Epidermal Patterning in Seedling Roots of Eudicotyledons

Annals of Botany 87: 649±654, 2001 doi:10.1006/anbo.2001.1385, available online at http://www.idealibrary.com on

Epidermal Patterning in Seedling Roots of Eudicotyledons L I A M . S. P E M B E R TO N , S H I N - L I N G T S A I , P E T E R H . LOV E L L and P H I L I P J . HA R R I S * School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand Received: 2 November 2000

Returned for revision: 17 December 2000 Accepted: 22 January 2001 Published electronically: 27 March 2001

Three types of epidermal patterning occur in roots of angiosperms: in Type 1, all the epidermal cells can potentially produce root hairs (hair cells); in Type 2, asymmetric cell divisions produce short cells that develop into hair cells and larger cells that do not (non-hair cells); and in Type 3, hair cells occur in ®les separated by one to three ®les of non-hair cells. In the present study we examined the epidermal patternings of seedling roots of 77 eudicotyledonous species from 43 families. We found that Type 1 patterning was the most common and no species had Type 2 patterning. Previously, Type 3 epidermal patterning had been described only in the family Brassicaceae. In addition to the Brassicaceae (including the Capparaceae), we found Type 3 patterning in the Brassicales families Limnanthaceae and Resedaceae, whereas the other Brassicales families we examined, Caricaceae and Tropaeolaceae, had Type 1 patterning. We also found Type 3 patterning in six families of the Caryophyllales sensu lato: Amaranthaceae, Basellaceae, Caryophyllaceae, Plumbaginaceae, Polygonaceae and Portulacaceae. However, the family Cactaceae, which is also in this order, had Type 1 patterning. Only one other species, Nemophila maculata (Boraginaceae), had Type 3 patterning; the other two species that we examined in this family had Type 1 patterning. Type 3 patterning thus occurs # 2001 Annals of Botany Company more widely in the eudicotyledons than was previously thought. Key words: Brassicales, Caryophyllales, eudicotyledons, epidermal patterning, phylogeny, root hairs, roots, seedlings.

I N T RO D U C T I O N Root epidermal cells make up the outermost layer of a root and can give rise to root hairs which are tubular outgrowths from these cells. Root hairs are important in aiding the uptake of nutrients and water and anchoring the plant in the soil by greatly increasing the surface area of the root (Hofer, 1996; Peterson and Farquhar, 1996; Ridge, 1996; Gilroy and Jones, 2000). Two types of epidermal cells can be recognized: those which develop a root hair (hair cells), and those which remain hairless (non-hair cells). In angiosperms, the hair and non-hair cells are arranged in three types of patterns (Dolan and Roberts, 1995; Dolan, 1996). In Type 1 patterning, all the root epidermal cells have the potential to produce root hairs, although this potential is not always realized. Except for the presence of hairs, these hair cells do not appear to be morphologically di€erent from non-hair cells. Type 2 patterning is characterized by asymmetric cell divisions of the epidermal cells in the meristematic zone. A root hair develops from the smaller of the two cells produced by this division; the larger cell remains hairless. In Type 3 patterning, the hair cells occur in ®les separated by one to three ®les of non-hair cells. The number of ®les of non-hair cells was shown to be ecotype-dependent in the roots of Arabidopsis thaliana (Berger et al., 1998). In this species, the hair cells are also shorter than non-hair cells and have denser cytoplasm (Dolan et al., 1993, 1994). The distribution of these di€erent types of root-epidermal patternings in di€erent angiosperm taxa has only been investigated to a limited extent (Dolan and Roberts, 1995; Dolan, 1996). Type 1 patterning appears to be the most * For correspondence. Fax ‡64-373-9-7416, e-mail p. harris@ auckland.ac.nz

0305-7364/01/050649+06 $35.00/00

widespread. Leavitt (1904) carried out a large survey of angiosperms and found Type 2 patterning in some families of monocotyledons, including the Poaceae, and in the dicotyledonous family Nymphaeaceae, but not in any of the families now known as the eudicotyledons (Angiosperm Phylogeny Group, 1998). Types 1 and 2 were also found in the Poaceae by Row and Reeder (1957). Type 3 patterning was ®rst described by Cormack (1935) in the brassicaceous species Brassica oleracea, B. alba (ˆSinapis alba), B. napus var. chinensis (ˆB. chinensis) and Raphanus sativus, and has since been found in two other species in this family: Lepidium sativum (BuÈnning, 1951) and the model plant Arabidopsis thaliana (Dolan et al., 1993, 1994). Cao et al. (1999) stated: `This cellular organisation of the root epidermis is a characteristic of most members of the Brassicaceae and has not to our knowledge been described for species outside this family'. In his survey of angiosperms, Leavitt (1904) recognized only Type 1 and 2 patternings. Type 3 patterning was probably overlooked at that time and recorded as Type 1. Indeed, Leavitt (1904) recorded two species of Brassicaceae, Cardamine hirsuta and Nasturtium ocinale (ˆRorippa nasturtium-aquaticum), as having Type 1 patterning. It is thus possible that Type 3 root-epidermal patterning occurs in angiosperm taxa other than the Brassicaceae, but has not been recognized to date. Further indirect evidence for this view was presented by Clowes (2000). His anatomical study of root apical meristems showed that trichoblasts ( precursors of hair cells) were arranged in a radial pattern in many families, including several families in the Brassicales. This type of trichoblast distribution could lead to a Type 3 pattern of root hairs. In the present study we examined the root-epidermal patterning of seedlings of 77 species in 43 families of # 2001 Annals of Botany Company

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eudicotyledons, including 17 species of the family Brassicaceae. In planning this survey, we used the Angiosperm Phylogeny Group (1998) classi®cation of angiosperms which is mostly based on recent molecular phylogenetic analyses. M AT E R I A L S A N D M E T H O D S Plant materials Four seeds of 77 species of eudicotyledons in 43 families were placed on two pieces of ®lter paper (No. 1, Whatman Ltd, Maidstone, UK) in 90 mm diameter glass Petri dishes. Sterilized distilled water (3 ml) was added to the Petri dishes, which were sealed with Para®lm M1 (American Can Company, Greenwich, CT, USA) to prevent water loss, and placed in the dark at 25 8C until the primary roots were up to 40 mm long. Seeds of the few species that did not germinate under these conditions were sown in 100 mm diameter pots in steam sterilized general potting mix (Watkins, Onehunga, Auckland) and kept at 23±25 8C in a glasshouse with natural lighting until the seeds had germinated and the primary roots were up to 60 mm long. The seedlings were removed from the ®lter paper or potting mix and excess soil was removed by gently washing with water. Microscopy Bright-®eld microscopy. The primary roots were cut, using a scalpel, from three to ®ve seedlings of each species, stained and examined by bright-®eld microscopy using a Zeiss KF2 microscope (Oberkochen, Germany) ®tted with a 20 W halogen quartz lamp; the type of epidermal patterning was recorded. When we started the survey, we stained roots in 0.05 % aqueous Toluidine Blue for 1 min, and then rinsed o€ excess stain. However, we later found that clearer results were obtained by staining the roots for a few seconds in black ink (Pelikan 4001, Brilliant Black, Germany) and removing excess ink with wet ®lter paper. Scanning electron microscopy (SEM). Roots of L. douglasii seedlings were used 3 d after emergence (approx. 40 mm long). Roots were cut 5 to 7 mm from the root tip, using a scalpel, and were stuck as quickly as possible with carbon tape onto an SEM stub. The root tips were then frozen by plunging them into a nitrogen slush. Preliminary work, with no partial freeze drying of the specimens, showed that there was a problem with ice crystals that formed on the surfaces of the roots and root hairs. To reduce the occurrence of these ice crystals, the stub was heated for 5 min to ÿ85 8C at 0.05 Torr in a special workchamber (Model SP2000, Emscope, Kent, UK) to allow the water to sublime. The stub was coated with gold under vacuum at ÿ185 8C using a sputter coater (Emscope) and inserted onto the cold stage of a scanning electron microscope (Model 505, Philips, Eindhoven, The Netherlands). Microscopy was done at 12 kV with the stage at ÿ185 8C. Photographs were taken on Ilford Plus 125-FP4 black and white ®lm. The negatives were scanned (Leafscan 45, Scitex, Massachusetts, USA) and the images

imported into Adobe Photoshop2 (Adobe Systems Inc., Mountain View, CA, USA). R E S U LT S The type of root-epidermal patterning of the seedling eudicotyledon species examined is shown in Table 1. Type 1 epidermal patterning was found in the majority of species, whereas Type 3 occurred only in the orders Brassicales and Caryophyllales sensu lato, and the family Boraginaceae. Type 2 root-epidermal patterning was not found in any of the species examined. In the Brassicales, all 17 species examined from 17 genera of the Brassicaceae had Type 3 root-epidermal patterning. These included Cleome spinosa, which was formerly placed in a separate family, the Capparaceae, but is now placed in the Brassicaceae (Angiosperm Phylogeny Group, 1998). Four other families of Brassicales were studied: Limnanthaceae, Resedaceae, Caricaceae and Tropaeolaceae. Type 3 patterning was found in Limnanthes douglasii (Limnanthaceae) and in Reseda alba and R. odorata (Resedaceae), but Type 1 patterning was present in Tropaeolum majus and T. peregrinum (Tropaeolaceae) and Carica papaya (Caricaceae). The root-epidermal patterning of seedlings of Limnanthes douglasii was examined by SEM as an example of a species from a family other than the Brassicaceae which we found, using bright-®eld light microscopy, had Type 3 patterning. This patterning is shown in Fig. 1. In the Caryophyllales sensu lato, Type 3 root-epidermal patterning was present in all the species examined except Mammillaria leucocentra (Cactaceae), which had Type 1 patterning (Table 1). The seedlings of this species had short, slowly elongating primary roots with extremely long hairs arising close to the apex. These long root hairs could be a compensating mechanism for successful seedling establishment in xerophytic conditions. Only one other species had Type 3 root-epidermal patterning: Nemophila maculata in the Boraginaceae (including the Hydrophyllaceae), a family in the Euasterids I not yet placed in an order (Angiosperm Phylogeny Group, 1998).

F I G . 1. Scanning electron micrograph of the surface of a seedling root of Limnanthes douglasii (Limnanthaceae) in the zone of root-hair elongation. The hair cells are in ®les separated by one or two ®les of non-hair cells, indicating Type 3 patterning. Bar ˆ 100 mm.

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T A B L E 1. The root-epidermal patterning types of the seedlings examined. The taxa are classi®ed according to the Angiosperm Phylogeny Group (1998) Taxa examined EUDICOTS Ranunculales Papaveraceae Papaver nudicaule L. Ranunculaceae Aquilegia vulgaris L. Nigella damascena L. CORE EUDICOTS Caryophyllales Aizoaceae Dorotheanthus bellidiformis (Burm. f.) N.E. Br. Amaranthaceae Amaranthus tricolor L. aAtriplex hortensis L. Celosia argentea var. cristata (L.) Kuntze Gomphrena globosa L. aSpinacia oleracea L. Basellaceae a Basella rubra L. Cactaceae b Mammillaria leucocentra Berg. Caryophyllaceae Dianthus caryophyllus L. Plumbaginaceae Limonium sinuatum (L.) Mill. Polygonaceae Rumex acetosa L. Portulacaceae Calandrinia umbellata (Ruiz & Pav.) DC. Claytonia perfoliata Donn ex Willd. ROSIDS Geraniales Geraniaceae Pelargonium  hortorum L.H. Bail. EUROSIDS I Cucurbitales Cucurbitaceae Cucurbita moschata Duchesne ex Poir. Fabales Fabaceae Trifolium repens L. Malpighiales Euphorbiaceae c Drypetes deplanchei subsp. anis Pax & K. Ho€m. Euphorbia variegata De¯ers Linaceae Linum usitatissimum L. Passi¯oraceae aPassi¯ora edulis Sieber ex Sims Violaceae Viola tricolor L. Rosales Rosaceae Geum chiloense Balb. ex Ser. EUROSIDS II Brassicales Brassicaceae Arabidopsis thaliana (L.) Heynh. ecotype Columbia Barbarea verna (Mill.) Asch. Brassica oleracea L. Cleome spinosa Jacq. Eruca sativa (Mill.) Thel. Erysimum cheiri (L.) Cranz. Hesperis matronalis L. Iberis umbellata L. aIsatis tinctoria L. Lepidium sativum L. Lobularia maritima (L.) Desv. Lunaria annua L. Malcolmia martima (L.) R. Br. Matthiola bicornis (Sm.) P. Ball Raphanus sativus L. Rorippa nasturtium-aquaticum (L.) Hayek Sinapis alba L. a

Patterning type

1 1 1

3 3 3 3 3 3 3 1 3 3 3 3 3

1

1 1 1 1 1 1 1 1

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Seeds germinated in potting compost. Roots 2 mm long when sampled. c Roots taken from a mature plant growing in potting compost. b

Taxa examined Caricaceae a Carica papaya L. Limnanthaceae Limnanthes douglasii R. Br. Resedaceae Reseda alba L. R. odorata L. Tropaeolaceae a Tropaeolum majus L. aT. peregrinum L. Malvales Malvaceae Althea ocinalis L. Hibiscus esculentus L. a Lavatera trimensis L. a Malva sylvestris L. aSidalcea malvi¯ora (DC) Benth. Myrtales Onagraceae aFuchsia denticulata Ruiz & Pav. Oenothera glazioviana Micheli ex Mart. Sapindales Rutaceae aRuta graveolens L. ASTERIDS Ericales Balsaminaceae Impatiens walleriana Hook. f. Polemoniaceae Phlox drummondii Hook. Primulaceae a Primula veris L. EUASTERIDS I Boraginaceae Echium plantagineum L. Phacelia campanularia A. Gray. Nemophila maculata Benth.ex Lindl. Gentianales Apocynaceae aAsclepias curassavica L. Rubiaceae Asperula orientalis Boiss & Hohen. Lamiales Bignoniaceae a Eccremocarpus scaber Ruiz & Pav. Lamiaceae Perilla frutescens var. crispa (L.) Britt. Scrophulariaceae Antirrhinum majus L. Verbenaceae a Verbena venosa Gill. & Hook. Solanales Convolvulaceae Convolvulus minor L. Solanaceae Lycopersicon esculentum Mill. Nolana paradoxa Lindl. EUASTERIDS II Apiales Apiaceae Daucus carota L. Asterales Asteraceae Echinops ritro L. Helianthus annuus L. Lactuca sativa L. Campanulaceae Campanula persicifolia L. Dipsacales Dipsacaceae Scabiosa stellata L. Valerianaceae a Valeriana ocinalis L.

Patterning type

1 3 3 3 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 3 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1

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Type 3 patterning varied slightly among the species examined: hair ®les and non-hair ®les alternated, for example in Raphanus sativus; one or two non-hair ®les occurred between the hair ®les, for example in Basella rubra, Calandrinia umbellata and Limnanthes douglasii; and two or three non-hair ®les occurred between the hair ®les in Nemophila maculata, Reseda alba and R. odorata. We also found the rare occurrence of two adjacent hair ®les in the seedling roots of Spinacia oleracea. The epidermal patterning on the roots of seedlings of Limnanthes douglasii with hair ®les separated by one or two non-hair cell ®les is shown in Fig. 1. DISCUSSION In our survey of seedling roots of eudicotyledons, epidermal patterning was either Type 1 or 3, with Type 1 being the most common; Type 2 patterning was not found, which is consistent with the survey of Leavitt (1904). Type 3 patterning occurred only in the orders Brassicales and Caryophyllales sensu lato, and in Nemophila maculata, which is a member of the Boraginaceae, a family in the Euasterids I not yet placed in an order (Angiosperm Phylogeny Group, 1998). In the Brassicales, all species examined within the family Brassicaceae had Type 3 epidermal patterning, including Rorippa nasturtium-aquaticum, which was described as Type 1 by Leavitt (1904). The Brassicaceae is a large, homogeneous family with 365 genera and 3250 species (Mabberley, 1997);

it is thus likely that all the species have this type of epidermal patterning. We found Type 3 patterning in other families of the Brassicales; this is the ®rst time this type of patterning has been described outside the Brassicaceae. Furthermore, the other families within the Brassicales that have Type 3 patterning appear phylogenetically to be the most closely related to the Brassicaceae. The phylogeny of families within the Brassicales has been investigated using morphological characters and the nucleotide sequences of rbcL and 18 S nrDNA (Rodman et al., 1996, 1998). A cladogram constructed by combining DNA sequence data from these two genes is shown in Fig. 2. The families we examined that have Type 3 patterning are present only in a subclade of the main Brassicales clade comprising species that have an extension to the 30 end of the rbcL gene and which does not occur elsewhere in the Brassicales. This subclade, which includes all the Brassicales families other than the Akaniaceae, Bretschneideraceae, Caricaceae, Moringaceae and Tropaeolaceae, is also synapomorphic for two morphological characters: onagrad embryogeny and dilated cisternae of the endoplasmic reticulum, which are unique to this clade (Rodman et al., 1998). The families that have Type 1 patterning, Caricaceae and Tropaeolaceae, are basal to the Limnanthaceae on the cladogram and are not part of this subclade. In addition to the families within Brassicales that we examined, the order contains the following small, often monogeneric families that have not been examined:

Arabidopsis Brassica Cleome Capparis Reseda Gyrostemon Tovaria Pentadiplandra Koeberlinia Batis Salvadora Setchellanthus Limnanthes Floerkea Carica Moringa Tropaeolum Bretschneidera Akania

Type 3 Type 3

Brassicaceae

Type 3 Type 3

Resedaceae Gyrostemonaceae Tovariaceae Pentadiplandraceae Koeberliniaceae Bataceae Salvadoraceae Brassicaceae

Type 3

Limnanthaceae

Type 1

Caricaceae Moringaceae

Type 1

Tropaeolaceae Bretschneideraceae Akaniaceae

F I G . 2. Cladogram modi®ed from Rodman et al. (1998) showing the phylogeny of the Brassicales constructed from the nucleotide sequences of rbcL and 18S nrDNA. Types 1 and 3 refer to the root-epidermal patterning of genera determined in the present study.

Pemberton et al.ÐRoot Epidermal Patterning Akaniaceae, Bataceae, Bretschneideraceae, Gyrostemonaceae, Koeberliniaceae, Moringaceae, Pentadiplandraceae, Salvadoraceae and Tovariaceae. From their positions on the cladogram, we predict that species in the families Bataceae, Gyrostemonaceae, Koeberliniaceae, Pentadiplandraceae, Salvadoraceae and Tovariaceae will have Type 3 rootepidermal patterning, whereas species in the other families will have Type 1 root-epidermal patterning. One feature shared by all 14 families of the Brassicales examined to date is the presence of mustard oil glycosides or glucosinolates (Rodman et al., 1996, 1998). Outside the Brassicales, these compounds have only been found in the genus Drypetes (Euphorbiaceae). However, phylogenetic studies have shown that the Euphorbiaceae is distant from the Brassicales; it is in the Eurosid I group rather than the Eurosid II group (Angiosperm Phylogeny Group, 1998). We found that Drypetes deplanchei and Euphorbia variegata had Type 1 patterning. We also found Type 3 patterning in all the species we examined belonging to the order Caryophyllales sensu lato except for Mammillaria leucocentra (Cactaceae) (Angiosperm Phylogeny Group, 1998). This order includes the 11 families recognized in the Caryophyllales sensu stricto (Cronquist, 1988): Achatocarpaceae, Aizoaceae, Amaranthaceae (including genera previously in the Chenopodiaceae), Basellaceae, Cactaceae, Caryophyllaceae, Didiereaceae, Molluginaceae, Nyctaginaceae, Phytolaccaceae and Portulacaceae. It also includes additional families whose DNA sequence data have recently shown them to be phylogenetically related to the Caryophyllales sensu stricto (Nandi et al., 1998): Ancistrocladaceae, Asteropeiaceae, Dioncophyllaceae, Droseraceae, Frankeniaceae, Nepenthaceae, Plumbaginaceae, Polygonaceae, Rhabdodendraceae, Simmondsiaceae and Tamaricaceae. For many years, the families of the Caryophyllales sensu stricto have been recognized as having a series of morphological and chemical features unknown in other angiosperms (Cronquist, 1988). These features include the production of betalains rather than anthocyanins as ¯ower pigments (except for the Caryophyllaceae and Molluginaceae) (Mabry, 1976; Cronquist, 1981), and having a characteristic type of sieve-tube plastid (Behnke, 1981). These families also have ester-linked ferulic acid in their unligni®ed primary cell walls (Hartley and Harris, 1981). This character has not been found in any other dicotyledonous order, but has been found in the commelinoid monocotyledons (Harris and Hartley, 1980; Harris, 2000). In addition, many of these families have a characteristic spherical, pantoporate type of pollen grain which is rare among other angiosperms (Cronquist, 1988). The new, enlarged grouping of the Caryophyllales sensu lato also has a range of features in common, including irregular secondary growth and an endosperm provided with starch grains which are not found at high frequencies in other taxa (Nandi et al., 1998). Type 3 patterning appears to be another feature that is common in this group. However, it is not present in the species of Cactaceae (Mammillaria leucocentra) we examined and more families need to be examined. The Type 3 root-epidermal patterning occurs in three phylogenetically well separated taxa of eudicotyledons: the Brassicales (Eurosids II), the Caryophyllales sensu lato

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(core eudicotyledons outside the Rosids and Asterids), and the Boraginaceae (Euasterids I). Thus, it appears that this type of patterning evolved at least three times in the eudicotyledons. The mechanisms controlling the development of Type 3 root-epidermal patterning in Arabidopsis thaliana have been studied extensively over the last decade, with some of the genes involved being characterized and models proposed for their interactions (Lee and Schiefelbein, 1999; Mendoza and Alvarez-Buylla, 2000). It would be interesting to determine whether similar genes control Type 3 root-epidermal patterning in other species of Brassicaceae and in the other families of Brassicales with Type 3 root-epidermal patterning. It would be even more interesting to determine if similar genes control Type 3 root-epidermal patterning in the Caryophyllales sensu lato and in Nemophila (Boraginaceae) which appear to have evolved the Type 3 root-epidermal patterning independently of one another and of the Brassicales. From the present study, root-hair patterning Types 1 and 3 occur in the eudicotyledons, but Type 2 root-hair patterning either does not occur or is very rare. However, this type of patterning occurs in the non-eudicotyledons: the Nymphaeaceae and many monocotyledonous families, including the Poaceae (Leavitt, 1904; Row and Reeder, 1957). As far as we are aware, Type 3 root-epidermal patterning has not been recorded in angiosperms other than the eudicotyledons. However, the only major survey of root-hair patterning which included the non-eudicotyledons (Levitt, 1904) did not recognize Type 3 patterning. Thus, Type 3 root-epidermal patterning may occur in this group of plants. Clowes (2000) carried out an anatomical examination of longitudinal and transverse sections of the root apical meristems of a range of angiosperm species. He examined these sections for the presence of trichoblasts which he de®ned as `a cell that is visibly recognizable as the precursor of a root hair cell'. He recognized two patterns of trichoblast di€erentiation: in vertical rows resulting from the unequal division of the mother cell, with the short cell later becoming the trichoblast; and a radial pattern, seen in transverse sections, in which trichoblasts arise from epidermal cells lying on the radii between the radial rows of cortical cells, with the epidermal cells on the same radii as the cortical cells not becoming the trichoblasts. These two patterns of trichoblast di€erentiation would be expected to result in the formation of Type 2 and 3 rootepidermal patternings, respectively. Interestingly, Clowes (2000) found that the vertical pattern of trichoblasts did not occur in the eudicotyledons, which is consistent with our ®nding no species with Type 2 patterning in this plant group. The vertical pattern of trichoblasts occurred in many species of monocotyledons, including the Poaceae, as does the Type 2 root-epidermal patterning. Furthermore, Clowes (2000) found the radial pattern of trichoblasts only in eudicotyledons. It occurred in the same Brassicales families in which we found Type 3 root-epidermal patterning: Brassicaceae (including the Capparaceae), Resedaceae and Limnanthaceae. It did not occur in the Tropaeolaceae, a family which has Type 1 root-epidermal patterning. Clowes (2000) also found the radial pattern of trichoblasts in all the families he examined,

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Pemberton et al.ÐRoot Epidermal Patterning

except Cactaceae, of the Caryophyllales sensu lato. This is consistent with our ®nding of Type 3 root-epidermal patterning in all families we examined in this order, except for the Cactaceae. Our ®nding of the Type 3 patterning in Nemophila maculata (Boraginaceae, including Hydrophyllaceae) is also consistent with the ®nding by Clowes (2000) of the radial pattern of trichoblasts in N. menziesii. However, Clowes (2000) also found this radial pattern in the two other species of the Boraginaceae he examined, whereas the two other species we examined in this family had Type 1 root-epidermal patterning. In contrast to our ®ndings on the distribution of the Type 3 patterning, Clowes (2000) found the radial pattern of trichoblasts in families outside the Brassicales, Caryophyllales sensu lato, and the Boraginaceae (including the Hydrophyllaceae): the Euphorbiaceae, Salicaceae and Urticaceae in Eurosids I; Onagraceae in Eurosids II; Balsaminaceae and Loasaceae in Asterids; and the Acanthaceae in Euasterids I (Angiosperm Phylogeny Group, 1998). However, two of the families had species with and species without trichoblasts: in the Euphorbiaceae, Euphorbia peplus had them, but Mercurialis perennis did not; in the Onagraceae, Epilobium parvi¯orum had them, but Circaea lutetiana did not. Furthermore, in the Urticaceae, Urtica dioica had trichoblasts, but Clowes (2000) recorded that `a few roots of Soleirolia (Urticaceae) lack trichoblasts'. Of these additional families in which Clowes (2000) found the radial pattern of trichoblasts, we examined taxa in the Euphorbiaceae, Onagraceae and Balsaminaceae, but, except for Impatiens, the genera we examined were di€erent to those examined by Clowes (2000). Thus, in the Brassicales, the Caryophyllales sensu lato, and Nemophila maculata (Boraginaceae), it appears that the presence of the radial pattern of trichoblasts is the precursor of Type 3 epidermal patterning in seedling roots. However, we have no evidence that this is the case for the other families in which the radial pattern of trichoblasts was found, or indeed for the other two species we examined in the Boraginaceae. It would be interesting to examine more species in these other families and to examine both the root apical meristems for trichoblasts and further back in the root for the type of patterning of the hair and non-hair cells. The presence of a radial pattern of trichoblasts as described by Clowes (2000) does not necessarily preclude subsequent development into Type 1 epidermal patterning. AC K N OW L E D G E M E N T S We thank Dr Ian Hallett, HortResearch Ltd, Auckland for assistance with scanning electron microscopy. L I T E R AT U R E C I T E D Angiosperm Phylogeny Group. 1998. An ordinal classi®cation for the families of ¯owering plants. Annals of the Missouri Botanical Garden 85: 531±553. Behnke H-D. 1981. Sieve-element characters. Nordic Journal of Botany 1: 381±400. Berger F, Hung C-Y, Dolan L, Schiefelbein J. 1998. Control of cell division in the root epidermis of Arabidopsis thaliana. Developmental Biology 194: 235±245.

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