Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis

Abstract

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.

Figure 1.

Differential distribution of xyloglucans (XyGs) in Arabidopsis wild-type shoot apices. A, Schematic structures of XyG subunits and specificity of XyG antibodies. Letters highlighted by red color mean higher affinity. B, Schematic structure of Arabidopsis SAM. C and D, Immunolocalization of XyGs in wild-type (Col) shoot apex sections (C) and whole mount tissues (D) labeled with LM15, LM25, and LM24 antibodies. Details are shown at bottom of (D). Scale bars = 20 µm.

Figure 2.

Altered distribution of XyGs in Arabidopsis XyG mutant shoot apices. A to C, Immunolocalization of XyGs in mutant backgrounds using LM15, LM25, and LM24 antibodies. Sections of xyl1-4 shoot apices (A), whole mount labeling of xyl1-4 (B), and sections of xxt1xxt2 shoot apices (C) are shown. Scale bars = 20 µm. D, Three-dimensional reconstruction image of the shoot apices prepared for XyG composition analysis. The buds are numbered according to their developmental stages (Smyth et al., 1990). Asterisk marks the flower bud at stage 3, which was not included for the sampling. Scale bar = 20 µm. E, MALDI-TOF MS analysis of XyGs in wild-type, xyl1-4, and xxt1xxt2 shoot apices. Gray areas of columns represent the proportion of acetylated subunits.

Figure 3.

Distribution of other wall components in xyl1-4 and xxt1xxt2 SAM. A and B, Distribution of methylesterified pectin in xyl1-4 (A) and xxt1xxt2 (B) SAMs labeled with JIM7 antibody. C and D, Distribution of unesterified pectin in xyl1-4 (C) and xxt1xxt2 (D) SAMs labeled with LM19 antibody. E, Distribution of crystalline cellulose in xyl1-4 and xxt1xxt2 SAMs labeled with CBM3a antibody. Scale bars = 30 µm. F to I, HPAEC-PAD analysis of relative amounts of cellulose (F), homogalacturonan (G), methylesterified pectin (H), and other polysaccharides (I) in XyG mutant inflorescences. Results are shown as mean ± sd obtained from 3 replications.

Figure 4.

Phyllotactic phenotype of XyG mutants. A, Representative image showing perturbation of phyllotaxis (indicated by arrowhead) in xyl1-4 and xxt1xxt2 mutants. Scale bar = 1 cm. B, Representative distribution angles of siliques on the inflorescence stem of Col, xyl1-4, and xxt1xxt2 plants. C, Distribution of divergence angles of siliques on the Col, xyl1-4, and xxt1xxt2 inflorescence stems. Orange lines denote the position of a divergence angle of 137°. Orange arrowheads mark the abnormal angle peaks; n = 649 angles from 20 Col plants; n = 683 angles from 21 xyl1-4 plants; n = 635 angles from 21 xxt1xxt2 plants. D, Diagram showing the method to measure the divergence angles (⍺) between successive primordia on confocal images of live meristems. Scale bar = 20 µm. E, Primordia distribution angles on Col, xyl1-4, and xxt1xxt2 meristems; n = 67 angles from 11 Col meristems; n = 55 angles from 6 xyl1-4 meristems; n = 46 angles from 8 xxt1xxt2 meristems. Asterisks denote statistically significant differences with wild type; *P < 0.05, Kolmogorov-Smirnov test.

Figure 5.

Meristem size and geometry of wild-type and XyG mutants. A, Overview of meristem size and geometry. Top, distribution map of cell area on Col and XyG mutant SAMs. Bottom, meristem curvature. All plants harbored the plasma membrane marker (35S:Lti6b-GFP). Images were postprocessed using the MorphoGraphX software. B, Cell area on meristem surface; n = 1409 cells from 4 meristems of 35S:Lti6b-GFP; n = 1469 cells from 4 meristems of xyl1-4 35S:Lti6b-GFP; n = 1028 cells from 4 meristems of xxt1xxt2 35S:Lti6b-GFP. Box plots display the interquartile range, split by the median; whiskers indicate the total range; outliers are plotted as individual points. C, Surface area of Col and XyG mutant meristems calculated from (B). D, Surface curvature of Col and XyGs mutant meristems; n = 11 for Col meristems; n = 10 for xyl1-4 meristems; n = 8 for xxt1xxt2 meristems. Mean values are represented with SD in (C) and (D).

Figure 6.

Young’s modulus of cell walls from Col, xyl1-4 and xxt1xxt2 SAMs. A, Representative map of indentation moduli on Col meristem surface. B, Quantification of Indentation modulus of Col, xyl1-4, and xxt1xxt2 SAMs; n = 6 for Col meristems; n = 7 for xyl1-4 meristems; n = 6 for xxt1xxt2 meristems. Mean values are shown with SD.

Figure 7.

Microtubule patterning on wild-type and XyG mutant SAMs. A, Representative microtubule patterning on 35S:GFP-MBD SAM. The orientation and length of magenta bars represent average microtubule orientation and degree of anisotropy in a single cell, respectively. Blue lines represent the radius of meristem. Details are enlarged at right. The α indicates the angle relative to radius. Scale bars = 10 µm. B and C, Quantifications of CMT orientation relative to the radius of xyl1-4 (B) and xxt1xxt2 (C) SAMs. d and E, Quantifications of CMT anisotropy in xyl1-4 (D) and xxt1xxt2 € SAMs. Statistic data in (B–E) was calculated from n = 1345 cells of 5 meristems of 35S:GFP-MBD, n = 1522 cells of 5 meristems of xyl1-4 35S:GFP-MBD, n = 1203 cells of 5 meristems of PDF1:mCitrine-MBD, and n = 998 cells of 5 meristems of xxt1xxt2 PDF1:mCitrine-MBD. P-values are calculated based on Kolmogorov-Smirnov test in (B–E). See also Supplemental Figure S9 for more details of CMT organization.

Figure 8.

CMT reactions to mechanical perturbation in wild-type, xyl1-4, and xxt1xxt2 SAMs. A, Time series of CMT patterning in 35S:GFP-MBD (wild type [WT]) and xyl1-4 35S:GFP-MBD SAMs after laser ablation at the meristem center. The orientation and the length of the red bar represent average CMT orientation and degree of CMT anisotropy respectively at cellular level. B and C, Quantification of CMT orientation angles relative to radius of wild type (B) and xyl1-4 (C) SAMs, 1 and 2 h after laser ablation; n = 167 cells from 4 wild-type meristems and n = 204 cells from 4 xyl1-4 meristems. D, Time series of CMT patterning on pPDF1:mCitrine-MBD (wild type) and xxt1xxt2 pPDF1:mCitrine-MBD SAMs after laser ablation at the meristem center. E and F, Quantification of CMT orientation angles relative to the SAM radius of wild type (E) and xxt1xxt2 (F) SAMs, 1 and 2 h after laser ablation; n = 164 cells from 4 wild-type meristems and n = 181 cells from 4 xxt1xxt2 meristems. ‘R’ in (A and D) represents radius of meristem. P-values are calculated based on Kolmogorov-Smirnov test. Scale bars = 20 µm.

Figure 9.

Phenotype of xyl1-4 bot1-7. A, Scanning Electron Microscope (sem) images of Col, xyl1-4, bot1-7, and xyl1-4 bot1-7 SAMs. B, Three dimensional reconstruction (left) and orthogonal view (right) of confocal image of xyl1-4 bot1-7 meristem. The arrowheads mark the points with negative curvatures on meristem surface, which are proposed to be organ boundaries. Scale bars = 50 µm (A and B). C, Representative image of silique distribution on xyl1-4 bot1-7 stem. Numbers denote the silique positions from bottom to top (old to young). D, Representative silique distribution angles on the inflorescence stem of xyl1-4 bot1-7. E, Distribution of divergence angles of siliques on the xyl1-4 bot1-7 inflorescence stems. Orange line denotes the position of angle around 137°; n = 504 angles from 10 plants.

Figure 10.

Phenotype of a triple mutant xxt1xxt2ktn1. A, Phenotype of representative Col-0, xxt1ktn1, and xxt1xxt2ktn1 plants grown in vitro for 3 weeks. Scale bar = 1 mm. B, Analysis of the progeny a xxt1−/− xxt2+/− ktn−/− plant. Among 147 plants, we identified 4 triple mutants.