Impairment in IQD8 function leads to defects in microtubule array orientation and formation

51 and IQD8 promoters overlapped partially, which further point to the partial redundancy in their functions. In addition, IQD6, IQD7, and IQD8 also showed some unique expression patterns, potentially reflecting tissue-specific functions. Data from our phenotypic analysis as well as broader localization pattern of IQD8 throughout out the cell-cycle, suggest its prominent role towards the phenotype.

In conclusion, our results revealed dual localization of IQD8-GFP at membranes (division site, cell plate) as well as at PPB and phragmoplast. We thus presumed that IQD8 at the plasma membrane plays a role in maintenance and organization of the division site, and when present at the phragmoplast, stabilizes phragmoplast microtubule and subsequently direct its expansion towards the division site.

2.2.3 Impairment in IQD8 function leads to defects in microtubule array

52 The degree of rotation was categorized into three groups; namely, 0°-5°, >5°≤15° and greater than 15°. The orientation of 0°-5° was considered as the ideal orientation of PPB.

96% of analyzed prophasic cells in WT showed ideal transverse orientation of PPB (Figure 2.12B). In contrast, the percentage of cells in ideal orientation was reduced to 62% and 73%

of the analyzed cells in iqd678 triple and iqd8-1 single mutants, respectively (Figure 2.12B).

Figure 2.10 Microtubule pattern analysis.

Analysis of microtubule arrays labeled with RFP-MBD in 7-day-old seedlings of iqd8-1 single, iqd678 triple mutants and Col-0. Microtubules organization during preprophase (A), metaphase/anaphase (B), and cytokinesis (C) stages of cell division in iqd8-1 single (second and third columns) and iqd678 triple mutants (fourth and fifth columns) compared to the Col-0 (first column). Red arrow indicates the position of PPB, red arrowheads indicate PPB orientation, brown arrowheads depict the PPB loss.

White and blue arrows indicate the orientation of spindle and phragmoplast, respectively. The images are representative of 8-10 seedlings from at least three independent experiments. Scale bars, 10 µm.

The PPB defects were considerably complemented by introgression of pIQD8:IQD8-GFP into the iqd678 triple mutant background (Figure 2.11D, Figure 2.12A, B). Our data showed that PPB defects in iqd678 triple mutants are more severe compared to iqd8-1 single mutant and WT (Figure 2.12A, B). Although milder, the presence of phenotype in iqd8-1 single mutant itself as well as phenotypic reversion in the complementation line point to the role of IQD8 primarily in positioning and formation of PPB microtubules for regulating the spatial control of cytokinesis. The loss of PPBs in iqd678 triple mutants is analogous to previously characterized cell division mutants like ton1, fass/ton2, pp2a, trm678 (Azimzadeh et al.,

53 2008; Camilleri et al., 2002; Schaefer et al., 2017; Zhang et al., 2016). These factors are known for their roles in PPB organization and formation, mutation in any of these factors results in severe loss of PPB. In addition, misorientation of PPBs in iqd678 triple mutants closely resemble the PPB orientation defect of phgap12 double mutants. Proper PPB formation and its orientation in theses mutants influence orientation of spindle and concomitantly, phragmoplast positioning for timely progression of mitosis (Ambrose and Cyr, 2008; Chan et al., 2005). This prompted us to further analyze microtubule organization in later division structures, the spindle and cytokinetic phragmoplast.

Figure 2.11 Immunolabeling showing microtubule patterns in iqd678 and iqd8-1 mutants compared to WT and complementation line.

Single-cell resolution of 7-day-old seedlings showing coimmunolocalization using tubulin-specific antibody. Nuclei were counterstained with 4‗, 6-diamidino-2-phenylindole (DAPI). Immunolabeling of tubulin was used to visualize microtubules in Col-0 (A), iqd8-1 (B) iqd678 (C) and pIQD8:IQD8-GFP/iqd678 complementation line (D). Images are single optical sections of Z-stacks images and represent results of at least two independent experiments. Arrows indicate the orientation of PPBs (yellow arrows), the position of spindles (white arrows) and position of phragmoplast (blue arrows).

Arrowheads depict the loss of PPB. Scale bars, 50 µm.

54 In general, organization of microtubules appeared to be normal in iqd678 triple mutants with occasional disturbance of spindle microtubule assembly. In iqd678, spindle microtubule occasionally appeared to be disorganized and collapsed. In very few cases, similar events also happened in WT but were more pronounced in iqd678 triple mutant (data not shown).

This suggests a potential role of IQD8 in bundling and crosslinking or bundling of spindle microtubules. However, this occasional disturbance of spindle reflects the presence of a redundant mechanism of spindle assembly. Unlike the Arabidopsis tan mutant, where the similar spindle defect causes a delay in metaphase progression (Martinez et al., 2019), in iqd678, metaphase progression appeared to be normal in most of the cases (Figure S 9). In most cases, spindle organization in iqd678 triple mutant was comparable to that of the WT, which is in turn reflected in normal metaphase progression in the iqd678 triple mutants.

During metaphase, the most obvious difference that we observed in the iqd678 and iqd8-1 mutants was the positioning of spindles (Figure 2.10B, Figure 2.11B, C). Approximately 44%

and 16% of cells in iqd678 triple and iqd8-1 single mutants, respectively, showed oblique orientation of spindles (Figure 2.12C). In contrast, 90% of the WT spindles, oriented in transverse orientation of 0°-5° (Figure 2.12C). However, around 3% of WT spindle also showed tilted orientations, which is consistent with results reported in the literature.

Altogether the data suggest that spindles misorientation in iqd678 triple and iqd8-1 single mutants are likely caused by defects in the formation and orientation of PPBs. A similar defect in spindle rotation has been observed in Arabidopsis mutants lacking TRM6-8, EB1C, AKT1 or AKT5 (Ambrose and Cyr, 2008, 2007; Komaki et al., 2010; Marcus et al., 2005).

Besides spindle defects in all these cytokinetic mutants, some produce severe defects in cell wall positioning while others have no apparent defects. The spindle rotation is a normal event that has also been reported in WT cells, which is corrected to the right plane during phragmoplast formation and orientation. However, the significant degree of orientation may not be corrected back to normal orientation during phragmoplast stage (reviewed in Rasmussen et al., 2013). Similar to cell wall positioning and PPB orientation, angles of spindles orientation were categorized into three different categories (, 0°-5°, >5°≤15°and

>15°). 0°-5° rotation of spindle was considered as ideal orientation. Our results showed that the loss of PPB iqd678 triple mutants leads to a major defect in the spindle orientation which potentially causes aberrant phragmoplast orientation.

55 Figure 2.12 Quantification of microtubule patterns.

Frequency distribution of microtubule orientation and organization in iqd8-1 single and iqd678 triple mutants compared to the WT and complementation line. Percentage frequency of cells showing loss of PPB (A), misorientation of PPBs (B), misorientation of spindles (C), misorientation of phragmoplasts (D) and altered phragmoplasts morphology (E) in respective genotypes. (n)= total number of cells analyzed per genotype. Total of 20-25 roots were used for the analysis. Data is representative of two independent experiments. Different colors represent the frequency of cells showing a specific pattern of microtubules which is indicated below each image.

56 Corroborated with the altered orientation of spindle, majority of cytokinetic cells in the iqd678 triple mutant showed oblique positioning of early and late phragmoplast (Figure 2.10C, Figure 2.11B,C). 12% and 40% of the total analyzed phragmoplasts in the iqd8-1 single and iqd678 triple mutants, respectively showed oblique orientation (Figure 2.12D) which was consistent with PPB loss in these mutants. This indicates that the phragmoplasts misorientation in the iqd mutants are most likely caused by a loss of phragmoplast guidance resulting from the loss of positional information of the PPB. Beside oblique orientation of phragmoplasts, iqd678 triple mutants and to some extent iqd8-1 single mutants also showed profound undulation of the phragmoplast microtubules (Figure 2.10C, Figure 2.11B, C), which is likely due to the lack of stability as well as hampered guidance mechanisms of phragmoplasts. During cytokinesis, we noticed both curved and straight phragmoplast in iqd678 and iqd8-1 mutants. However, curved phragmoplasts have never been reported in WT cells. In contrast to WT phragmoplasts, in the iqd678 triple mutants, 31% of analyzed phragmoplasts were curved (Figure 2.12E). The effect occurred less frequently in iqd8-1 single mutants (11% of the total phragmoplast), albeit more often than in the WT (Figure 2.12E). The effect of iqd678 impairment on mitotic microtubule organization raised the question, whether the organization of microtubule during interphase is also affected or not.

Our analysis revealed that as in the epidermis of WT, the vast majority of interphase cortical microtubules in iqd8-1 single and iqd678 triple mutants, aligned in transverse orientations to the elongation axis of cells (Figure 2.13).

Figure 2.13 Organization of interphasic microtubule arrays in the iqd8-1, iqd678 and Col-0.

(A-C) Analysis of interphasic corticalmicrotubule arrays in root epidermal cells of 7-day-old seedlings.

Microtubules are visualized by introducing RFP-MBD marker in Col-0 (A), iqd8-1 (B) andiqd678 triple mutants (C).Images are single optical section and represent two independent experiments from 8-10 roots per genotype. Scale bars, 10 µm.

57 Our results are similar to reports on other cell division mutants that show loss of PPB without any noticeable effects on the organization of interphasic cortical microtubule arrays (Schaefer et al., 2017; Zhang et al., 2016). Together, these observations suggest that IQD8 is only required for the formation of PPBs but not for the overall organization of interphasic microtubule arrays.

To monitor the duration of cell division pattern in Col-0, and iqd678 triple mutants, we additionally performed time-lapse microscopy of the RFP-MBD marker in the living root tips of the plants (Figure S 9). We observed the tendency of faster progression from preprophase to metaphase stage of cell division in iqd678 triple mutant compared to WT. However, we noticed variations in other cases in the iqd678 triple mutant. Therefore, at this point, any conclusion could not be drawn concerning the effects of mutations in iqd678 triple mutants on the duration of the cell cycle.

Microtubule pattern analysis revealed that in contrast to PPB loss, all subsequent mitotic microtubule arrays were present in the iqd8-1 and iqd678 mutants. Moreover, microtubule pattern analysis of iqd678 triple and iqd8-1 single mutants revealed that IQD8 is primarily required for organization and formation and positioning of PPB, and IQD6 and IQD7 additionally facilitate the function. Our result demonstrates multifunction of IQD8, and its impairment leads to a defect during several stages of cell wall positioning, which is consistent with the observed differential localization pattern of IQD8.