The Role of the Primary Cell Wall in Plant Morphogenesis

Abstract

Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress⁻strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector, with direction defined by Hechtian adhesion sites, has a magnitude of a few piconewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress-activated plasma membrane Ca²⁺ channels and auxin-activated H⁺-ATPase. The proton pump dissociates periplasmic AGP-glycomodules that bind Ca²⁺. Thus, as the immediate source of cytosolic Ca²⁺, an AGP-Ca²⁺ capacitor directs the vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto, these components comprise the Hechtian oscillator and also the gravisensor. Thus, interdependent auxin and Ca²⁺ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca²⁺ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, the evolutionary origins of the Hechtian oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life.

Keywords: H+-ATPase; calcium signaling; cell wall protein; hechtian oscillator; morphogenesis.

Figure 1

Hechtian strand occurrence: from algae to chloroplast guard cells. (A) Closterium desmid in 12% sucrose: reprinted from [22], Scale bar = 2.1 µm; (B) Penium margaritaceum: reprinted from [23], Scale bar = 2 µm; (C). Ginkgo cells plasmolysed in 0.3 M NaCl reprinted from [24], Scale bar is 10 µm; Arrows in (A–C) show location of Hechtian strands. (D1–3) Guard cell H⁺-ATPase and its translocator PATROL (proton ATPase translocation control) in Arabidopsis. The plasma membrane ATPase AHA1 and its translocator PATROL1 co-localise at the tips of Hechtian strands in plasmolyzed guard cells transformed with GFP-PATROL1 (D1) and RFP–AHA1 (D2) merged in (D3). Enlarged images of the region enclosed in the yellow square are not shown here. Reprinted from [25], Scale bars = 5 µm.

Figure 2

The Hechtian growth oscillator. Regulators of the plasma membrane H⁺-ATPase reflect its two major roles: 1. It maintains negative inner membrane potential and enhances anion exit and cation entry; 2. It dissociates AGP-Ca²⁺ as a source of cytosolic Ca²⁺. Green arrows indicate upregulation or red represent downregulation, with exceptions where the mechanism remains to be elucidated. Auxin activates H⁺-ATPase. cf. Figure 3: Plasma membrane H⁺-ATPase regulation is central to plant biology [38]; the effects of high steady state auxin levels on root cell elongation in Brachypodium [39]; auxin-driven morphogenetic patterns depend on unidirectional fluxes [40]; the evolution of auxin signaling and PIN proteins [41]; what initiates auxin biosynthesis remains unknown [42]; auxin activates the plasma membrane H⁺-ATPase via phosphorylation [38]. Abscisic acid negative regulation: decreases steady-state levels of phosphorylated H⁺-ATPase possibly by promoting dephosphorylation via a protein phosphatase [43] and suppresses hypocotyl elongation in Arabidopsis [43]; abscisic acid stress signalling evolved in algal progenitors [44]. Blue light: the blue light photoreceptor pigment phototropin increases cytosolic Ca²⁺ [45]. Brassinosteroids: Increase cytosolic Ca²⁺ via increased auxin levels [46]. Cytokinins enhance cell division by unknown mechanisms. Ethylene upregulates auxin biosynthesis in the Arabidopsis root apex and inhibits root cell expansion [47]; thus, anthranilate synthase mutants yield ethylene-insensitive root growth phenotypes. Ethylene specifically inhibits the most rapid growth phase of expanding cells—normally the root hair initiation zone but ethylene moves it much closer to the tip. Auxin and ethylene act synergistically to control root elongation, root hair formation, lateral root formation and hypocotyl elongation [48]. Ethylene modulates root elongation through altering auxin transport: Ethylene binds to receptor proteins such as ETR1 and EIN2 controlling transcription factor EIN3 that targets ERF1, the ethylene response factor that regulates diverse genes, most likely tissue-specific. In shoots, auxin moves from the apex to the base [49]. PIN1 mutants decrease auxin transport in inflorescences while PIN2, PIN3, and PIN7 mutants decrease polar auxin transport in roots. In roots, auxin moves rootward via PIN1, PIN3, and PIN7 in the central cylinder and shootward in the outer cell layers via PIN2 which mediates gravitropism. Ethylene-induced auxin synthesis involves the α and β subunits of anthranilate synthase [47]. Fusicoccin: The fungal toxin fusicoccin fills a cavity in the interaction surface between PM H⁺-ATPase and 14-3-3 proteins to form a tight bridge that activates the ATPase irreversibly [38]. Gibberellins increase cytosolic Ca²⁺ via a DELLA-independent signalling pathway [50]. Gravity: in root gravitropism, auxin regulates root curvature via apoplastic pH and a Ca²⁺-dependent signalling pathway [51]; PIN proteins responsible for polar auxin transport and gravitropism reviewed in [42]; in microgravity, cucumber seedling PIN protein distribution is parallel to the minor root axis. However, a 1 g force re-aligns PIN1 to the lower side of the endodermis thus facilitating auxin transport from the upper side of the root to the lower side. Similarly, PIN3 and PIN7 of the gravity sensing columella also re-align to the lower side [52]. Nitric oxide: high apical levels of reactive oxygen species (ROS) in Arabidopsis root hairs suggest possible activation of a Ca²⁺ channel that modulates root hair-tip growth [53]. Red light: the elevation of auxin levels is well established as an early event, in response to response to prolonged shade. During an initial triggering phase, phytochrome interacting factors (PIFs) bind to the promoters of auxin synthesis genes and generate a burst of auxin that promotes growth [54]. ROS: NADPH oxidase generates active oxygen species that activate Ca²⁺ channels and regulate cell expansion and root morphogenesis [55].

Figure 3

Posttranslational regulation of the PM H⁺-ATPase alternates between two states: reprinted from [38]. Red = downregulated pumps hydrolyze ATP with low efficiency, hence the low transport of H⁺. Green = upregulated pumps with high ATPase efficiency, and high H⁺ transport rates. C-terminal regulatory domains control transition between the two states. The phosphorylation (P) of the C-terminal penultimate threonine residue (Thr-947 in pump AHA2) creates a binding site for a 14-3-3 protein that stabilises the pump. The binding of lysophosphatidylcholine (Lyso-PC) and phosphorylation at Thr-881 also activates the PM H⁺-ATPase independently of phosphorylation and 14-3-3 protein binding. It is not known whether phosphorylation at Thr-881 in the C-terminal domain interferes with or depends on 14-3-3 binding (as indicated by the question mark). Phosphorylation at Ser-899 or Ser-931 inactivates the pump. Phosphorylation at Ser-931 blocks the binding of 14-3-3 protein, but is not known for Ser-899. Only well-characterized regulatory events are shown.

Figure 4

Embryogenesis in Arabidopsis: Cell lineages, PIN protein localization, and auxin response maxima (reprinted from [71]). Auxin response maxima and PIN protein localization follow a regular cell division pattern. Thin lines show lineages between stages: PIN protein localization is shown as follows: red (PIN1), blue (PIN4), and green (PIN7), and DR5 reporter is pink. (A) After the division of the zygote, one and two-cell embryos express PIN7 in the basal daughter cell (bc), and the apical cell (ac) expresses the DR5 reporter. After two more cell-divisions, all proembryo (pe) cells express PIN1 and DR5 reporter. Basal suspensor (sus) cells express PIN7 localized on the proembryo side. At the globular stage, central lower cells of the proembryo establish basal PIN1 polarity while PIN1 localizes apically in outer protoderm (pd) cells. At the same time, PIN7 polarity reverses in suspensor cells and PIN4 is activated in the uppermost suspensor cell that now expresses the DR5 reporter and is specified as hypophysis (hyp). During the transition stage, the PIN1 polarity at the flanks of the apical embryo half converges in adjacent cells, accompanied by the appearance of new DR5 maxima. These sites mark the initiation of the cotyledons; (B) The torpedo stage shows discrete regions of the embryo: RAM (root apical meristem), green, root apical meristem (white, future quiescent centre); hypo, blue, hypocotyl; Cot, yellow, cotyledons; and SAM (shoot apical meristem), red, the shoot apical meristem.

Figure 5

PIN proteins maintain their polarity at the cell wall of plasmolysed cells. Plasmolysis shows that apical (PIN2-GFP and PIN1-GFP-3) and basal (PIN1-GFP-2) proteins remain attached to the cell wall by their polar domains. (A–D) Note—yellow arrowheads = nonpolar PIN-GFP signal at the plasma membrane. HS = Hechtian strands with PIN-GFP-connections to the wall. White arrowheads = a strong persistent PIN-GFP signal at the cell wall–plasma membrane interface. Scale bars = 10 µm. Reprinted from Figure 4 of [91].

Figure 6

Reprinted with permission from Sato et al. [49]. Root gravitropism in Arabidopsis. (A) Gravity perception in Arabidopsis thaliana. At time point 0, roots grow vertically. After a 90° turn, the following events occur: (1) At 10 s, statoliths are still at the old bottom of the cell. After 3 min, statoliths move towards the new bottom of the cell to be uniformly distributed at 5 min [92]; (2) PIN3 and PIN7 relocalization is achieved 2 min after the gravity stimulus and, in consequence, a lateral auxin gradient is generated between the upper and lower side of the root (thin and thick orange arrows respectively) [100]; (3) the development of differential extracellular pH levels between the upper (acidic) and lower (alkaline) side of the gravistimulated root [51]; (B) Gravity signal transduction and transmission via auxin transport and redistribution. Black arrows show that AUX1 and PIN2 channel auxin from the shoot to the root tip. Blue arrows show how PIN4 distributes auxin efflux through the vascular tissue to the columella cells. PIN3 and PIN7 set up the auxin flow (green arrows), with an accumulation on the lower side of the root. PIN2 and AUX1 transport auxin through the lateral root cap to the epidermal cells in the elongation zone (orange arrows) where the actual growth response will occur.

Figure 7

Stress vectors in the shoot apical meristem. The suggested changing meristem stress patterns cause auxin depletion in the boundary region as primordia form [26]. Note that the magnitude and direction of the stress vector are strongly anisotropic (red arrows) in the boundary region between the primordium and shoot apical meristem apex. Reprinted from [85] with permission from AAAS).

Figure 8

Chilly Brook chalk cliffs with E. huxleyi. Coccolithophores such as E. huxleyi (inset) constructed massive soft chalk deposits of calcium carbonate during the cretaceous period (145–65 MYA), now seen here from across the Chilly Brook water meadows dominated by Ranunculus acris, adjacent to the River Ouse in the South Downs National Park at Lewes, UK. This view encapsulates the entire evolutionary progression from the simplest unicellular protists such as E. huxleyi to advanced dicots such as R. acris at the pinnacle of alternation of generations, all dependent on calcium. (Photo: DTAL).

Figure 9

Metaphyte origins. Progression from single cells to filaments that align, forming a flat thallus. The examples here are the biflagellate Chlamydomonas, Spirogyra and the red arrow points to Coleochaete scutata.

Figure 10

Coleochaete hydroxyproline glycosides. Alkaline hydrolysates of Coleochaete scutata cell walls fractionated on a cation exchange column gave a Hyp-glycoside profile that appeared similar to the profile of cell walls isolated from higher plants. However, closer inspection and quantitative sugar analyses showed a striking difference; in higher plants, the Hyp-glycosides are simple arabinosides, whereas Coleochaete shows heterooligosaccharides of up to seven sugar residues that include galactose, glucose and arabinose, compositions remarkably similar to the profile of cell walls isolated from Chlamydomonas [119] but different from other members of the plant kingdom [120]. Presented at the 11th Cell Wall Meeting, Copenhagen [121].

Figure 11

The moss Bryum capillare exemplifies the alternation of generations. Bryum capillare with attached diploid sporophytes dependent on the haploid gametophyte generation. (Photo: DTAL).