Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

Abstract

Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation.

Keywords:

• cell expansion
• cellulose microfibril
• cellulose synthase interacting1
• microtubule orientation
• primary cell wall
• procuste1
• radially swollen1
• thanatos

Cortical Microtubule Patterning is Aberrant in the Fast Elongation Zone of Primary Cell Wall Mutants

Anisotropic cell expansion relies on wall reinforcement due to deposition of cellulose microfibrils transversely to the growth axis. In primary walls of expanding cells, cellulose is produced by cellulose synthase complexes, consisting mainly of Cellulose SynthaseA1 (CesA1), CesA3 and CesA6,¹ which are connected to cortical microtubules by Cellulose Synthase Interacting1 protein (CSI1).^2,3 Cortical microtubules guide the movement of cellulose synthase complexes and, ultimately, regulate microfibril orientation.⁴ Besides the role of cortical microtubules in cell wall structure, cell wall itself influences microtubule organization.⁵ Hence, defects in cellulose synthesis have been reported to be related with alterations of microtubule orientation.^6-9 We recently showed that in Arabidopsis thaliana cellulose-deficient thanatos and pom2–4 mutants, of AtCesA3 and AtCSI1 respectively, suppression of cell expansion results in cortical microtubule reorientation in the fast elongation zone.¹⁰ As both genes are involved in cellulose synthesis in primary cell walls, we examined the orientation of microtubules in 2 additional primary wall-related mutants, radially swollen1 (rsw1)^11,12 and procuste1 (prc1)¹³ of AtCesA1 and AtCesA6, respectively.

The orientation of cortical microtubules in the fast elongation zone of 5 days-old wild-type roots, kept at either 22°C or 31°C for 24 h, was transverse ( Fig. 1A ). On the contrary, in the fast elongation zone of rsw1 roots, temperature-induced suppression of cell expansion resulted in alteration of cortical microtubule orientation from a transverse to a random pattern ( Fig. 1B ). Likewise, cortical microtubules were not transverse in the fast elongation zone of prc1 roots ( Fig. 1C ). These findings, in agreement with the recently reported results in than (AtCesA3) and pom2–4 (AtCSI1) roots,¹⁰ indicate that in fast elongating cells of primary cell wall mutants the orientation of microtubules is aberrant. Nevertheless, in the cells of the meristematic and transition zones of the deficient in primary wall cellulose synthase complex mutants rsw1, than and prc1 or in the cellulose-deficient pom2–4 mutant, cortical microtubules were transverse as in wild-type roots ( Figs. 1 and 2 ; ref. 10). Taken together, cortical microtubule patterning is consistent throughout the developmental zones of primary cell wall mutant roots.

Figure 1. Maximum projections of consecutive CLSM sections after α-tubulin immunostaining, as described in ref. 10, through the transition and elongation zones of wild-type (A) and rsw1 roots kept for 24 h at 31°C (B), or prc1 (C) roots at 22°C. Cortical microtubules are transversely oriented in transition zone cells (arrowheads) in all roots. In cells of the elongation zone (arrows), microtubule orientation is not transverse in the mutants, compared to the wild-type (B, C; cf. A). All plant materials were purchased from NASC (http://arabidopsis.info/). Scale bar: 20μm.

Figure 2. CLSM sections after α-tubulin immunostaining, as described in ref. 10, through the meristematic zone of wild-type (A) and rsw1 roots kept for 24 h at 31°C (B), or prc1 roots at 22°C (C). Cortical microtubules are transverse (arrows) in interphase meristematic cells. Scale bar: 20 μm.

Microtubule Orientation and Cell Expansion: It Takes Two to Tango

The term “microtubule-microfibril syndrome”¹⁴ refers to a dynamic seque2ce of events driven by the cooperation between microtubules and cell wall elements, to accomplish growth and morphogenesis of plant organs. Any malfunction of either partner, microtubules or cell wall elements, inevitably affects the other and ultimately the development of the whole plant. As root growth is guided by gravity, anisotropy establishes an inherent bias toward transverse orientation of cortical microtubules.¹⁵ Cell expansion, slow or fast, requires deposition of new cellulose microfibrils, as the preexisting microfibrils separate.¹⁶ Concomitantly, new microtubules are recruited under the expanding plasma membrane, parallel to these that already exist, aiming to maintain the microtubule density of the cortical array. While cellulose synthase complexes “glide” on these microtubules, they deposit new transverse microfibrils in the expanding wall. Cell expansion commences at low rate in the meristematic zone and remains moderate in the transition zone, whereas in the fast elongation zone it accelerates.¹⁷ As cells acquire their final length in growth terminating zone, the newly deposited microfibrils are no more strictly transverse and thereby cortical microtubules are reoriented.¹⁸

The supply of newly synthesized microfibrils has to keep pace with wall expansion throughout cell elongation. It has been suggested that cellulose deficiency in primary wall mutants is probably not severe enough in slowly expanding cells of the meristematic and transition zones.¹⁰ Thus, as the cells within these zones keep elongating, the orientation of microtubules remains transverse ( Fig. 3 ). On the contrary, cellulose shortage restricts cell expansion in the fast elongation zone, as the newly synthesized microfibrils deposited in the expanding cell wall may not be enough for cell elongation. Cessation of expansion at this stage is similar to that of fully expanded wild-type cells and results in premature microtubule reorientation ( Fig. 3 ).

Figure 3. A graphical model illustrating the effect of cellulose-deficient primary cell wall mutants on cortical microtubule orientation in Arabidopsis thaliana root. The short primary root phenotype of pom2–4, prc1, rsw1 and than mutants is caused by reduction of anisotropic cell expansion, originating from decreased cellulose microfibril deposition. Suppression of cell expansion results in alteration of cortical microtubule orientation from a transverse (black lines) to a random (red lines) pattern in the fast elongation zone. On the contrary, in the meristematic and transition zones cortical microtubules exhibit transverse orientation (black lines) reminiscent of the pattern in wild-type roots. The mutants are: pom2–4 of Cellulose Synthase Interacting1 protein (CSI1),³ prc1 (procuste1 of CesA6),¹³ rsw1 (radially swollen1 of CesA1),^11,12 and than (thanatos of CesA3).¹⁹

As recently reported,¹⁰ inhibition of cell expansion, irrespective of the cause, induces the reorientation of cortical microtubules, since there is no demand for deposition of more transversely oriented microfibrils in the cell wall. Therefore, it seems plausible that a signal of biophysical nature is transduced from the cell wall to the protoplast, triggering the shift of microtubule orientation from transverse to random in cells ceasing elongation. The components involved in this process remain still unknown and further research is required to determine these factors. Likewise, the cues involved in the initial establishment of transverse microtubule orientation in the meristematic root cells need to be elucidated.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Prof. Polydefkis Hatzopoulos for critical reading and suggestions on the manuscript.

Funding

This work was supported by the Research Committee of Aristotle University of Thessaloniki (grant no. 89348). Gerasimos Daras acknowledges support from IKY fellowships of excellence for postgraduate studies in Greece-Siemens program.