ERECTA family genes regulate development of ... - Wiley Online Library

FEBS Letters 588 (2014) 3912–3917

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ERECTA family genes regulate development of cotyledons during embryogenesis Ming-Kun Chen, Elena D. Shpak ⇑ Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA

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Article history: Received 18 August 2014 Revised 8 September 2014 Accepted 9 September 2014 Available online 19 September 2014 Edited by Julian Schroeder Keywords: Embryo Cotyledon Arabidopsis Stomata TOO MANY MOUTHS Cell proliferation

a b s t r a c t Receptor-like kinases are important regulators of plant growth. Often a single receptor is involved in regulation of multiple developmental processes in a variety of tissues. ERECTA family (ERf) receptors have previously been linked with stomata development, above-ground organ elongation, shoot apical meristem function, flower differentiation and biotic/abiotic stresses. Here we explore the role of these genes during embryogenesis. ERfs are expressed in the developing embryo, where their expression is progressively limited to the upper half of the embryo. During embryogenesis ERfs redundantly stimulate the growth of cotyledons by promoting cell proliferation and inhibiting premature stomata differentiation. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction During development a multicellular organism must coordinate the proliferation pattern and specialization behavior of its cells. Cell cooperation in such an organism is dependent upon communications between cells mediated by molecular messages. Receptorlike kinases are primarily plasma membrane localized proteins that can sense extracellular molecules. Many of these receptors are involved in sensing signals originating from other cells. ERf receptor-like kinases appeared early in land plant evolution and already existed in the time of bryophytes [1]. All analyzed angiosperms have at least two genes belonging to this family, but in many plant species further gene duplications occurred. Arabidopsis has three such genes: ERECTA (ER), ERECTA LIKE1 (ERL1) and ERECTA LIKE2 (ERL2) [2]. These ERf receptors sense secreted cysteine-rich peptides from the EPF/EPFL family, enabling cell–cell communications in various plant tissues [3].

Abbreviations: SAM, shoot apical meristem; ERf, ERECTA family; ER, ERECTA; ERL1, ERECTA LIKE1; ERL2, ERECTA LIKE2; MMC, meristemoid mother cells; GMC, guard mother cell; TMM, TOO MANY MOUTHS ⇑ Corresponding author at: Department of Biochemistry, Cellular and Molecular Biology, M407 Walters Life Sciences, 1414 Cumberland Avenue, University of Tennessee, Knoxville, TN 37996, USA. Fax: +1 865 974 6306. E-mail address: [email protected] (E.D. Shpak).

The ERf family is involved in regulation of multiple aspects of plant development [4]. ERf receptors regulate the growth of aboveground plant biomass where they promote organ elongation, shoot apical meristem function, and cell specification in the epidermis. The er mutants have a compact inflorescence due to reduced elongation of internodes and pedicels, round flowers, and short siliques and petioles [5,6]. A temporal study of pedicel development demonstrated that during early stages of organ growth ER promotes elongation of epidermal and cortex cells along the proximodistal axis which accelerates the cell cycle [7]. The elongation of aboveground organs is further reduced when ERL1 and ERL2 are mutated in the er background [2]. But the most dramatic phenotype is observed in the er erl1 erl2 mutant. In addition to severely reduced organ elongation, the er erl1 erl2 mutant has novel phenotypes that cannot be observed in single or double mutants. The er erl1 erl2 plants are sterile; in their flowers the stem cell population is often misplaced, ovules and anthers do not differentiate properly, and sepals often have stigmatic papillae on top [2,8]. This homeotic conversion of sepals might be a consequence of ectopic expression of the transcription factor AGAMOUS [8]. The er erl1 erl2 seedlings also have bigger shoot apical meristems (SAM) and form leaves at a slower rate and with abnormal phyllotaxy [9,10]. The changes in SAM structure and function correlate with increased sensitivity to cytokinins and with changes in auxin distribution and transport [9,10]. Finally, an analysis of er erl1 erl2 epidermis demonstrated

http://dx.doi.org/10.1016/j.febslet.2014.09.002 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

M.-K. Chen, E.D. Shpak / FEBS Letters 588 (2014) 3912–3917

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that ERfs inhibit stomata formation and regulate their spacing [11]. While multiple functions of ERfs have been described, the involvement of this gene family in regulation of embryogenesis has not been investigated prior to this study. Plant embryogenesis can be divided into two phases: morphogenesis and maturation. During the first phase three tissue layers (the protoderm, ground tissue, and procambium) are formed and the basic body plan is created. During the second phase the embryo grows and prepares to enter metabolic quiescence. In Arabidopsis a series of stages named after the shape of the embryo is recognized: preglobular, globular, heart, torpedo, and walking stick [12]. The morphogenesis phase of embryogenesis continues until the heart stage when patterning of embryo is completed and the shoot apical meristem, the cotyledons, the hypocotyl, root, and the root apical meristem are established. The maturation phase begins during the heart stage when proplastids in the epidermis of hypocotyls differentiate into chloroplasts [13]. Analysis of er erl1 erl2 embryo development demonstrated that beginning from the heart stage ERfs promote cotyledon growth and inhibit differentiation of epidermis.

hypophysis. Beginning from the heart stage, the activity of all three promoters was consistently limited to the upper half of the embryo (Fig. 1B, G, and L). This confinement of ERf gene expression to the apical end became especially noticeable during the early torpedo stage (Fig. 1C, H, and M). In the early walking stick stage the expression of ERf genes is restricted to the shoot apical meristem (SAM) and cotyledons (Fig. 1D, I, and N), and later in that stage it subsides, with the longest expression in the SAM (Fig. 1E, J, and O). TOO MANY MOUTHS (TMM) is a receptor-like protein that is able to form heterodimers with ERfs [15]. It is known to be expressed in shoot epidermal cells [16]. Our analysis of TMM expression demonstrated that during embryogenesis TMM is expressed much later compared to ERfs, at the end of the torpedo stage or in the early walking stick embryo, mostly in developing cotyledons (Fig. 1S). In a short time its expression becomes limited to stomatal lineage cells which has not been previously observed for ERfs (Fig. 1T). Thus, all four genes analyzed are expressed during embryogenesis, preferentially in the upper part of the embryo. And while there is an overlap in their expression, the expression pattern of TMM and ERfs are quite distinct.

2. Results

2.2. ERfs stimulate cotyledon growth during embryogenesis

2.1. Expression of ERf genes during embryo development

High expression of ERf genes in the developing embryo prompted us to investigate the role these genes play during embryogenesis. Since ER, ERL1, and ERL2 are expressed in a highly overlapping pattern at this stage of plant development, we anticipated some redundancy in their function. Consequently, we did not detect a conspicuous phenotype in single or double mutants. As er erl1 erl2 mutants are infertile, we analyzed embryogenesis in er erl2 erl1/+ plants and compared them to the wild type. We observed that 26.6% of embryos (Ntotal = 94) produced by er erl2 erl1/+ plants developed differently beginning from the early torpedo stage, and these embryos were considered to be er erl1 erl2 triple mutants. In these embryos, cotyledons grew more slowly than the axis and never reached the expected size (Fig. 2C, F, I, and L). In the WT the length of cotyledons from the torpedo stage to embryo maturity is correlated with the length of the axis and amounts to 60–75% of it (Fig. 2M). In the triple mutant the length of cotyledons was only 30–40% of the axis length. We did not observe this phenotype in any WT embryos, and the remaining 73.4% of embryos developing from er erl2 erl1/+ plants were indistinguishable from the WT (Fig. 2N). At the end of embryo growth, in the late walking stick stage, the cotyledons of mutant embryos were considerably shorter (234 ± 8 lm; mean ± standard deviation here and below) than either WT (353 ± 18 lm) or their siblings (351 ± 23 lm), while their axis was longer (618 ± 25 lm in mutants versus 535 ± 27 lm in siblings and 471 ± 25 lm in WT). The phenotype observed in er erl1 erl2 was not present in tmm-1 embryos. The tmm-1 embryos had a regular shape where the fractional length of cotyledons to axis was 72 ± 6% (n = 10, measured in early walking stick stage). Previously it has been reported that ERfs regulate SAM size [9,10]. Analysis of er erl1 erl2 embryo development suggest that the SAM size is already increased during early stages of embryogenesis (compare Fig. 2A and C). However, decreased growth of cotyledons in er erl1 erl2 is not likely to be a direct consequence of changes in SAM size. In clv3 mutants the embryo SAM size is increased [17] but our analysis of clv3-9 embryogenesis did not detect any changes in cotyledon elongation and the ratio of cotyledon to axis length was 71.7 ± 7.5% (n = 10, measured in the late walking stick stage).

Analysis of microarray data [14] suggests that ERfs are expressed in the embryo proper beginning from the globular stage and tapering off by the end of embryo maturation. To verify these data we analyzed the expression of ER, ERL1, and ERL2 promoterGUS fusions [2]. As anticipated, activity of all three promoters was detected in the embryo proper from the globular stage to the maturation stage (Fig. 1). In the globular embryo the genes were expressed everywhere except in cells originated from the

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Fig. 1. Transcriptional reporters of ERfs are expressed during embryogenesis in the upper portion of the embryo. The promoters of ER, ERL1, ERL2, and TMM were fused to GUS and their activity was monitored in developing embryos. Bar = 20 lm for (A) and bar = 50 lm for (D). Images for (A)–(C), (F)–(H), (K)–(M), (P)–(R), and images (D), (E), (I), (J), (N), (O), (S), and (T) are at the same magnification. Stages of embryo development are indicated on top.

2.3. ERfs promote cell proliferation in cotyledons The decreased size of er erl1 erl2 cotyledons was easily noticeable immediately after seed germination (Fig. 3A and B). To

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Axis length (µm) Fig. 2. The elongation of cotyledons during embryogenesis is decreased in er erl2 erl1. (A–L) DIC images of different stages of embryogenesis: early torpedo (A–C), late torpedo (D–F), early walking stick (G–I), and late waking stick embryos (J–L). The columns represent the same type of plant: wild type on the left, er erl2 or er erl2 erl1/+ in the center, and er erl2 erl1 on the right. All images in a row are at the same magnification. (M and N) Morphometric analysis of embryo growth in WT, in er erl2 or er erl2 erl1/+, and in er erl1 erl2. (M) Gives the average length of cotyledons relative to the length of axis at different stages of embryogenesis. Error bars are ± S.D., n = 8–15. (N) Shows the correlation between the length of cotyledon and length of axis for individual seedlings. The linear regression lines demonstrate that the growth of WT embryos is almost identical to that of er erl2 and er erl2 erl1/+ but significantly different from er erl1 erl2. Torpedo and walking stick embryos were used for analysis.

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Axis length (µm) Fig. 3. The reduced size of er erl1 erl2 cotyledons correlates with decreased cell proliferation. (A) The size of er erl1 erl2 cotyledons is smaller compared to WT in 2-day old seedlings. Bar = 100 lm. (B) Both average length (L) and width (W) of cotyledons is reduced in er erl1 erl2 versus WT in 2 day old seedlings (n = 5). (C) The average length and width of epidermal and cortex cells is unchanged in er erl1 erl2 compared to WT. The calculated value is a mean measured in 5 cotyledons, 50 cells each. (D) The predicted number of epidermal and cortex cells in a row along the proximodistal (L) and mediolateral (W) axes is significantly reduced in er erl1 erl2 cotyledons compared to WT, n = 5. (A–D) The age of the seedling is given from the moment they were transferred to the growth room. Error bars are ± S.D. (E) The number of cells in the developing cotyledons of er erl1 erl2 embryos (in two different shades of blue) is smaller compared to er erl2 or er erl2 erl1/+ siblings (in pink and red). Each data point corresponds to a number of cells in a longitudinal row in an individual cotyledon. Ep – epidermis, C – cortex, ad – adaxial, ab – abaxial.

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understand whether the decreased growth of cotyledons was due to reduced cell size or reduced cell number, we analyzed epidermal and cortex cells on the abaxial size of cotyledons in 2-day old seedlings (just before or at the time of germination). While cotyledons increase in size approximately two times during imbibition, most of the growth occurs due to cell elongation with cell division playing a secondary role [18,19]. Thus, the number of cotyledon cells on day two is approximately equal to the number of cells constituting the cotyledon at the end of embryogenesis. The length of er erl1 erl2 cotyledon cells in epidermis and cortex along both the proximodistal and mediolateral axes did not differ from the WT (Fig. 3C), suggesting that the difference in size is due to different numbers of cells. Based on cotyledon length and cell size we estimated that the number of cells in a proximodistal row of either the epidermis or cortex in the mutant is approximately half that of the WT (Fig. 3D). The reduction along the mediolateral axis was slightly less severe (70%). Analysis of cotyledon growth during earlier stages of embryo development was consistent with importance of ERfs for cell proliferation (Fig. 3E). We analyzed the number of cells in epidermis and cortex, on both abaxial and adaxial sides of cotyledons, and in each developmental stage the number of cells in er erl1 erl2 was reduced compared to the WT. These data suggest that ERfs stimulate cotyledon growth by promoting cell proliferation. 2.4. ERfs delay stomata formation in cotyledons during embryogenesis Next we analyzed stomata formation in cotyledons at day two. In cotyledons the timing of stomata development is uniform along the proximal–distal axis, in contrast to the gradual development of stomata from tip to base in leaves [19]. Formation of stomata starts with differentiation of meristemoid mother cells (MMC). A MMC divides asymmetrically, forming a small triangular meristemoid and a bigger Stomatal Linage Ground Cell. The meristemoid directly or after several rounds of asymmetric divisions differentiates into an oval guard mother cell (GMC). A GMC divides symmetrically to form two bean shaped guard cells. Usually only meristemoids form during embryogenesis and are present in dry seeds, with the first stomata appearing around day two [19] concurrent with seed germination. On day two we observed formation of some stomata in the WT (8.4% of the total number of epidermal cells), as well as meristemoids (4.6%) and GMCs (4.7%) (Fig. 4). At the same time, in er erl1 erl2 there was a dramatic increase in stomata number (35.7%), but a much reduced number of meristemoids (1.8%) and GMCs (0.9%) (Fig. 4). These data suggest that ERf genes delay formation of stomata during embryogenesis.

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2.5. Analysis of auxin distribution and transport in er erl1 erl2 embryos Auxin transport plays a key role in establishment of cotyledons [20]. Previously we have shown that ERfs are important for correct auxin distribution and PIN1 expression in the SAM and leaf primordia [9]. However, analysis of TAA1pro:GFP-TAA1, PIN1pro:PIN1-GFP, PIN3pro:PIN3-GFP, and DR5rev:ER-GFP expression during embryogenesis did not identify an obvious change in auxin biosynthesis, transport, or accumulation in er erl1 erl2 embryos (Fig. 5). Thus, ERfs most likely regulate cotyledon growth independently of auxin. This is consistent with the observation that the embryonic phenotype of er erl1 erl2 (reduced growth of cotyledons) is different from phenotypes often observed in auxin-related mutants (abnormal number and shape of cotyledons; cotyledon fusion). 3. Discussion A simple comparison of walnut, pea and Arabidopsis embryos makes it evident that in dicots cotyledons can vary dramatically in size and shape. However, the genetic mechanisms responsible for that variance are unknown. Cotyledons and true leaves have different origins (cotyledons develop from the apical domain of the embryo and true leaves from SAM) but they have similar morphology and share many common developmental features [21]. Hypothetically, some genes regulating growth of those two organs might be in common. Here we show that ERf genes promote cotyledon growth during embryogenesis. The decreased cotyledon length in er erl1 erl2 is linked with a reduced number of cells in both the epidermis and cortex. The role of ERfs in cotyledon growth is consistent with their expression pattern in that region. While ERfs control cotyledon elongation, they do not play a significant role in their initiation or separation nor do they have any noticeable effect on auxin transport in the vasculature during embryogenesis. Thus, the pathways controlling the formation of auxin maxima during cotyledon and leaf initiation might be different. In addition ERfs inhibit premature differentiation of stomata in the epidermis of cotyledons. Usually only meristemoids form during embryogenesis [19], but in er erl1 erl2 most meristemoids differentiate into stomata before seed germination, and there is a significant increase in asymmetric cell divisions leading to stomata formation. While ERfs and TMM both regulate stomata formation during embryogenesis their role in this process is not identical. TMM prevents excessive entry into the stomata lineage pathway but it is not involved in inhibition of meristemoid differentiation [22]. Interestingly, at the end of embryogenesis it is TMM and not ERf that is present in the stomatal lineage cells. We speculate

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Fig. 4. Stomata form prematurely in er erl1 erl2 cotyledons. (A) The average percentage of different cell types in abaxial epidermis of cotyledons (n = 6) on day two. M – meristemoid, GMC – guard mother cell, S – stomata. Error bars are ± S.D. Values significantly different from the control are indicated by asterisks (P < 0.02). (B) Tracing of cotyledon epidermis in two day old seedlings based on DIC images. Meristemoids and guard mother cells are in gray, and stomata are in black. Bar = 10 lm.

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the samples were fixed in cold 90% acetone for 20 min, rinsed with cold water, vacuum infiltrated with standard staining buffer (containing 2 mM x-gluc) and incubated for 24 h, at 37 °C. After staining, samples were incubated in an ethanol: acetic acid (1:1) solution for over 4 h and then cleared in a chloral hydrate solution (chloral hydrate:H2O:glycerol 8:1:1) overnight. Differential interference contrast (DIC) optics were used to observe GUS staining. To analyze embryo development and to measure cortex and epidermis cell size in cotyledons, samples were fixed overnight with ethanol:acetic acid (9:1). After fixation, samples were rehydrated with an ethanol series to 50% ethanol and cleared in chloral hydrate solution as above. Microscopic observations were done using a Nikon Eclipse 80i microscope with DIC optics, pictures obtained with a 12 megapixel cooled color DXM-1200c (Nikon) camera, and NIS-Elements BR imaging software (Nikon) was used for measurements. To observe the GFP signal we used a C-FL B-2A (Nikon) filter cube. Arabidopsis Genome Initiative numbers for the genes discussed in this article are as follows: ER (At2g26330), ERL1 (At5g62230), ERL2 (At5g07180), TMM (At1g80080).

Funding Fig. 5. Similar expression pattern of auxin related markers in embryos of siblings/ wild type and er erl1 erl2. During embryogenesis DR5rev:ER-GFP is expressed in the root apical meristem, at the tips, and in vasculature of cotyledons (A); TAA1pro:GFPTAA1 in the L1 layer of the SAM, in vasculature, and in the quiescent center (B); PIN1pro:PIN1-GFP in the vasculature of hypocotyls and cotyledons (C); PIN3pro:PIN3-GFP in the root apical meristem, at the tips of cotyledons, and in vasculature of hypocotyls (D). Bar = 100 lm.

that inhibition of meristemoid differentiation is achieved in the earlier stages and does not require continuous expression of ERfs. Recently several papers have linked stomata differentiation after germination with auxin transport and signaling [23–25]. In the future it would be interesting to investigate whether ERf mediated inhibition of stomata differentiation during embryogenesis involves modification of auxin signaling or transport through components other then PIN1 and PIN3. 4. Materials and methods 4.1. Plant materials and growth conditions The Arabidopsis ecotype Columbia was used as a WT. The er-105 erl1-2/+ erl2-1 and tmm-1 mutants have been described previously [2,16]. The transgenic plants expressing TAA1pro:GFP-TAA1, PIN1pro:PIN1-GFP, and DR5rev:ER-GFP in er erl1 erl2 background are described elsewhere [9]. PIN3pro:PIN3-GFP plants [26] were crossed with er erl1/+ erl2; the er erl1 erl2 plants containing the described construct were identified in the F3 generation based on the phenotype and the presence of GFP fluorescence. Plants were grown on modified Murashige-Skoog media plates supplemented with 1 Gamborg B5 vitamins and 1% w/v sucrose. For all experiments, seeds were stratified for 2 days at 4 °C before germination. 4.2. GUS histochemical analysis and microscopy ERpro:GUS, ERL1pro:GUS, ERL2pro:GUS, and TMMpro:GUS transgenic plants have been described earlier [2,16]. Promoter regions cloned were of the following length: 1.68 kb for ER, 4.1 kb for ERL1, 4.4 kb for ERL2, 0.51 kb for TMM. Three independent transgenic lines were used for analysis of ER, ERL1 and ERL2 promoters; one available line was used to characterize TMM expression. Promoter GUS analysis was performed according to [27] with minor modifications. Siliques were slit longitudinally and then

This work was supported by the National Science Foundation [IOS-0843340 to E.S.]. Acknowledgments We thank Anna Stepanova, Albrecht von Arnim, Eva Benkova, and Zachary Nimchuk for sharing with us TAA1pro:GFP-TAA1, PIN1pro:PIN1-GFP, DR5rev:ER-GFP; PIN3pro:PIN3-GFP, and clv3-9 seeds.

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