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tus, the host nucleus migrates towards the infection site. (Genre et al. 2008). Once highly branched arbuscules form, the nucleus then moves from the periphery ...
Plant, Cell and Environment (2011) 34, 1577–1585

doi: 10.1111/j.1365-3040.2011.02354.x

Mycorrhizal symbiosis stimulates endoreduplication in angiosperms pce_2354

1577..1585

L. D. BAINARD1*, J. D. BAINARD1*, S. G. NEWMASTER1 & J. N. KLIRONOMOS2 1 Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 and 2Biology and Physical Geography Unit, University of British Columbia, 3333 University Way, Kelowna, British Columbia, Canada V1V 1V7

ABSTRACT Symbiotic and parasitic relationships can alter the degree of endoreduplication in plant cells, and a limited number of studies have documented this occurrence in root cells colonized by arbuscular mycorrhizal (AM) fungi. However, this phenomenon has not been tested in a wide range of plant species, including species that are non-endopolyploid and those that do not associate with AM fungi. We grew 37 species belonging to 16 plant families, with a range of genome sizes and a range in the degree of endopolyploidy. The endoreduplication index (EI) was compared between plants that were inoculated with Glomus irregulare and plants that were not inoculated. Of the species colonized with AM fungi, 22 of the 25 species had a significant increase in endopolyploid root nuclei over non-mycorrhizal plants, including species that do not normally exhibit endopolyploidy. Changes in the EI were strongly correlated (R2 = 0.619) with the proportion of root length colonized by arbuscules. No change was detected in the EI for the 12 non-mycorrhizal species. This work indicates that colonization by symbiotic fungi involves a mechanism to increase nuclear DNA content in roots across many angiosperm groups and is likely linked to increased metabolism and protein production. Key-words: Glomus irregulare; arbuscular mycorrhizal fungi; DNA content; endopolyploidy; genome size.

INTRODUCTION Endoreduplication occurs when DNA synthesis is not followed by cytokinesis in the mitotic cell cycle. Repeated endoreduplication cycles result in an increase in the DNA content of cells at distinct ploidy levels (e.g. 4C, 8C, 16C, etc.), and nuclei that experience this process are said to be endopolyploid (also referred to as somatic polyploidy or, less commonly, hypertrophy). Endopolyploidy is common in many plant and animal groups and is often associated with specific organs or tissues (Nagl 1976). In angiosperms, Correspondence: L. D. Bainard. E-mail: [email protected] *Contributed equally to this work. © 2011 Blackwell Publishing Ltd

endopolyploidy has also been strongly correlated with family and weakly correlated with genome size and life history strategy (Barow & Meister 2003). Environmental variables can influence the degree of endopolyploidy in an individual plant. In particular, biotic interactions can change the degree of endopolyploidy in the host organism. For example, endoreduplication was increased in maize seedlings infected with Ustilago maydis (Callow 1975), whereas Phytophthora nicotianae var. parasitica infection of tomato roots reduced host endopolyploidy (Lingua et al. 2001a). Induction of host endopolyploidy has also been found to confer a beneficial role in biotrophs that develop a sustained site of nutrient acquisition [e.g. powdery mildew (Chandran et al. 2010), rhizobia (Vinardell et al. 2003; Maunoury et al. 2010) and parasitic nematodes (Gheysen & Mitchum 2009)]. There is evidence that arbuscular mycorrhizal (AM) fungal colonization can increase the degree of endopolyploidy in root cortex cells. Berta et al. (2000) found that Glomus mosseae modified the ploidy distribution of nuclei in Lycopersicon esculentum L. roots. They found an increased number of 2C and 8C nuclei and a decreased number of 4C nuclei in G. mosseae-colonized roots compared with uncolonized control roots. Additionally, small increases in endopolyploid nuclei were found in the roots of Pisum sativum L. (Repetto et al. 2007) and Allium porrum L. (Fusconi et al. 2005) when inoculated with G. mosseae. In contrast, previous studies (Blair, Peterson & Bowley 1988; Berta et al. 1990) found that AM fungal colonization had no effect on the ploidy level of root nuclei. This could be due to poor levels of colonization, as Blair et al. (1988) only grew plants for 20 days before analysis, and neither paper provides clear colonization data. Physiological changes of the host cell have been observed in association with AM fungal colonization, including changes to the position and size of the nucleus. When AM fungi infect root cortical cells via a pre-penetration apparatus, the host nucleus migrates towards the infection site (Genre et al. 2008). Once highly branched arbuscules form, the nucleus then moves from the periphery of the cell back towards the centre, accompanied by an increase in nuclear size and chromatin decondensation (Balestrini, Berta & Bonfante 1992). It has been suggested that these changes are due to modification of gene expression during the 1577

1578 L. D. Bainard et al. establishment of the mycorrhizal symbiosis (Balestrini et al. 1992; Lingua, Fusconi & Berta 2001b). Nuclear enlargement has also been observed in inner cortical cells containing the pre-penetration apparatus, suggesting endoreduplication is part of pre-invasion cell preparation (Genre et al. 2005, 2008). Additional cellular alterations observed after establishment of the arbuscule include changes to the morphology of the large vacuole and the cell wall, and increasing amounts of cytoplasm and organelles, such as mitochondria and plastids (Kinden & Brown 1975; Bonfante & Perotto 1995; Gianinazzi-Pearson et al. 1996; Lingua et al. 2001b). Many of the physiological changes associated with AM fungal colonization are also related to metabolism and nutrient exchange, which is the primary role of the AM fungal symbiosis (Smith & Read 2008). In root cells colonized with AM fungi, Berta et al. (2000) observed higher levels of metabolic activity associated with an increase in nuclear DNA content, compared with controls. An increase in endoreduplication at or near sites of nutrient exchange has also been observed in other symbiotic and parasitic relationships (Wildermuth 2010). In turn, increased protein production and gene transcription has been found to accompany AM fungal colonization (Journet et al. 2002; Delp et al. 2003; Gomez et al. 2009), although more evidence is needed. While there is evidence to suggest that AM fungi affects endopolyploidy in plant root cells, this has only been tested in few species and results have been conflicting, making it unclear how common this phenomenon is across angiosperms. In this study, we first test the hypothesis that AM fungal colonization induces endoreduplication in plant roots, by surveying a broad group of common angiosperm species that associate with AM fungi. Secondly, we test if AM fungal colonization can induce changes in the nuclear ploidy of root cells of plant species that typically do not exhibit endopolyploidy, such as species in the Asteraceae (Barow & Meister 2003). Thirdly, we test if the presence of AM fungi has an effect on plant species that do not typically form an association with AM fungi, including species from the Amaranthaceae, Brassicaceae, Caryophyllaceae and Polygonaceae families (Brundrett 2009). Using correlative analysis, we also determined whether certain AM fungal structures (i.e. arbuscules, vesicles, or hyphae) are more likely to be responsible for changes in endoreduplication in root nuclei. These hypotheses were tested using controlled experiments with 37 plant species inoculated with or without Glomus irregulare (previously Glomus intraradices isolate DAOM 197198; Sokolski et al. 2010), a common mycorrhizal fungus.

MATERIALS AND METHODS Experimental design Seeds from 37 plant species, representing 16 families, were collected from local populations in Guelph, Ontario, Canada. Seeds were surface sterilized (incubated for 1 min in 10% bleach and 95% ethanol solutions) and added to

pots (Deepots, Stuewe & Sons, Tangent, OR, USA) with sterile potting soil (Sunshine Mix #4, Sun Gro Horticulture, Vancouver, BC, Canada). The potting soil was autoclaved twice (45 min at 121 °C) to eliminate the biotic components of the soil. Half of the pots were inoculated with G. irregulare (isolate DAOM 197198) and the other half (control pots) remained free of inoculum. The inoculum consisted of A. porrum roots that were colonized by G. irregulare, which were placed in a layer below the seeds. To neutralize any effect of the A. porrum roots, control pots received a layer of uncolonized A. porrum roots. After adding the seeds, a layer of sterile turface (Profile Products LLC, Buffalo Grove, IL, USA) was placed over the seeds. Pots were placed randomly on a bench at the University of Guelph Phytotron, watered as required and fertilized every 2 weeks. Growing temperatures were maintained at 22–24 °C during the day and 16–18 °C at night, with a 16 h photoperiod. After emergence, seedlings were thinned to one plant per pot. Plant species were grown until the flowering stage, which ranged between 12 and 36 weeks, depending on the species. Several perennial species did not mature beyond the rosette stage and were harvested prior to flowering. The time period provided sufficient time for G. irregulare to successfully colonize roots in the mycorrhizal treatment.

Colonization measurements At the time of harvest, roots were washed with distilled water to remove soil, and then chopped into small segments (approximately 2 cm), avoiding root tips. Roots from each plant were combined and homogenized separately, and divided into three subsamples. One subsample was used to quantify the level of colonization by G. irregulare and two subsamples were used for flow cytometric analysis. Roots were stained with Chlorazol Black E (Sigma-Aldrich, Oakville, ON, Canada) (Brundrett 1994) and assessed for arbuscular, vesicular and hyphal (total) colonization using the magnified intersects method (McGonigle et al. 1990).

Flow cytometry Root samples were chopped in 1.2 mL cold LB01 buffer, in the presence of 100 mg mL-1 propidium iodide (SigmaAldrich, Oakville, ON, Canada) and 0.5 mg mL-1 RNase A (Qiagen, Toronto, ON, Canada) (methods determined by preliminary tests). The resulting homogenate was filtered through a 30 mm mesh, resulting in approximately 1.0 mL of sample. Samples were incubated on ice for 20 min, and for each sample, at least 1500 nuclei were analysed. Flow cytometric analysis was completed on a Partec CyFlow SL (Partec GmbH, Münster, Germany) equipped with a blue solid-state laser tuned at 20 mW and operating at 488 nm. Before each use, the instrument was calibrated using 3 mm calibration beads (Partec). To measure the degree of endopolyploidy, the number of nuclei (n) in each ploidy level was counted by observing the nuclear fluorescence on a log scale. The fluorescence was also plotted versus side scatter (a measure of surface

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1577–1585

AM fungi stimulates endoreduplication 1579 complexity) and polygons were drawn around the scatter plots of nuclei to further isolate the nuclei in each ploidy level. This was carried out using FloMax Software (version 2.52; Partec). The degree of endopolyploidy was quantified by calculating the cycle value, or endoreduplication index (EI), which is a measure of the number of endoreduplication cycles per nucleus that occurred in the nuclei measured. This is calculated according to the following formula (Barow & Meister 2003):

EI = (0 × n2 c + 1 × n4 c + 2 × n8 c + 3 × n16 c . . .) ( n2 c + n4 c + n8 c + n16 c . . .) Genome size (2C value) was estimated using young leaf tissue. Of the six individuals growing for each species, three plants were randomly selected, and genome size was estimated for each plant on two separate days. Nuclei extraction followed the procedure given above, except that the leaf tissue was co-chopped with fresh leaf tissue from an appropriate plant standard. Seeds for standards with known DNA content were acquired from the Laboratory of Molecular Cytogenetics and Cytometry, Olomouc, Czech Republic, and the standards were grown in the University of Guelph Phytotron. Relative nuclear fluorescence was measured at 488 nm on a linear scale on a Partec CyFlow SL. Over 1000 nuclei were acquired for both the standard and sample peak, and coefficients of variation averaged below 5% for both peaks. The six genome-size estimates were averaged to produce one estimate for each species.

Data analysis The mean EI values for control and G. irregulare-colonized roots were compared for each plant species individually using Student’s t-test. The data for all species met the assumptions of this statistical test except Geranium robertianum. The data for G. robertianum did not exhibit homogeneity of variances and, therefore, mean values were compared using a Mann–Whitney U-test. The relationship between change in EI value and proportion of root length colonized by arbuscules, vesicles and hyphae was analysed using linear regression. Change in EI value was calculated by subtracting the control (uncolonized) EI value from the mycorrhizal colonized EI value for each species. In addition, the relationship between genome size and proportion of root length colonized by arbuscules, vesicles and hyphae was analysed using linear regression. Only plant species that were colonized by G. irregulare were included in the linear regression analyses. G. robertianum was excluded from the regression analyses as the roots were highly pigmented, which prevented accurately assessing the colonization of the different mycorrhizal structures. Arbuscule, vesicle and hyphae colonization data were arcsine squareroot transformed prior to statistical analysis. Student’s t-test and Mann–Whitney U-test were performed in SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and regression analyses were performed in SigmaPlot 10.0 (Systat Software, San Jose, CA, USA).

RESULTS AND DISCUSSION We analysed the degree of endopolyploidy, genome size and proportion of root length colonized by G. irregulare of 37 plant species belonging to 16 angiosperm families to determine (1) if AM fungal colonization induces endoreduplication in plant roots; (2) if AM fungal colonization induces changes in root endoreduplication in nonendopolyploid species; (3) if the presence of AM fungi influences root endoreduplication in non-colonized plants; and (4) which AM fungal structures are responsible for changes in root endoreduplication. The plant species used in this study, along with their mycorrhizal status and genome-size estimates, can be found in Table 1. AM fungal colonization varied among the plant species; 25 species were colonized by G. irregulare and 12 species were not colonized. There is evidence that species belonging to commonly non-mycorrhizal families (e.g. Amaranthaceae, Brassicaceae, Caryophyllaceae, Polygonaceae, etc.) can have a low level of AM fungal colonization, including the formation of vesicles and arbuscules (Hirrel, Mehravaran & Gerdemann 1978; Smith & Read 2008). However, none of the species belonging to these families were colonized by G. irregulare as no AM fungal structures were observed in their root tissues. Flow cytometric analysis was used to determine the ploidy levels of the nuclei in control and G. irregulareinoculated roots (Fig. 1). The degree of endopolyploidy was quantified by calculating the EI, which is a measure of the average number of endoreduplication cycles per nucleus measured (Barow & Meister 2003). Nuclei in the roots ranged from 2C to 16C ploidy levels. There was a wide variation in the degree of endopolyploidy among the plant species in the control treatment, with EI values ranging from 0.01 to 0.87. In addition, flow cytometry was used to estimate genome size, and there was a wide variation of genome size (2C) estimates, with 2C values ranging from 0.43 to 9.72 picograms (pg). Similar to previous studies (Nagl 1976; Barow & Meister 2003), we found that plant species with small genomes had a range in degree of endopolyploidy, whereas plant species with larger genomes had a lower degree of endopolyploidy (Fig. 2). The roots of all species colonized by G. irregulare had an increase in endoreduplication compared with those from the control treatment, with 22 of the 25 species having a significant (P < 0.05) increase in EI (Fig. 3). In general, the increase in endoreduplication of G. irregulare-colonized roots corresponded with an increase of nuclei in the highest ploidy level found in control roots. However, nuclei were detected in higher ploidy levels than were found in control roots for eight plant species colonized by G. irregulare. In contrast, none of the 12 non-mycorrhizal plant species had a significant (P > 0.05) increase in endoreduplication in the AM fungal treatment over the control. In general, AM fungal-colonized plant species tended to have a low degree of endopolyploidy, with only three plant species having an EI greater than 0.2 (Fig. 2).All non-mycorrhizal species had an EI greater than 0.1, which is the value that must be

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1577–1585

1580 L. D. Bainard et al. Table 1. Genome size and AM fungal status of plant species used in this study

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. a

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Chenopodium album L. Daucus carota L. Asclepias syriaca L. Arctium minus (Hill) Bernh. Cichorium intybus L. Cirsium arvense (L.) Scop. Cirsium vulgare (Savi) Ten. Crepis tectorum L. Erigeron philadelphicus L. Lactuca serriola L. Tripleurospermum inodorum (L.) Sch. Bip. Solidago flexicaulis L. Symphyotrichum lanceolatum (Willd.)G.L. Nesom Taraxacum officinale F.H. Wigg. Cynoglossum officinale L. Brassica nigra (L.) W.D.J. Koch Erucastrum gallicum (Willd.) O.E. Schulz Erysimum cheiranthoides L. Lepidium campestre (L.) W.T. Aiton Thlaspi arvense L. Cerastium fontanum Baumg. Silene latifolia Poir. Medicago lupulina L. Medicago sativa L. subsp. sativa Trifolium pratense L. Geranium robertianum L. Leonurus cardiaca L. Nepeta cataria L. Epilobium parviflorum Schreb. Oenothera biennis L. Plantago lanceolata L. Plantago major L. Setaria viridis (L.) P. Beauv. Persicaria maculosa Gray. Rumex crispus L. Linaria vulgaris Mill. Urtica dioica L.

Amaranthaceae Apiaceae Apocynaceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Boraginaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Caryophyllaceae Caryophyllaceae Fabaceae Fabaceae Fabaceae Geraniaceae Lamiaceae Lamiaceae Onagraceae Onagraceae Plantaginaceae Plantaginaceae Poaceae Polygonaceae Polygonaceae Scrophulariaceae Urticaceae

3.80 ⫾ 0.003 1.04 ⫾ 0.013 0.86 ⫾ 0.000 4.41 ⫾ 0.027 2.76 ⫾ 0.052 3.02 ⫾ 0.006 5.45 ⫾ 0.012 6.51 ⫾ 0.022 4.61 ⫾ 0.008 5.91 ⫾ 0.013 9.72 ⫾ 0.104 4.00 ⫾ 0.025 4.17 ⫾ 0.040 2.64 ⫾ 0.011 1.25 ⫾ 0.009 1.11 ⫾ 0.006 2.12 ⫾ 0.026 0.43 ⫾ 0.003 0.71 ⫾ 0.005 1.10 ⫾ 0.005 5.69 ⫾ 0.022 5.97 ⫾ 0.008 1.41 ⫾ 0.008 3.73 ⫾ 0.021 1.00 ⫾ 0.002 2.44 ⫾ 0.005 1.68 ⫾ 0.003 1.23 ⫾ 0.005 0.77 ⫾ 0.004 2.28 ⫾ 0.001 2.85 ⫾ 0.079 1.46 ⫾ 0.005 1.10 ⫾ 0.005 3.71 ⫾ 0.026 4.59 ⫾ 0.007 1.97 ⫾ 0.013 1.17 ⫾ 0.006

+ + + + + + + + + + + + + + + + + + + + + + + + + -

Mean genome size (2C) and standard error (n = 3). AM fungal status based on this study.

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exceeded to be considered to have endopolyploid nuclei (Barow & Meister 2003; Jovtchev et al. 2006). Importantly, we found evidence that AM fungal colonization can induce the endoreduplication of root nuclei in plants that are otherwise not thought to be endopolyploid. Asteraceae is considered to be a family that lacks endopolyploid nuclei (Barow & Meister 2003); however, when colonized by G. irregulare, there was a significant increase in the number of 4C nuclei. In the control treatment, all plant species belonging to the Asteraceae had an EI of 0.02, which corresponds with 2% or less of the nuclei having a ploidy level of 4C. When colonized by G. irregulare, the cycle values of all the Asteraceae species increased significantly with EI values as high as 0.095 (9.5% of nuclei in 4C ploidy level). G. irregulare also increased the degree of endopolyploidy of several other non-endopolyploid species including Plantago major, Epilobium parviflorum, Oenothera biennis, Daucus carota and Linaria vulgaris.

To explore which component of AM fungal colonization contributed most to endoreduplication, the relationship between change in EI (G. irregulare-colonized EI value minus control EI value) and proportion of root length colonized by arbuscules, vesicles and hyphae was analysed (Fig. 4).All of the AM fungal structures formed a significant (P < 0.05) positive linear relationship with the increase in endoreduplication associated with G. irregulare-colonized roots. This indicates that as the level of colonization of each structure increases in the roots, so does the degree of endopolyploidy. However, arbuscular colonization had the strongest relationship with the change in cycle value (R2 = 0.619, P < 0.0001), providing evidence that G. irregulare arbuscular colonization is strongly correlated with an increase in the degree of endopolyploidy. Upon removal of Setaria viridis, which appears to be an outlier in the data, the correlation becomes even stronger (R2 = 0.718). Using static cytometry, Berta et al. (2000) have shown that arbuscules

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1577–1585

AM fungi stimulates endoreduplication 1581 (a)

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can alter the ploidy distribution of nuclei in tomato (L. esculentum L.) roots colonized by G. mosseae. They found that cortex cells hosting arbuscules had an increase of 8C nuclei and decrease of 2C and 4C nuclei compared with non-hosting cells. The variation in endoreduplication increase among G. irregulare-colonized plant species could be due to differences in the morphology of the mycorrhizal colonization, in addition to the variation of colonization levels. Arbuscular mycorrhizas can be classified into two main types, Arum and Paris mycorrhizas (Dickson 2004). Arum- and Paristype mycorrhizas vary in morphology and metabolic activity, with a continuum of intermediate forms (Dickson 2004; Van Aarle et al. 2005). In Arum-type mycorrhizas, the arbuscules are the primary site for nutrient exchange, whereas in Paris-type mycorrhizas, nutrient exchange occurs at both

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control and Glomus irregulare-inoculated Arctium minus (a), Asclepias syriaca (b) and Lepidium campestre (c) roots. A. minus and A. syriaca roots were colonized by G. irregulare and L. campestre roots were not. Peaks represent number of nuclei in each ploidy level.

hyphal and arbusculate coils (Van Aarle et al. 2005). Further investigation is required to determine the effect of Arumand Paris-type mycorrhizas on the endoreduplication process in AM fungal-colonized root cortex cells. There are several reasons why the endopolyploid response to AM fungal colonization is significant. Firstly, there is evidence to suggest that there is an increase in gene expression correlated with the increase in cellular ploidy level. Ploidy-induced changes in gene expression have been documented in yeast (Galitski et al. 1999), Arabidopsis (Chen et al. 2004) and maize (Riddle et al. 2010). Secondly, metabolic activity is increased at sites of AM fungal infection (Smith & Read 2008), and there are high levels of nutrient exchange in colonized cells. In turn, cells colonized by AM fungi show an up-regulation of genes relating to protein synthesis, metabolism and abiotic stimuli (Journet et al. 2002;

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1577–1585

1582 L. D. Bainard et al.

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Figure 3. Degree of endopolyploidization of control (white bars) and Glomus irregulare-inoculated (black and grey bars) angiosperm roots. Bars represent the mean endoreduplication index and standard error. Asterisks indicate a significant difference (P < 0.05) between treatments. Black bars represent plant species that were colonized by G. irregulare, and grey bars represent plant species that were not colonized by G. irregulare.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1577–1585

AM fungi stimulates endoreduplication 1583 0.16

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Figure 4. The relationship between the change in endoreduplication index of Glomus irregulare-colonized roots relative to control roots and the proportion of root length colonized by (a) arbuscules (y = 0.015 + 0.001x), (b) vesicles (y = 0.029 + 0.001x) and (c) hyphae (y = 0.012 + 0.001x). Arbuscule, vesicle and hyphae (total) colonization data were arcsine square-root transformed prior to statistical analysis. Significance of the regression equations are indicated as *P < 0.05, **P < 0.01, ***P < 0.0001.

Delp et al. 2003; Gomez et al. 2009). All of these processes would be facilitated by a ploidy increase in DNA content via endoreduplication. The third possible advantage involves the relationship between nuclear size and cell size. While endopolyploidy does not always result in an increase in cell size, an increase in nuclear ploidy is often correlated with cellular expansion (Sugimoto-Shirasu & Roberts 2003). As

arbuscules take up a certain amount of space within the host cell, a larger host cell may be advantageous for arbuscule formation, and, in addition, the larger cell surface area allows for increased transport of nutrients (Wildermuth 2010). As increased cortical cell sizes have been found when arbuscules are present (Balestrini, Cosgrove & Bonfante 2005), there is evidence to suggest that these factors are related.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1577–1585

1584 L. D. Bainard et al. This study presents compelling evidence of the widespread response of endoreduplication in root cells colonized by AM fungi. Future research should involve continued surveys of plant groups with varying degrees and forms of colonization. In turn, it is necessary to explore the response of endoreduplication in root cells to a range of phylogenetically diverse AM fungal taxa that exhibit different life history strategies (Hart, Reader & Klironomos 2001). More research is needed to determine the control of the endoreduplication process in roots with regard to specific cues and the timing of the response. More importantly, determining the plant functional response to root cell endopolyploidization will elucidate if this process gives a selective advantage to plants that have a greater increase in endoreduplication. Additionally, arbuscules are short-lived and there is variation in the duration of the arbuscular cycle among plant species (Alexander et al. 1988, 1989; Brundrett & Kendrick 1990). Given this variation, it is interesting to consider the function of the endopolyploid nucleus once the arbuscule is no longer in the host cell. Although endoreduplication is not typically considered to be a reversible process, there is some evidence in Arabidopsis thaliana to suggest that endopolyploid cells can actually re-enter mitosis (Weinl et al. 2005). Research in this area may give additional insight into the control of endoreduplication in AM fungal-colonized cells.

ACKNOWLEDGMENTS Funding for this research was provided by the Natural Sciences and Engineering Research Council (Discovery grant to J.N.K.; CRD grant to S.G.N.; PGSD to L.D.B and J.D.B.), the Canadian Foundation for Innovation (LOF to S.G.N.) and the Government of Ontario (OGS to L.D.B.). We thank Thomas Henry, Kelsey O’Brien and Benjamin Yim for lab assistance.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Ploidy level distribution of nuclei from control (open bars) and Glomus irregulare-inoculated (grey bars) plant roots. The number in each histogram corresponds to the plant species number listed in Table 1. Bars represent the mean proportion of nuclei in each ploidy level (i.e. 2C, 4C, 8C and 16C) and standard error. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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