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Control of Microtubule Nucleation and Dynamics in Plant Cells

Final Report Summary - COMNADIPC (Control of Microtubule Nucleation and Dynamics in Plant Cells)

Research Objectives:
During land plant evolution, genetic and functional changes of regulatory mechanisms drive morphological innovations. One such innovation is the loss of centrioles and the gain of properties of plant microtubules that allow them to self-nucleate in acentriolar cells. The nature of this proposed research is to study the evolution of the microtubule-organizing gene network during the conquest of land by plants. In particular, it is still unclear how the components of the complex system of genes that control microtubule nucleation and dynamics have changed and the relationship between these changes and the loss of centrioles. One approach to solving this is to study transitional forms that combine aspects of both types of microtubule organisation. The moss Physcomitrella patens contains both acentriolar cells and centriolar cells. Recently novel land plant-specific microtubule-associated proteins (MAPs), EDE1, have been identified from an angiosperm Arabidopsis thaliana and P. patens by the Doonan group (Pignocchi et al., 2009). The EDE1 is a good candidate for probing microtubule organisation in acentriolar cells and centriolar cells due to its role on microtubule nucleation.
Buildling on this preparatory work, the current proposal will address two main goals: firstly, to understand the biological and molecular functions of PpEDE1, particularly in acentriolar cells and centriolar cells, and secondly to estimate functional changes of plant MAPs including PpEDE1 related to innovations of the microtubule organisation in plant cells. This will be done using two different species, the moss P. patens and the angiosperm A. thaliana, and a range of approaches, from the reverse genetics to the biochemical technique, by addressing the following objectives:
1.1 How does PpEDE1 affect microtubule function?
1.2 Does PpEDE1 localise on cytoplasmic or mitotic MTs in the moss?
1.3 How does PpEDE1 affect microtubule dynamics?
1.4. Is EDE1 functionally conserved between mosses and angiosperms?
1.5 What is the evolutionary distribution of other plant-specific MAPs?

Results and Discussion:
Recent progress suggests EDE1 is a plant-specific member of augmin complex (Nakaoka et al., 2012). Therefore, four PpEDE1, PpEDE2, PpEDE3, and PpEDE4 genes described in the proposal were re-named as AUG8a, AUG8b, AUG8c, and AUG8d, respectively.

“1.1How does PpEDE1 affect microtubule function?”
To observe MT dynamics in living cells, MT-reporter lines expressing sGFP-α-tubulin or tagRFP-α-tubulin fusion proteins under the control of the constitutive KINID1a promoter were produced (Hiwatashi et al., 2014). The lines expressing sGFP-α-tubulin or tagRFP-α-tubulin fusion protein were named GTU and tRTU, respectively. The citrine gene, encoding a modified yellow fluorescence protein, was integrated into the EB1b gene locus by gene targeting to generate the lines expressing the EB1b-citrine fusion protein. These lines were named EY. Furthermore, the lines expressing both the tagRFP-α-tubulin and EB1b-citrine fusion proteins were established by inserting the citrine gene into the genomic EB1b gene locus of the tagRFP-α-tubulin line (Hiwatashi et al., 2014). The line was designated as EYRT. In addition, inducible overexpression and RNA interference system has been established (Kubo et al., 2013; Nakaoka et al., 2012).
To assess whether AUG8 play roles on cell expansion and division, knock-out mutants of the AUG8 gene were generated. In the GTU line, each AUG8 gene was replaced with an antibiotic-resistant marker cassette by gene-targeting to make null mutants. The single and double deletion mutants were obtained. Moreover, the aug8a aug8b aug8c and aug8a aug8c aug8d triple deletion mutants were generated. Two kinds of triple deletion mutants (aug8a aug8b aug8d and aug8b aug8c aug8d) as well as a quadruple deletion mutant (aug8a aug8b aug8c aug9d) were not obtained. The single and double deletion mutants of AUG8 genes exhibited no detectable alternations on the morphological characters including cell length and shape. The aug8a aug8b aug8c triple deletion mutants exhibited morphological alternations of colony shape, while the aug8a aug8c aug8d triple mutant did not. The colony shape of the triple deletion mutants was different from that of the GTU line, and rather was similar to that of the control GTU line treated with MT-depolymerising drugs, suggesting that the triple deletion mutants had defects in the MT organisation in acentriolar cells.
The failure to recover quadruple deletion mutants suggests that deletion of all four AUG8 genes leads to the lethality at the protonemal stage.

“1.2 Does PpEDE1 localise on cytoplasmic or mitotic MTs in the moss?”
Accumulation of the AUG8 transcripts in the haploid tissue and the sporophyte-like tissue derived from the haploid tissue by apogamy was investigated using the digital gene expression analysis with the next generation sequencer. The transcripts of all four AUG8 genes were detected in both tissues, suggesting that all AUG8 transcripts are expressed in both haploid and diploid generation. Among them, the AUG8c transcripts were most abundant in both tissue types.
To observe the cellular localisation of AUG8 in protonemata, the sGFP gene was inserted just after start codon of AUG8a, AUG8b, and AUG8c genes by gene targeting, to make the lines expressing the sGFP-AUG8 fusion proteins. Similar, the citrine gene was inserted just before stop codon of the AUG8d gene to generate the lines expressing a AUG8d-citrine fusion protein. The live-imaging analysis with the sGFP-AUG8a, sGFP-AUG8b, sGFP-AUG8c, and AUG8d-citirne lines revealed that all AUG8 fusion proteins were detected in cytoplasmic MTs and further mitotic and phragmoplast MTs in protonemata. The signals of the AUG8c protein were more intense than those of other fusion proteins, which is consistent with abundance of AUG8c transcripts in protonemata. The localisation of the AUG8c fusion proteins on MTs was highly dynamic in a punctate manner. The AUG8c fusion protein was localised to the base points of branching MTs on cytoplasmic MTs, suggesting that the AUG8c protein are associated with MT nucleation on the existing MTs in cytoplasm.

“1.3 How does PpEDE1 affect MT dynamics?”
To assess whether the AUG8 proteins regulate MT stability in vivo, the deletion mutants was treated with the MT-depolymerising drugs and observed the growth of the plants. An application of the MT-depolymerising drug cremart showed that the aug8a aug8c aug8d triple deletion mutants were hypersensitive to the drug, suggesting that the MTs are more unstable and/or less generated in that triple deletion mutants.
To assess whether the AUG8 protein is associated with the augmin complex in P. patens, immunoprecipitation followed by Mass Spectrography using the AUG4, another member of the augmin complex was done. Immunoprecipitation followed by MS analysis indicated that the AUG8 protein was detected together with other at least 5 augmin subunits, suggesting that AUG8 was a subunit of the augmin complex.
The timelapse observation of sGFP-α-tubulin in the single, double, and triple deletion mutants revealed that their MT dynamics at interphase as well as M-phase was similar to that in the control line GTU.

“1.4. Is EDE1 functionally conserved between mosses and angiosperms?”
To test whether the AUG8 genes can fully substitute for EDE1 function, it was planned that A. thaliana mutants lacking EDE1 function such as ede1-1 plants were transformed with AUG8a, AUG8b, AUG8c, and AUG8d genes driven by the constitutive CaMV35S promoter. Binary vectors carrying the AUG8b, AUG8c, and AUG1d fused with the GFP gene, were made. The ede1-1 mutants were transformed with the aforementioned binary vectors. The T3 plants were identified and preliminary results indicate that all 4 moss AUG8 genes can complement all aspects of the ede1-1 phenotype, such as the root growth, inflorescence morphology and seed set. This result suggests that the AUG8 function is conserved between A. thaliana and P. patens.

“1.5 What is the evolutionary distribution of other plant-specific MAPs?”
The phylogenetic trees of 33 land plant MAPs genes were constructed as a collaboration work (Banks et al., 2011). Of land plant MAPs, two different subgroups of plant-specific kinesins and one subgroup of ubiquitin-like domain protein were analysed deeply.
One of two kinesin groups was a plant-specific orphan kinesin, named KINID1a and KINID1b (for kinesin for interdigitated microtubule 1a and 1b). These kinesins function in the bundling of phragmoplast MTs at cytokinesis (Hiwatashi et al., 2008). The KINID1a and KINID1b proteins also accumulate in growing tips of protonemal apical cells, which exhibit tip-growth. To elucidate MT dynamics and its underlying mechanism responsible for moss tip growth, the dynamics of MTs and the KINID1 proteins, and phenotype of the kinid1a kinid1b-deletion mutants was investigated. Functional analysis of KINID1a and KINID1b revealed that these kinesins regulated the maintenance of the MT bundles in the apical expansion zone of the polarised growth in the moss. It was found that A. thaliana had one orthologous gene named PAKRP2 to KINID1a. The PAKRP2 protein was associated with the cytokinetic apparatus, phragmoplast, during cell division, and was not likely associated with polarised growth. This result suggested that function of the kinesin subgroup including KINID1a was not completely conserved between P. patens and A. thaliana (Hiwatashi et al., 2014).
Another kinesin was KAC1 and KAC2 (for KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 and 2), which were required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts in A. thaliana. To assess whether function of KAC1 and KAC2 proteins is conserved among land plants, the KAC1 and KAC2 proteins of the fern Adiantum capillus-veneris and P. patens were identified. The chloroplast movement was examined in the knock-down mutants of A. capillus-veneris and the deletion mutants of P. patens. The mutant analysis revealed that KAC orthologs (AcKAC1 and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern A. capillus-veneris and the moss P. patens, and chloroplast positioning and movement were mediated through the activities of KAC proteins, which were conserved in land plants (Suetsugu et al., 2012).
The ubiquitin-like domain protein ULD1a and ULD1b are involved in the loss of the phragmoplast MTs. To investigate the turnover of MTs at cytokinesis, deletion of both the ULD1a and ULD1b genes were deleted in the GTU line and EY line. The imaging of MT dynamics during cytokinesis of the double deletion mutants revealed that these ubiquitin-like domain proteins regulate the MT depolymerisation by repressing the growth activity of the MT plus-ends at the phragmoplast equator. Novel MAPs, which are associated with the ULD1a and ULD1b protens were searched using the ULD1b-sGFP fusion protein as a tag. The candidate MAPs were immune-precipitated using anti-GFP antibody with an extract from the line expressing the ULD1b-sGFP fusion protein and followed by MS analysis. The protein profile was summarised.
To identify the new MAPs in land plants, the INTACT system was applied. In the INTACT method, the tissue-specific promoters were cloned in order to express the NTF (nuclear targeting fusion protein) in the cell-specific manner in P. patens. The expression cassettes were built.

This study indicates that the augmin complex has common functions in MT generation between acentriole plant cells and centriole animal cells, providing new insights into the evolution of MT organisation. Other aspects of MT organisation in these primative plants were also clarified, indicating that functions involved in seed development and root growth actually arose early in plant evolution and that those functions have not diverged significantly since. This also indicates that the mechanisms that primative plants use for cell division and growth are also largely conserved and this knowledge will inform various areas of plant biotech, including the directed manipulation of plant development. In the medium to long term, the ability to re-engineer plant form has far reaching implications.

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