), 16 to 20 in C. velutina and 13 to 18 in C. walqueriana. In all species, the vascular cylinder was limited by an O-thickened endodermis (Fig. 2B) impregnated with lipids (Fig. 2C) and lignins (Fig. 2D), except in passage cells. The cortical parenchyma had cells with different types of cell wall thickenings, such as uniform thickenings - always impregnated with lipids and/or lignins (Fig. 2C-E), or phi-thickenings in C. schilleriana, C. walqueriana and C. velutina (Fig. 2F). The exodermis externally limits the cortical parenchyma and, just like the endodermis, had cell walls impregnated with lipids and/or lignins (Fig. 2G-J).

Figure 2
Root anatomy of four different species of Cattleya. A. Overview of a vascular cylinder in C. schilleriana showing 13 xylem and phloem strands. B. Detail of a vascular cylinder, with xylem and phloem strands and O-thickened endodermis. C. Cross sections stained with Sudan III (red color) in C. nobilior roots. O-thickenings in the endodermis (arrows) and isolated cells in the cortical parenchyma with uniform thickenings (*) impregnated with lipids. D-E. Lignins detected with a fluorescent microscope using a DAPI filter. D. O-thickenings (arrows) in the endodermis, and E. uniform thickenings (*) in the parenchymatic cortex. F. Parenchymatic cortex with phi-thickenings (arrows). G. Overview of a C. nobilior root. H. Detail of the exoderm with U-thickenings in C. schilleriana. I. U-thickened exodermis and velamen cells impregnated with lignin (image using DAPI filter) in C. velutina. J. U-thickened exodermis impregnated with lipids in C. schilleriana. (ED - endodermis, Ex - exodermis, PC - parenchymatic cortex, V - velamen, VC - vascular cylinder).

The velamen occupied about 33 % of the total root area in C. schilleriana and C. velutina, and almost 50 % of it in C. nobilior and C. walkeriana (Tab. 1).The number of velamen cell layers varied with the position around the circumference of the root; the regions flattening with the substrate had fewer numbers of layers, while the free growing areas showed a higher number of layers. Four to eight cell layers were observed in C. nobilior, two to six in C. schilleriana, four to nine in C. velutina, and four to seven in C. walqueriana.

The RAM comprised the “closed” type with a slight bulge of the outer cortical initials towards the lateral rootcap, curving along the base of the columella (Fig. 3A, B, C). The cells of the protodermis divided periclinally still under the root cap, with many layers under its lateral margins (Fig. 3B). Lignins and lipids were not detected in the velamen cells close to the RAM, but were gradativelly deposited in the velamen cell walls, lipids first and mainly around the exodermis, and lignins later in all cell walls of the velamen. The mature cell walls of all studied species had similar thickening patterns, with many pores (Fig. 3D, E, F). Still in the green region of the apex (developing region), about 1 cm from the RAM, it was possible to observe many dead cells in the velamen forming white patches (Fig. 1A). At approximately 12 mm from the RAM, the velamen was fully mature in all species, with all cells dead, completely filled with air and of white color.

Figure 3
Root apical meristem (RAM) (in longitudinal sections) and mature velamen (transverse sections using scanning electron microscopy) in Cattleya. A. Overview of the apex of the C. velutina root. B. Detail of the young velamen still protected by the root cap. C. Detail of the RAM of the closed type. D. Mature velamen with dead cells in C. nobilior. E. Detail of the velamen cells with wall thickenings and many pores in C. nobilior. F. Detail of the velamen cells with wall thickenings in C. velutina.

Inmunocytochemical analysis: distribution and degree of methylesterification of pectins in the velamen

The epitopes of HGs did not label the RAM in any of the species of Cattleya, but labeled the young and mature tissue. Except for C. nobilior, HG epitopes with a high degree of methylesterification recognized by JIM7 labeled the velamen cells in young regions. In mature roots of C. nobilior, these HGs with a high degree of methylesterification weakly labeled the cell junctions of the velamen (Fig. 4A), but strongly labeled the region around all cell walls of C. velutina (Fig. 4B) and C. walqueriana. No epitopes were recognized by JIM7 in the velamen cells of Cattleya schilleriana. About 1 cm from the apex, when the velamen cells began to die, the epitopes of HGs with up to 40% methylesterification begins to appear, weakly recognized by JIM5, in the cellular junctions of C. nobilior (Fig. 4C). In completely mature roots, these epitopes recognized by JIM5 strongly labeled the velamen cells of C. walqueriana, C. velutina (Fig. 4D) and C. nobilior. In C. schilleriana, we only observed lower intensity of labeling in velamen cells (Fig. 4E).

Figure 4
Homogalacturonans of high and low methylesterification labeled by JIM7 and JIM5 antibodies, respectively, in roots of Cattleya. The positive reactions are represented by the green color in all images. The blue color (A, C and E) comes from an overlap processing using DAPI filter and shows lignin. A. Positive result for JIM7 at the cellular junctions of the velamen in C. nobilior in the developing region of the roots. B. Positive result for JIM7 in the mature region of the velamen in C. velutina roots. C. Very weak result for JIM5 in the developing region of C. nobilior roots. D. Positive result for JIM5 in the mature region of the velamen in C. velutina. E. Weak result for JIM5 in the mature region of the velamen in C. schilleriana. (Ex - exodermis, PC - parenchymatic cortex, V - velamen).

The presence of lipids in the velamen cell walls

Histochemical assays showed deposition of lipids in the cell walls during the development of the velamen (Fig. 5). Deposition gradually begins near the exodermis (Fig. 5A), increasing throughout the velamen with maturation, except in the epivelamen (Fig. 5B). In C. velutina, red color for lipids was observed in all cell walls of the velamen, even with maturation (Fig. 5B-C). However, in the other three species, the construction of the secondary cell wall seemed to hide this coloration. In this case, the lipds were conserved in the lamella media (mainly in contact with the exodermis - Fig. 5D-F). In C. schilleriana, lipid deposition occured later than in the other orchid analyzed.

Figure 5
Histochemical test with Sudan III for the detection of lipids in Cattleya roots. A positive reaction is indicated by the red color. A. Velamen with centrifugal deposition of lipids in the developing region of the C. schilleriana roots. B-C. Lipids in mature roots of C. velutina. (B) An overview of the velamen and exodermis and (C) a detail showing the velamen cell walls and the media lamella of the exodermis. D. Detail of the velamen cells with a positive result in the developing region of the roots of C. nobilior. E. Positive results in the velamen cell of the mature region of C. walqueriana. F. Positive results in the velamen cell of the mature region of C. schilleriana. (Ex - exodermis, PC - parenchymatic cortex, V - velamen).

Secondary wall development - deposition of lignins in velamen cells

Using fluorescence microscope with a DAPI filter (Fig. 6), we observed that velamen lignification began near the exodermis (centrifuge), where maturation occurred first (as observed in C. schilleriana - Fig. 6A-D). The deposition of lignins in the velamen was gradual throughout the root apex and restricted to the secondary cell walls (Fig. 6E-I). We could not detect lignin deposition in regions near RAM (Fig. 6I) and we first detected it in cellular junctions of C. velutina (Fig. 6G-H) and C. nobilior. Despite the later deposition of lipids in C. schilleriana when compared to the other species, in this species, lignins were detected early in a younger velamen, prominently in the developing region. In mature roots with complete cell wall development, lignins were detected throughout the velamen as well as in cells of the epivelamen in all studied species.

Figure 6
Lignin fluorescence using a DAPI filter in cross sections of Catlleya roots. A-D. Centrifugal deposition of lignins in the velamen cells of C. schilleriana. The red arrows represent the direction of lignin deposition and tissue maturation. A. Schematic drawing representing the mature roots. B-C. Developing region. D. Mature velamen. E-I. Detection of lignins at different times of maturation in C. velutina roots. E. Schematic drawing representing the root apex and the direction of tissue maturation (red arrow). F. Mature velamen. G-H. Developing regions. I. Root apex meristem (RAM). (Ex - exodermis, PC - parenchymatic cortex, V - velamen).

Discussion

All species of Cattleya roots studied here had similar tissue composition, as described for other species of epiphytic orchids (Krauss 1948; Moreira & Isaias 2008; Moreira et al. 2013; Joca et al. 2017). In all roots, the division of the protodermal cells in the RAM was of the closed type (according to Heimsch et al. (2008) classification). We detected pectins, lipids and lignins during the development of the velamen cell wall. HGs with a high methylesterification degree labeled the velamen cells in young regions, mainly in C. velutina and C. walqueriana. About one centimeter from the apex, when the velamen cells began to die, the epitopes of HGs with up to 40 % methylesterification began to appear, strongly labeled in mature roots of C. walqueriana, C. velutina and C. schilleriana. Cell wall complexity increased during root differentiation, with lipids appearing first, followed by lignins. The deposition of these substances was centrifugal, initiating near the exodermis in the direction to the epivelamen.

Development of the velamen in Cattleya roots

The velamen is a tissue that undergoes programmed cell death (Pridgeon 1987), being mature when all its cells are dead. In the four species studied, the formation of the velamen began with the division of the protodermal cells still under the root cap at the apex of the root. This is not a pattern for all orchid species, as shown in Anathallis sclerophylla in which the cell divisions for velamen formation occurs only after the disappearance of the root cap (Kedrovski & Sajo 2019). As observed for other species of orchids (Heimsch et al. 2008), the apical meristems had a closed-type architecture, with the initials of the root cap, cortex and vascular cylinder separated. The epidermal initials were associated with the cortex and not attached to the root cap initials.

Cattleya velutina had more calibrous roots than the other species studied, but C. nobilior and C. walkeriana were the species that showed more investment in this tissue (area occupied by the velamen). The only species that seemed to show different timing in maturation of the velamen cells was C. schileriana. This species did not show a wider velamen diameter, indicating that there is no relation between these traits and the timing of maturation of velamen cells. Some strategies such as a larger number of xylem strands aligned with the endodermal passage cells, cortical thickenings and the investment in velamen can maximize the apoplastic flow in the direction to the xylem (Joca et al. 2017). Thus, the rapid maturation of the velamen (including lipid and lignin deposition) can be interesting in order to maximize water uptake. In addition, root caliber is not indicative of investment in the velamen, but it is known that the greater investment in this tissue, the higher water and nutrient uptake (Joca et al. 2017). Thin roots such as those of C. nobilior and C. walkeriana had about 50 % of their area occupied by the velamen, demonstrating the importance of this tissue for resource absorption.

Analysis of the velamen cell wall composition

During the development of the velamen, chemical and structural changes in the cell walls occur until this structure reaches stability in the mature root. In this study, we detected a spatial-temporal variation in the deposition of cell wall components during the development of the velamen in four species of Cattleya. The differential distribution of pectins, lipids and lignins, in a general way, guarantees the mechanical resistance of plant tissues, being also involved in apoplastic and symplastic water flow (Albersheim et al. 1996; Albersheim et al. 2010; Joca et al. 2017). In addition, hydrophobic compounds in the velamen may be related to water retention inside the organ (Kedrovski & Sajo 2019) and to protection against photooxidation (Chomicki et al. 2015).

The plant cell wall formation is based on nanocomposites of cellulose microfibrils incorporated into different matrix polymers (Gierlinger et al. 2013; Fahey et al. 2017; Kang et al. 2019). The celluloses are rigid and are embedded in a matrix rich in water, hemicellulose, pectins and proteins, that together constitute the primary cell wall (Pridgeon 1987; Macedo 2013). Cellulose is a polysaccharide composed of (1 → 4) -β-D-glucopyranose units (Newman 2004) and is considered to be a crystalline polymer. This crystalline polymer is associated with pectins, and ogether increasing cell porosity, adhesion and flexibility (Jarvis 1984; Albersheim et al. 2010). One of the most abundant kinds of pectins, the HGs, has been associated with cellular development, cell wall porosity and adhesion, as well as rigidity (Wolf et al. 2009). HGs are produced in the Golgi complex in a highly methylesterified condition (exhibiting up to 80 % methyl esterification) (Ralet et al. 2008, Wolf et al. 2009). In the current study, HGs were not detected in the RAM of the four species of Cattleya. However, HGs were detected in highly methylesterified forms as the cells of velamen reached maturation. During the process of maturation, the HGs are de-methylesterified, and the low methylesterified forms of HGs then become predominant in the mature tissue. The presence of HGs with low and high methylesterification in the velamen cells could maintain tissue stability and the necessary flexibility during the processes of development and next water and nutrient uptake. However, as observed in many other orchid species the pettern of HG distribution was not the same for all species (Joca et al. 2020).

Non-methylesterified pectins can lead to Ca²⁺ binding and the formation of gels which impart greater stiffness to the wall (Wolf et al. 2009). In addition to biophysical changes in the cell wall, changes in the degree of methyl-esterification may lead to the formation of pectic oligogalacturonide (small products of HG breakdown). These molecules act as analog hormone signals that neutralize the action of auxins, regulating cellular development (Ridley et al. 2001). In this context, the regulation of enzymatic activity in the degree of methylesterification of HGs may play some role in the control of cell growth (Wolf et al. 2009). In the velamen, in regions where the cells begin to die (about one centimeter from the apex), low labeling by JIM5 antibody (for HG epitopes with up to 40 % methylesterification) was observed, possibly indicating that HGs are signals of the final stages of maturation of these cells.

In the mature velamen, HGs occur sometimes in association with lipids and usually with lignins, which represent the development of the secondary cell wall. Lipids and lignins were detected in the cells of the exodermis and were gradually deposited throughout the velamen (obeying a centrifugal direction). Suberin is the main lipid described for the cell walls of the velamen and is hydrophobic and, just like lignins, is responsible for preventing the free flow of water, ions and pathogens by apoplast flow across the exodermis (Schreiber 2010; Joca et al. 2017). However, in the velamen, the secondary wall with thick and large highly hydrophobic pores guarantees mechanical resistance and efficient water flow via the apoplasts (Benzing 1986). The large amount of parietal thickenings, representing the secondary walls, ensure a solid surface with several pores (see Fig. 3) which promote a better transport of water throug the velamen, since the transport occurs by mass flow and passively, allowing the root to mobilize water reserves and minerals (Benzing et al. 1982).

Main considerations

A still developing green tip was observed in the root apex of the four Catlleya species, taking on a white coloration with velamen maturation and filling with air. Close to the RAM, the purple color was derived from the presence of anthocyanins in the vacuole of cortical cells, while the green color (in the subsequent region) was due to the differentiation of chloroplasts. The development of the primary wall led to the deposition of pectins as highly methylesterified HGs, with root maturation undergoing demethylesterification. The deposition of lipids in the velamen cells, and subsequently lignin deposition, marked a transition stage to secondary wall formation, efficiently directing water and nutrients across the simplast of the exodermis. The deposition of lipids and lignins began near the exodermis in the direction of the epivelamen. Before maturation, dead and living cells were intercalated and living cells with a protoplast gave a translucent appearance to the velamen, permitting visualization of the parenchymatic cortex (with chlorophyll). At this stage, wall thickenings were already visible and lignin deposition gave rigidity to the tissue.

Acknowledgements

We thank Universidade Federal de Uberlândia (UFU), the Graduate Program in Plant Biology, the laboratories of Instituto de Biologia (Laboratório de Fisiologia Vegetal and Laboratório de Anatomia, Desenvolvimento Vegetal e Interações). We also thank the Colégio de Aplicação COLUNI of Universidade Federal de Viçosa for kindly supplying the plants. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

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Publication Dates

• Publication in this collection
  25 Jan 2021
• Date of issue
  Oct-Dec 2020

History

• Received
  08 Apr 2020
• Accepted
  20 July 2020