Mechanisms for shaping, orienting, positioning and patterning plant secondary cell walls

Abstract

Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that—as for the growth of all plant organs—are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls.

Xylem formation has been one of the key steps of plant evolution. These physically strong tube cells allowed plants to colonize land by reinforcing their upright position against gravity and resisting desiccation by permitting water conduction throughout the plant body. This double role is fulfilled by specific conducting wood cells—the tracheary elements (TEs). These cells represent the cellular units of the adjustable plant vasculature, which relies on the three structural characteristics of TEs: (1) these cells develop a secondary cell wall to resist pressure exerted by the sap they will conducted, (2) these cells undergo programmed cell death (PCD) to hollow out their entire cytoplasmic content to form a conduit for the sap and (3) these cells will undergo a terminal perforation at their basal end (with respect to the corresponding meristem) to form a complete functional vascular cylinder which will connect with the underlying vascular vessels once terminally differentiated.^1,2 TEs are further characterized by a diversity of organizational pattern described by their secondary cell wall, which can be annular or spiral (referred to as protoxylem-type ornamentations) reticulate or pitted (referred to as metaxylem-type ornamentations).^3,4 These differently ornamented TEs are developmentally regulated and for protoxylemtype TEs appear during the development of early primary tissues (annular TEs are mostly observed in developing embryos) while metaxylem-type TEs appear in the later development of primary and secondary tissues (they represent the TEs present in wood). Annular and spiral TEs are first formed in organs undergoing primary growth and are considered to be “extendable” (their pattern in rings and spirals does not oppose further extension of the TE cell) during the growth of this organ. Once the growing organ has attained a certain size these TEs will be crushed by the surrounding tissue whilst the more heavily reinforced reticulate and pitted TEs will form to insure the vascular flow and strengthen the entire organ. In short, the modularity and plasticity of this plant vascular system is directly dependant on the differentiation and the type of cell wall ornamentation of its constituent TEs. The establishment of such regular patterning of secondary cell walls has been attributed to the underlying cortical microtubule array that predefines the cell wall depositions (reviewed in ref. 2). Pharmacological modulation of microtubule properties in both whole plants and in vitro TE differentiating systems leads to severe defects in the patterning, orientation, smoothness and deposition of TE secondary cell walls (reviewed in ref. 2).

Live Cell Imaging of TE Formation

The development of real-time live cell imaging has allowed great progress in the understanding of the chronological sequence of many dynamic processes in plant cell biology.⁵ Very few studies have dealt with TE development and then only focused on very short time periods. For example, Obara et al.⁶ estimated the time of programmed cell death in TEs, which been previously estimated in the Zinnia TE system to be less than 10 min, but this meant that the overall process of secondary cell wall deposition was unmonitored. The development of a new in vitro system from the transformable, sequenced genetic model Arabidopsis has allowed longer time acquisitions.^7,8 This is illustrated in Figure 1 showing real-time live-cell imaging of Arabidopsis cells, which 48 h prior to this, were hormonally induced to become TEs. The cells were stably expressing constitutive Histone-2A-GFP fusion and were monitored for 2 days with one frame every 10 min (Fig. 1A–R). Secondary cell wall formation was detected using wheat-germ agglutinin coupled to an Alexa-633 fluorescent probe (boxed area in black and white in Fig. 1A–R) as previously used in reference ⁷ and ⁹. Fluorescent stains for cellulose such as Calcofluor, Evans Blue and Congo Red have unfortunately been shown to disturb cellulose formation in bacteria, algae and plants.^10,11 Live cell imaging of the TE formation confirmed that secondary cell wall is laid down after cell elongation (Fig. 1A–D) as previously reported for Zinnia TEs.¹² The complete deposition of TE secondary cell wall, which is at least 10-times thicker than the primary cell wall,² takes 10 to 12 h (Fig. 1E–L) and is then followed by a phase where nuclear movement is stopped although cyclosis still occurs for 2 to 6 h (Fig. 1L–O) prior to programmed cell death, which then proceeds in less than 10 min (Fig. 1O–P). Generally, lysis associated with programmed cell death is so rapid and powerful that the entire cell contracts on itself (Fig. 1P–R). The extreme speed of secondary cell wall synthesis can be explained by the greater density and number of cellulose synthesizing particles underneath the forming secondary cell wall deposition.¹³ While the speed of CesA particle movement in primary walls has been quoted at 0.33 ± 0.065 µm min⁻¹,¹⁴ and confirmed by Desprez et al.,¹⁵ Wightman and Turner¹³ have used a novel means of calculation that suggests secondary wall CESA move at the surprising speed of 420 ± 30 µmmin⁻¹. With 10 times more cellulose being laid down in 8–12 h, it seems that the entire cellular machinery of TEs is diverted to only produce secondary cell wall polymers.

Figure 1.

Live cell imaging of te differentiation. (A–R) Chronological sequence of TE differentiation at 2-h intervals. The GFP-tagged nuclear signal is shown in green while xylan cell wall fluorescence is presented in the boxed area at the top right corner of every image. Note that secondary cell wall formation starts when cell elongation has finished (D), progresses linearly for 10 h and then plateaus for 6 h until programmed cell death is signalled by the absence of nuclear signal (P). Bars = 8 µm. (S) Kymographic analysis of secondary cell wall formation is shown on top; below this the movement of the GFP-tagged nucleus is shown along the orange line drawn in (A and R). Note that the nucleus stops moving and remains in the upper end during the plateau phase preceding programmed cell death. (T) Relative fluorescence of the secondary cellulosic cell wall during TE differentiation. Full movie can be viewed at www.upsc.se/edouard_pesquet.

TE-Specific Microtubule Associated Proteins

The scaffold necessary to guide and control this extremely rapid secondary cell wall deposition is formed of microtubules (reviewed in ref. 2). But the overall properties, dynamics and organization of the microtubules themselves are regulated by specific microtubule associated proteins (MAPs) which can have all kinds of properties such as bunching or severing or stabilizing, or destabilizing microtubules while some MAPs carry cargo along microtubules. To identify MAPs specifically associated with secondary cell wall formation in xylem, we analysed the gene expression of all known plant MAPs co-regulated with secondary cell wall-specific cellulose synthases (CesAs¹⁶) using two independent publicly available micro-array datasets: (1) the Zhao et al.¹⁷ dataset on Arabidopsis vascular tissue expression specificity and (2) the Kubo et al.¹⁸ dataset on Arabidopsis in vitro TE differentiation. As expected, all three secondary cell wall CesAs (including IRX3/AtCesA7, IRX1/AtCesA8 and IRX5/AtCesA4) are specifically expressed in xylem tissue (Fig. 2A) and are induced in differentiating TE cell cultures whilst the cells are depositing their secondary cell wall (Fig. 2B). Amongst all MAPs studied, only one was specific to xylem (Fig. 2C) and co-regulated with CesAs during TE differentiation (Fig. 2D): MAP70-5. The expression of the other four MAP70 isoforms appears not to be linked to xylem formation (Fig. 2C and D). Further reinforcing this link between the MAP70-5 isoform and the secondary cell wall synthesis, a gene co-regulation network using the ATTED-II database showed that MAP70-5 is only separated by two genes from the various component of the secondary cell wall synthesis machinery (either by At1g27380 and At3g50220 or by At2g29130 and At3g15050). Together, this strongly underlines that MAP70-5 is one of the few vascular-specific MAPs associated with secondary cell wall formation.^2,8

Figure 2.

Microarray expression analysis of secondary cell wall cellulose synthases and MAP70s. (A and C) Specificity of secondary cell wall cellulose synthases (CesA) (A) and MAP70s (C) in vascular and non-vascular tissue; micro-array expression data provided in reference ¹⁷. Note the high level of MAP70-5 expression in xylem. (B and D) Micro-array expression analysis of secondary cell wall cellulose synthases (CesA) (B) and MAP70s (D) during in vitro differentiation of Arabidopsis TEs, derived from Affymetrix expression data provided in reference ¹⁸. TE differentiation is indicated as a red dotted line (adapted from from ref. 18). Note, the expression of MAP70-5 is similar to that of secondary cell wall CesAs. (E) Gene co-expression network linking MAP70-5 to the secondary cell wall synthesis machinery using ATTED-II database (atted.jp). Note that only two genes (At3g50220-At1g27380/ At3g15050-At2g29130) are separating MAP70-5 from secondary cell wall synthesis machinery.

MAP70s correspond to a distinct class of MAPs of around 70 kDa that are exclusively present in land plants (both in bryophytes or mosses and tracheophytes or vascular plants) as no true homologous genes can be found in the currently sequenced animals, fungus, bacteria or algae (Fig. 3B). MAP70s are capable of interacting with themselves, with other MAP70 isoforms and other component of the microtubular cytoskeleton such as gamma-tubulin (Fig. 3A). Interestingly, a recent paper suggested that AtMAP70 may be related to animal intermediate filaments.¹⁹ In plants, MAP70s are organized into a small multigenic family ranging from two isoforms in Selaginella sp. to ten in Glycine max (Fig. 3B). Although all MAP70s share common structural features characterized by a succession of five coiled-coil regions (indicated in green in Fig. 3C), they can be further subdivided into two major clades (Fig. 3B): the MAP70-1 and the MAP70-5 clades centered on specific MAP70 isoforms (Fig. 3C). MAP701-type and MAP70-5-type proteins only share a maximum of 37% identity within one species, while each member of one type of subcategory presents an overall conserved structure, sharing from 70 to 80% identity within one species (Fig. 3C). Between species in each specific clade, MAP70-1s share a minimum of 55% identity while MAP70-5s share a minimum of 52% identity at the protein sequence level. Interestingly, the MAP70-1 clade includes most MAP70 isoforms (four out of the five present in Arabidopsis thaliana and six out of the ten present in Glycine max) and is present in all land plants from mosses to angiosperms while the MAP70-5 clade appears to be exclusive to angiosperms (Fig. 3B).

Figure 3.

MAP70 isoforms in plants and algae. (A) Yeast two-hybrid experiment of MAP70-5, MAP70-1 and gamma-tubulin protein interactions, swapping activating (pGaD) and binding domains (pGBW). (B) phylogenetic cladogram of MAP70 proteins in algae (Cr-Chlamydomonas reinhardtii/Vc-Volvox carteri), mosses (Pp-Physcomitrella patens), ferns/spikemosses (Sm-Selaginella moellendorffii), dicotyledons (Vv-vitis vinifera/ Pt-Populus trichocarpa/At-Arabidopsis thaliana/Al-Arabidopsis lyrata/Rc-Ricinus communis/Gm-Glycine max/Bo-Brassica oleracea/Ac-Aquilegia coerulea/ Mg-Minulus guttatus/Cp-Carica papaya/Ppe-Prunus persica/Cs-Cucumis sativus/Mt-Medicago truncatula/Me-Manihot esculenta) and monocotyledons (Os-Oryza sativa/Zm-Zea mays/Bs-Brachypodium sylvaticum/Bd-Brachypodium distachyon/Sb-Sorghum bicolor/Si-Setaria italica) using ClustaIX for protein alignment and plotted using Njplot. All sequences were obtained from Phytozome v6.0 (www.phytozome.net). (C) schematic consensus structure of MAP70-1-like and MAP70-5-like protein in plants with conserved domains between and within clade (in grey and blue respectively). The position of coiled-coil domains (in green) was determined using “Coiled-coil predictions”²¹ (www.russell.embl.de/cgi-bin/coils-svr.pl); the microtubule-binding domain was previously determined by reference ²².

Impact of MAP70 Downregulation on Secondary Cell Wall

The silencing of either MAP70-5 or its interacting partner MAP70-1 caused similar types of defect in TEs: a mild effect causing part of the secondary cell wall to traverse the cell's interior whilst still maintaining an overall regular cell wall pattern, and a more severe defect causing complete loss of cell wall patterning and an overall collapse of the secondary cell wall into the cell (Fig. 4A–E). Although cortically-positioned secondary cell walls of MAP70-silenced TEs were not significantly different in thickness than non-transgenic TEs, the variation in thickness between the different thickenings within a same cell was much greater in MAP70-silenced TEs (Fig. 4F). Furthermore, as illustrated in Figure 4G, the intruding secondary cell wall strands were also extremely variable in dimension and could attain sizes equivalent to normal secondary cell wall thickenings themselves (Fig. 4H). Moreover, modulation of the quantities of MAP70s directly affects the overall pattern of TE secondary cell wall. On the one hand, an increase of MAP70s leads to more protoxylem-type patterning while on the other hand its reduction leads to more metaxylem-type TEs.⁸ Altogether these results confirm that not only are MAP70-5/MAP70-1 essential for the proper cortical positioning of TE secondary cell wall but that they also define the overall pattern described by this secondary cell wall.

Figure 4.

Impact of MAP70-5/-1 silencing on secondary cell wall organization in Arabidopsis TEs in vitro. (A–E) Confocal imaging of MAP70-5-RNAi TEs exhibiting cell wall internalization. (A) overall maximal projection; (B) maximal projection of cell interior showing internalized wall/MT strand; (C–E) cross sections through orange dotted lines illustrated in (B). Bars = 8 µm. (F) Histogram showing the thickness of primary (marked iary) and the cortically-positioned secondary cell wall (marked iiary) in undifferentiated cells, for control and MAP70-RNAi Tes. (G) Transmission electron microscopy images of MAP70-RNAi Tes exhibiting internalized secondary cell wall strands, bars = 2 µm. Cell wall internalizations are indicated with red arrows. (H) Histogram showing length (L) and width (W) of the internalized secondary cell wall strands in control and MAP70-RNAi TEs.

Conclusions

MAPs specific to xylem-vessels, such as MAP70-5 and the recently describe MIDD1,²⁰ will shape, organize, position and dictate the behavior of the TE cortical microtubule array to form the correct template for a defined secondary cell wall pattern. A specific function of the land plant MAP70, and more specifically of the vascular plant MAP70-5, is the correct position of the secondary cell wall at the cell cortex.

Addendum to: Pesquet E, Korolev AV, Calder G, Lloyd CW. The Microtubule-Associated Protein AtMAP70-5 Regulates Secondary Wall Patterning in Arabidopsis Wood Cells. Current Biology. 2010;20:744–749. doi: 10.1016/j. cub.2010.0.