Live imaging of fluorescently tagged cellulose synthase complexes (CSCs) in the Arabidopsis VND7-inducible protoxylem system (Watanabe et al., Science 2015) established that secondary cell wall deposition involves fast, densely packed CSCs restricted to narrow membrane domains, but left a set of explicit unresolved issues. The quantitative core of that account remains open: (1) no study has quantified CSC insertion events at secondary wall domains or partitioned the final CSC density between delivery rate and plasma-membrane residence time; (2) CSC velocity is still used as a proxy for cellulose synthesis rate, because the glucan-chain polymerization rate per complex has never been measured directly, for either primary- or secondary-wall CSCs; (3) the molecular mechanism that restricts CSC and SmaCC delivery to the plasma-membrane domains underlying cortical microtubule bands is only partially identified, with the anterograde-versus-recycling role of SmaCCs still unresolved; and (4) the identity of the associated vesicles and the quantitative conservation of these dynamics across native xylem vessels and fibers remain unestablished.

Background

Plant cells are wrapped in a polysaccharide-rich primary cell wall; many specialized cells later deposit a thick secondary cell wall inside it, which constitutes the xylem wood of vascular plants and the vast majority of terrestrial biomass. The load-bearing component of both wall types is cellulose, a bundle of crystalline glucan chains synthesized at the plasma membrane by the cellulose synthase complex (CSC), a six-lobed rosette-shaped assembly of cellulose synthase (CESA) proteins. Different CESA isoform sets build primary versus secondary walls: in Arabidopsis, CESA1/3/6-like isoforms act in primary walls, while CESA4, CESA7 and CESA8 are required for secondary walls. Each rosette moves through the membrane as it extrudes a cellulose microfibril into the wall; how fast the complex moves (its velocity) is widely used as a readout of how fast it synthesizes cellulose, although the number of glucan chains per rosette (18 versus 24) is itself still debated.

Because developing xylem cells lie deep inside tissues, secondary-wall CSCs long resisted live imaging. Two routes overcame this: imaging CESA7-containing compartments in native root protoxylem vessels (an early water-transport cell type with spiral wall thickenings) at reduced resolution, and ectopic induction of protoxylem identity in epidermal cells. Watanabe et al. (2015) used the latter — the VND7::GR system, in which a dexamethasone-inducible fusion of the master xylem transcription factor VND7 to the glucocorticoid receptor triggers transdifferentiation of epidermal cells into protoxylem-like cells — together with a functional YFP-tagged CESA7 in a cesa7 null background. They measured secondary-wall CSC velocity (265 ± 75 nm/min, higher than primary-wall CSCs under identical conditions), documented a redistribution of CSCs into narrow membrane domains co-aligned with cortical microtubule bands, observed delivery events from small CESA-containing compartments (SmaCCs; when microtubule-associated these are termed MASCs, microtubule-associated cellulose synthase compartments) by FRAP (fluorescence recovery after photobleaching, a method in which a membrane region is bleached with a laser and the reappearance of fluorescence reports insertion of new particles), and found that microtubule depolymerization disorganizes CSC tracks without changing velocity or total cellulose content.

The paper closed with six unresolved issues: (i) the VND7::GR induction system may not fully replicate native xylem differentiation, since VND7 levels differ from endogenous ones; (ii) the number of CSC insertion events at secondary wall domains was not quantified, so the relative contribution of delivery versus plasma-membrane residence time to final CSC density is unknown; (iii) CSC velocity is only a proxy — direct measurement of glucan-chain polymerization per complex was not achieved; (iv) the molecular mechanisms that target CSCs and SmaCCs specifically to the membrane domains underlying microtubule bands are unknown; (v) the nature and function of the electron-lucent vesicles seen in electron microscopy require characterization; and (vi) it is unknown whether the observed dynamics are conserved in other secondary-wall-producing cell types, such as native xylem vessels and fibers.

Since 2015, several of these strands have advanced: CSI1/POM2 has been identified as the microtubule tether for secondary-wall CSCs, the Golgi-derived origin of SmaCC compartments has been revealed, native protoxylem vessels have become imageable, and catalytically active CesA trimers have been reconstituted in vitro.

Problem Statement

The problem is to complete the quantitative, mechanistic account of cellulose synthase complex (CSC) dynamics during secondary cell wall deposition that Watanabe et al. (2015) initiated, by resolving the following open issues:

  1. CSC flux at secondary wall domains: quantify the rate of CSC insertion (delivery) events at the plasma-membrane domains underlying microtubule bands, and determine the relative contributions of insertion rate versus plasma-membrane residence time (lifetime) to the final, high CSC density observed at secondary wall thickenings.
  2. Per-complex synthesis rate: measure directly the rate at which an individual CSC polymerizes glucan chains, so that CSC velocity can be either validated or replaced as a proxy for cellulose synthesis rate, and so that the claimed activity difference between primary-wall and secondary-wall CSCs can be settled directly.
  3. Targeting mechanism: complete the molecular mechanism that delivers CSCs and SmaCCs specifically to the plasma-membrane domains beneath cortical microtubule bands — beyond the identified CSI1/POM2 tether (CELLULOSE SYNTHASE INTERACTIVE PROTEIN 1, also encoded by the POM-POM 2 locus, the linker protein that binds both CESAs and cortical microtubules) and the exocyst components (the octameric vesicle-tethering complex that captures secretory vesicles at the plasma membrane), this requires establishing whether SmaCCs act as the anterograde carriers that insert secondary-wall CSCs at thickenings (as opposed to recycling or endocytic compartments), and what restricts insertion to the band domains.
  4. Vesicle identity: determine the nature and function of the CESA-associated post-Golgi vesicles observed in electron microscopy of secondary-wall-forming cells (the electron-lucent vesicles; the population structure and cargo of SmaCCs/MASCs).
  5. Generality: establish, with matched quantitative measurements, whether these delivery, activity and targeting dynamics are conserved across secondary-wall-producing cell types in native tissues — xylem vessels beyond protoxylem and wood fibers — and thereby bound the validity of the VND7::GR transdifferentiation system as a model for native xylem differentiation.

These facets share one scientific objective: a closed quantitative account of how CSC number, lifetime and synthetic activity at wall domains produce the rapid, patterned cellulose deposition of secondary walls. Facets 1 and 2 supply the flux and rate terms, facet 3 the targeting mechanism that localizes them, facet 4 the carrier identity, and facet 5 the domain of validity. All five facets are jointly posed as one problem and together constitute this single account.

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