Plant Cell Rep DOI 10.1007/s00299-014-1571-1

ORIGINAL PAPER

New phenotypic characteristics of three tmm alleles in Arabidopsis thaliana Longfeng Yan • Xi Cheng • Ruiling Jia • Qianqian Qin • Liping Guan • Hang Du • Suiwen Hou

Received: 17 November 2013 / Revised: 13 January 2014 / Accepted: 15 January 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Key message Three new tmm mutants were isolated and showed differential phenotypes from tmm-1, and TMM overexpression led to abnormal leaf trichomes. Abstract TOO MANY MOUTH (TMM) plays a significant role in the stomatal signal transduction pathway, which involves in the regulation of stomatal distribution and patterning. Three mutants with clustered stomata were isolated and identified as new alleles of tmm. tmm-4 mutation included a base transversion from adenine to thymidine in position 1,033 of the TMM coding region and resulted in premature termination of translation at position 345 of TMM. tmm-5 had a base transition from cytosine to thymidine in 244 of TMM and translated 82 amino acids before premature termination. tmm-6 mutation took a base transition from guanine to adenine in 463 of TMM and changed a glycine (Gly) to an arginine (Arg) in position 155 of the protein. tmm-6 had an evident reduction of stomatal clusters and fewer stomata in cluster compared with other tmm alleles, possibly due to decreased level of entry divisions in cells next to two stomata or their precursors. tmm-5 and tmm-6 were hypersensitive to abscisic Communicated by K. Chong. L. Yan, X. Cheng, and R. Jia contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1571-1) contains supplementary material, which is available to authorized users. L. Yan  X. Cheng  R. Jia  Q. Qin  L. Guan  H. Du  S. Hou (&) Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, 730000 Gansu, China e-mail: [email protected]

acid (ABA) in seedling growth and seed germination, while tmm-4 was defective in response to ABA during seed dormancy, suggesting that TMM was involved in ABA signaling transduction. Interestingly, overexpression of TMM resulted in the reduction of leaf trichomes and their branches, and this might reveal a new function of TMM in trichome development. Keywords tmm alleles  Stomata patterning  ABA response  Trichome development

Introduction Stomata are small pores on the surfaces of flowering plants and play an essential role in regulation of gas exchange between the aerial organs epidermis and atmosphere by controlling the aperture of a pore bordered by two guard cells. In Arabidopsis thaliana, a series of asymmetric and symmetric cell divisions generate different types of cells and regulate the patterning of stomata on the organic surface (Bergmann and Sack 2007; Nadeau and Sack 2002a; Sachs 1991). Many genes have been identified to regulate stomatal development through cell–cell signaling, and a model has been proposed in which a receptor complex composed of TOO MANY MOUTH (TMM) and ERECTA (ER)-family receptor-like kinase is activated by EPIDERMAL PATTERNING FACTOR 1 (EPF1), and the activated receptor signals to downstream MAPK cascade through YODA to control stomatal formation and distribution (Bergmann et al. 2004; Hara et al. 2007; Nadeau and Sack 2002b; Pillitteri et al. 2007). TMM is identified as the first component for regulating stomatal patterning and development. TMM encodes a putative leucine-rich repeat (LRR)-containing receptor-like protein which is located at

123

Plant Cell Rep

cell surface and responds to the positional signals to control the number and orientation of division (Nadeau and Sack 2002b). Mutations in TMM, for example tmm-1, disrupt ‘‘at least one-celled spacing rule’’ and lead to formation of excess stomata and stomatal clusters in leaves, which is due to the failure of orient spacing and amplifying division and the prohibition entry division of adjacent cells to two or more stomata or precursors (Yang and Sack 1995). As is well known, abscisic acid (ABA) plays a predominant role in stomata closure regulation, and ABA may interact with jasmonic acid (JA) in this process (Daszkowska-Golec and Szarejko 2013). On the other hand, ABA is also known as the main factor involved in seed dormancy and germination, by antagonizing the action of GA or ethylene (Shu et al. 2013; Wang et al. 2013), or coordinating with auxin (Liu et al. 2013). ABA-responsive genes, such as RD29A, MYB2 and PP2C (including ABI1 and ABI2), participate mainly in stress adaption, while ABI3, ABI4 and ABI5 play a dominant role in seed germination and dormancy (Dai et al. 2013). Up to now, only one study reported that tmm-1 and Atrlp17-1, a null T-DNA insertion mutant, showed slightly reduced growth and decreased chlorosis compared with wild type in response to exogenous ABA (Wang et al. 2008). However, COI1, a protein containing 16 LRRs and an F-box motif, was found to play an indispensible role in stomata closure induced by MeJA by activating S-type anion channels (Munemasa et al. 2007; Xie et al. 1998). As an LRR-containing protein, TMM is expected to respond in a similar way as COI1 to ABA in stomata closure induction, and potentially seed dormancy and germination. The mature Arabidopsis leaf trichome consists of a unicellular structure with a stalk and three to four branches. About 70 trichome mutants have been identified, and over 50 genes have been isolated, which can be classified as several groups, including transcription factors, cell cytoskeleton organizers, cell cycle factors and so on (Balkunde et al. 2010; Marks et al. 2009). However, among the reported genes, the LRR-containing proteins have not been found till now. Here, we present a description of three putative new alleles of tmm mutants: tmm-4, tmm- 5 and tmm-6. New phenotypic characteristics have been observed to supplement and certify the functions of TMM in regulation of stomatal development, ABA response, and trichome formation.

trap line S4, which expresses GFP in mature guard cells (Hou et al. 2006), were mutagenized by ethyl methanesulfonate (EMS, Sigma). In brief, seeds were treated with 0.13 % EMS for 16 h at 22 °C and vernalized, then were sown into a mixture with peat and perlite under 16 h light/ 8 h darkness at 19–23 °C. M2 seeds were harvested from individual M1 plants and planted for screening as mentioned above. Mutant screening Dental resin impression was used to examine the abaxial epidermis of cotyledons and primary leaves during their developmental process (Geisler et al. 2000). Differential interference contrast (DIC) microscope and confocal laser scanning microscope (Zeiss, Germany) were used to observe stomatal characteristic of putative mutant seedlings. All mutants were backcrossed with S4 plants three times to purify the genetic background. Identification of putative mutants To identify whether three putative mutants were the tmm alleles, tmm-1 was used to cross with them for complementation test. To determine mutant sites, the TMM gene was amplified by PCR from genomic DNA of mutants with primers 50 -GCACGATATGAATTCTTCC-30 and 50 -GCAGTTTTATCATCCCTCC-30 . The amplified fragments were cloned and sequenced. Real-time RT-PCR To detect the expression levels of TMM and trichome marker genes, total RNA was isolated from 50 mg of 10-day-old seedlings on MS by TaKaRa miniBEST Plant RNA Extraction Kit, and first-strand cDNAs were generated by First-Strand cDNA Synthesis Kit (TaKaRa) and Oligo dT Primers using 1.0 lg RNA according to manufacture’s instructions. To detect the expression levels of ABA-responsive genes, 10-day-old seedlings on MS were transferred to KCl–MES buffer (30 mM KCl, 10 mM MES, pH 6.2) with or without 10 lM ABA and incubated under continuous white light for 5 h, and then total RNA was isolated as mentioned above. Real-time RT-PCR was performed on Agilent technologies Stratagene Max3005p Real-time PCR machine, using the SYBR premix EX Taq II (TaKaTa) and primers listed in Table S1.

Methods and materials Quantitative characterization of phenotypes Plant material and growth conditions Arabidopsis thaliana ecotype Columbia (Col) was used in all experiments. Seeds of a popular GAL4-GFP enhancer

123

Six randomly selected square areas of 0.0324 mm2 were counted per organ for determining stomatal density (number of stomata per square millimeter), stomatal index

Plant Cell Rep

[number of stomata/(number of epidermal cells ? number of stomata)], density of stomatal clusters (number of stomatal clusters per square millimeter), percentage of stomata in clusters (number of stomata in clusters/number of overall stomata 9 100) and stomata in each cluster size. For detecting these stomatal parameters during the developmental process, abaxial surface of leaf was chosen to collect data, and the same leaves were measured four times when they were 4-, 7-, 11- and 16-day-old, respectively. At least five individual plants were randomly chosen. Picture drawing To illuminate the difference of stomata distribution among tmm mutants, successive dental resin impressions were observed every 24 h from the same primary leaf of 7-dayold seedling. At least five plants were randomly chosen for examination and observed for 4 days. The sketch map was drawn based on the observations.

detect seed dormancy (Parcy et al. 1997) with three repeats. The germination percentage was determined after placing 3 days in 1/2 MS media with 0, 0.5, 1, 5, or 10 lM ABA, respectively. The germination was scored after an obvious protrusion of the radical through the seed coat. This assay was repeated three times. Water loss analysis and drought treatment Five rosette leaves were detached from plants grown at the same developmental stage and environmental condition, and weighed immediately as fresh weight, then placed into an illumination incubator and weighed at indicated time intervals. The water loss rate was measured as percentage of initial fresh weight (Pandey and Assmann 2004). 4-week-old plants were drought treated, and observed after withholding water till plants died. Each assay was repeated three times.

Leaf cross section

Plasmid constructs and transformation

Mature rosette leaves were fixed in a solution of 5 % (v/v) formaldehyde, 63 % ethyl alcohol, and 5 % acetic acid for 24 h. Then, paraffin section technique was used to get the cross sections of leaves. Sections with 8–10 lm were stained with 1 % safranin O and 0.1 % fast green.

For overexpression of TMM, a 1,491-bp genomic sequence consisting of the complete coding region was PCR-amplified from Columbia genome with primers 50 -GGGGTAC CATGGCACGATATGAATTC-30 and 50 -CGAGCTCGCT CTAACTAGATATTAGC-30 . The amplified fragment was cloned into pUCm-T and sequenced, then was put downstream of the super promoter of modified pCAMBIA1300 (Ni et al. 1995) to obtain the overexpression construct 1300-TMM, which was transformed into the wild type using the flower-dipping method (Clough and Bent 1998). Meanwhile, overexpression lines of different tmm mutation version were constructed, including 1300-tmm4, 1300-tmm5 and 1300-tmm6. To rescue the phenotypes, the constructs of TMMpro::TMM-GFP and 1300-TMM were transformed into tmm mutants (Nadeau and Sack 2002b).

ABA assay Analyses of seedling growth and stomatal response to ABA treatment were performed according to Wang et al. (2008) and Pei et al. (1997) with slight modification. In brief, for seedling growth analysis, seeds were surface sterilized and sown on the plates containing 1/2 Murashige and Skoog (MS) media, 1 % (w/v) sucrose and 1 % agar with or without 0.5 lM ABA, stored at 4 °C in dark for 48 h. Photos were got after 10-day growth under a 16-h photoperiod at 22 °C. For the stomata closure assay, 4-week-old rosette leaves were broken in pieces and incubated in KCl–MES buffer under continuous white light for 90 min to open the stomata. Then, one half of pretreated leaf pieces was transferred to the KCl–MES buffer with 10 lM ABA to test ABA sensitivity, with the left leaf pieces as control. After incubated 2 h under light, the width and length of stomatal aperture were measured for 30 randomly selected stomata. The two assays were repeated three times. Seed dormancy and germination The seeds of the third and fourth siliques were collected and germinated on water-soaked filter paper for 10 days to

Screening and phenotype analysis of transgenic plants Transgenic plants T1 lines were selected from 1/2 MS media with 25 mg/L hygromycin (Solarbio, Beijing) and their phenotypes were observed. Phenotypic details were obtained from hygromycin-resistant T2 lines. Stomatal density and index were collected from abaxial epidermis of 2-week-old primary leaves. Since the density of trichomes on leaves obviously increases during the transition from vegetative to reproductive growth (Telfer et al. 1997), so the numbers of trichomes and their branches were obtained from the third true leaves in vegetative growth (3-weekold) and in reproductive growth (6-week-old), respectively. At least six of the third leaves were analyzed at different developmental stages. The average length of detected

123

Plant Cell Rep Fig. 1 The stomata distribution and structure of tmm allelic mutants. (a–d) Confocal images of mature guard cell (green) in the leaves epidermis: a S4, b tmm-4, c tmm-5 and d tmm-6. (e–j) DIC images of abaxial epidermis in leaves: e wild type, f S4, g tmm-1, h tmm-4, i tmm-5 and j tmm-6, stomatal cluster was indicated by brackets, bars represented 20 lm. (k–l) Light microscopy of cross sections in rosette leaves: k wild type and l tmm-4, asterisk marked substomatal cavities, arrow indicated stoma, bars represented 50 lm(color figure online)

leaves was about 0.3 cm in 3-week-old plant and 1 cm in 6-week-old plant, respectively.

Results Identification and analysis of three new tmm alleles Over 3,000 M2 seedlings from about 1,000 parental (M1) plants mutated with EMS were screened, and three putative mutants with clustered stomatal phenotype had been obtained. The mutational phenotypes were similar to that of tmm-1 (Nadeau and Sack 2002b; Yang and Sack 1995), and then these three mutants were designated as tmm-4, tmm-5 and tmm-6, respectively. The stomatal clusters generally appeared in tmm-4 and tmm-5 but seldom in tmm-6 (Fig. 1b–d), and the cluster usually consisted of 4–5 stomata in tmm-1, tmm-4 and tmm-5 in comparison with two stomata in tmm-6 (Fig. 1g–j). Cross section of rosette leaf showed that large substomatal cavities were present at clustered instead of single stomata (Fig. 1k–i), which was the unique trait of tmm mutants in internal leaf tissues, suggesting that a common larger substomatal cavity was shared by clustered stomata, or many substomatic chambers were in close contact with each other. Sequencing results revealed that three tmm alleles mutated at different sites in TMM coding region: tmm-4 (A–T at

123

Fig. 2 Mutational sites and expression analysis of tmm alleles. a Delineation of mutational sites for different tmm mutants in TMM protein. Mutational sites were indicated by arrows: black arrow sites induced premature termination of translation; red arrow sites produced amino acids replacement. b Expression of TMM detected by real-time RT-PCR. ACTIN was as control(color figure online)

Plant Cell Rep Fig. 3 Stomatal parameters in different developmental stages of cotyledon and rosette leaves. Bars represented SE, n = 30

1,033 nt), tmm-5 (C to T at 244 nt), and tmm-6 (G–A at 463 nt). In tmm-4 and tmm-5, nonsense mutations led to premature termination of TMM translation at 345 and 82 aa, respectively. However, in tmm-6, missense mutation induced a glycine (Gly) to an arginine (Arg) at 155 aa (Fig. 2a). Realtime RT-PCR analysis showed that TMM transcripts could be normally accumulated in tmm mutants with slight increase in tmm-1 and tmm-4 and decrease in tmm-5 and tmm-6, indicating that the transcription of TMM was basically not subject to the difference of mutational sites (Fig. 2b). Considering the phenotypic and genetic characterization of three mutants, we believed that they were new alleles of tmm.

The tmm-6 shows different stomata patterning from other tmm alleles To estimate how tmm alleles affected stomata patterning, stomatal distribution was observed at different developmental stages of cotyledon and rosette leaves, respectively. The stomatal density and index were increased twofold to threefold in tmm mutants as compared to wild type during the cotyledon developmental process (Fig. 3, left panel). It was noteworthy that stomatal density of tmm-6 was higher than that of three other mutants at the 11th day of cotyledons growth, and then decreased as others when the leaves

123

Plant Cell Rep

Fig. 4 Size distribution of stomatal clusters in tmm-1 and tmm-6 for cotyledon and rosette leaves. Bars represented SE, n = 30. Data, including SE, were obtained from at least five 16-day-old leaves

stopped expanding. However, unlike other tmm mutants, the cluster density of tmm-6 was obviously lower when the cotyledon started expanding, but increased following the cotyledon growth (Fig. 3, left panel). Interestingly, there were only about 23 % stomata formed clusters in tmm-6 in comparison to over 40 % in other allelic mutants at the 4th day, nevertheless, the percentage of stomata in clusters seemed not to be correlated with cotyledon developmental stages. As for true leaves, stomatal density and index were still higher in tmm mutants; however, tmm-6 was easily distinguished from other tmm mutants by obviously lower stomatal density in the initial developmental stage (Fig. 3, right panel). Like in cotyledon, the clustered stomata were rarely formed in tmm-6 rosette leaves, since the density of stomatal clusters and percentage of stomata in clusters were significantly decreased in tmm-6 as compared to other tmm mutants during the developmental process of primary leaves (Fig. 3, right panel). Since the stomata clusters in tmm-6 seemed to be different from other mutants, the cluster size and size distribution were measured. The results showed that both in cotyledons and rosette leaves, cluster containing two stomata was the most predominant component in tmm-6; on the contrary, cluster size was relatively comparable in tmm1. Meanwhile, the extra-large cluster consisted of 13 or 9 stomata in cotyledon and rosette leaves, respectively, occurred only in tmm-1 instead of tmm-6 (Fig. 4). Furthermore, the interactions between tmm-6 and its allelic mutants were analyzed by hybridization. The stomatal phenotypes of F1 progenies from tmm-1 and tmm-4 or tmm1 and tmm-5 were similar to their parents. However, all stomatal parameters in F1 of tmm-1 and tmm-6 were lower than that in tmm-1 and higher than that in tmm-6 (Fig. S1a).

123

The stomatal density of F1 was almost equal to the mean of that of tmm-1 and tmm-6, although the stomatal index and percentage of stomata in clusters of F1 were close to tmm6. Especially, the clusters mainly consisted of 2 or 3 stomata in F1 plants, but mostly formed by two stomata in tmm-6 and usually contained more than three stomata in tmm-1 (Fig. S1b). To estimate whether the differences of stomatal phenotypes existed in other organs, stomatal density was measured in tmm mutants and wild type (Table 1). Obviously different from the wild type, stomata on the stem were lost both in tmm-1 and tmm-6. Unlike tmm-1, the stomata density of sepal and silique in tmm-6 showed similar level to that in wild type. In the case of stomatal density of cauline leaves, there was no significant difference between tmm-1 and tmm-6 for narrow cauline leaves, while both mutants were significantly different for wide cauline leaves. Though stomatal density of rosette leaves increased both in tmm-1 and tmm-6 compared with wild type, it was significantly lower in tmm-6 than in tmm-1. TMM differentially participates in physiological processes regulated by ABA To authenticate the relationship between TMM and ABA, physiological processes affected by ABA, such as germination, seed dormancy, and stomata closure, were tested in tmm alleles (Fig. 5). In the absence of ABA, the seedlings of tmm alleles could normally develop and were indistinguishable from the wild type. Compared with non-treated seedlings, application of ABA induced chlorosis in all genotypes. However, in comparison to other tmm mutants, the growth of

Plant Cell Rep Table 1 Stomatal density in various organs of wild type and tmm mutants Organ

WT

Sepal

155.35 ± 11.11b

Stem

S4

a

60.70 ± 5.10

a

67.90 ± 5.51

277.78 ± 17.87a

165.64 ± 14.71b

b

0b

0

135.80 ± 6.66

128.60 ± 4.78

173.87 ± 11.80

139.63 ± 10.25b

Cauline leaf (wide)

299.38 ± 11.08c

304.53 ± 10.61c

533.95 ± 32.29a

400.21 ± 13.75b

Rosette leaf

b

287.04 ± 7.99

c

286.01 ± 8.64

b

tmm-6

Silique Cauline leaf (narrow)

b

166.67 ± 11.22b

tmm-1

b

279.84 ± 6.55

c

272.63 ± 6.75

a

a

344.65 ± 12.69a

360.08 ± 8.69

a

412.55 ± 23.68

370.37 ± 15.20b

Data, including S.E., were obtained from at least five 6-week-old plants. The superscript letters represent statistical significance at p \ 0.05 (Duncan test), and values with the same letter are not statistically different

tmm-5 and tmm-6 was severely inhibited in the media with 0.5 lM ABA (Fig. 5a). This indicated that different sensitivity of ABA responses existed among various tmm alleles. Meanwhile, the germination rate was remarkably decreased in tmm-5 and tmm-6 under a series of ABA concentration, whereas the tmm-1 and tmm-4 seeds showed no evident difference as compared to the wild type (Fig. 5b). This result was consistent with the seedling growth inhibition and suggested that mutations of TMM in tmm-5 and tmm-6 led to ABA hypersensitive response. Seed dormancy was mainly controlled by ABA (Ni and Bradford 1993), however, freshly harvested tmm-4 seeds germinated more than wild type under the same condition (Fig. 5c), suggesting that tmm-4 mutant was defective in seed dormancy. As a stress hormone, ABA controls stomata aperture and further water loss. The application of exogenous ABA led to stomatal closure at roughly similar levels in tmm mutants and wild type (Fig. 5d). On the other hand, the water loss rate in detached leaves showed no evident difference in tmm-1 and tmm-6 compared with the wild type, but slightly higher in tmm-4 and tmm-5 (Fig. 5e). And the survival days of different allelic mutants were same as wild type after stopping water. These results suggested that TMM may not directly participate in ABA-induced stomata closure or stress adaptability. To uncover the differences among the tmm alleles in ABA signaling pathway, the expression levels of ABA-inducible genes were detected. The results showed that the transcripts of MYB2, PP2C, RD29A and ABI5 were obviously accumulated with ABA treatment in tmm alleles (Fig. 6), suggesting that the ABA signaling pathway was operating normally. However, the response levels of these genes were varied between tmms, hinting that the expression changes of detected four genes might not be the direct reason leading to the ABA hypersensitivity in tmm-5 and tmm-6. Meanwhile, the expression level of ABI5, which represses the seed germination (Lopez-Molina et al. 2002), was lower in tmm-4 than in other lines seedlings, perhaps leading to the defective dormancy in tmm-4. In a word, TMM could play an important role in developmental processes such as germination and seed dormancy, instead of stress response induced by ABA.

Overexpression of TMM affects trichome development As expected, stomatal density in tmm-4 and tmm-5 transformed with TMMpro::TMM-GFP was completely rescued, however, clusters with 2–3 stomata still occasionally appeared in tmm-4 rescued plants, which indicated that TMMpro::TMM-GFP could lead to substantial but incomplete complementation in tmm-4. Similarly, TMM overexpression rescued tmm-6 phenotype as well (Fig. S2). We also transformed original or mutational version of TMM into the wild type, and found that there was no difference in leaf stomatal distribution (including stomatal density and stomatal index) between transgenic lines and the wild type (Table S2). These observations indicated that neither normal nor mutational TMM overexpression could affect normal stomatal patterning. Unexpectedly, in TMM overexpression plant leaves, the number of trichome and branch on rosette leaf was obviously distinctive from the wild type and tmm mutants (Fig. 7a; Table 2). Compared to the wild type, the trichome number in TMM overexpression (TMM-OX) line T2-1 decreased about fourfold at the stage of vegetative growth, but reduced dramatically 12-fold after transition to reproductive growth (Table 2). In general, trichomes on rosette leaves have three branches (Folkers et al. 1997). In accordance with the observation, we found that about 80 % trichomes had three branches throughout the whole development process for wild type and three tmm alleles. However, in TMM-OX T2-1 line, \10 % trichomes had three branches at vegetative growth stage, and this percentage increased to nearly 15 % in reproductive growth stage. Meanwhile, 60 and 33 % unbranched trichomes from TMM overexpression plants were observed during vegetative growth and reproductive stage, respectively. Similarly, the number of trichomes and branches was also remarkably decreased in other two TMM-OX lines T2–2 and T2–3 at different developmental stages (Table 2). To validate the expression of trichome marker genes, we performed real-time RT-PCR, and found that in three TMM-OX lines, the expression levels of GL1 and KIS were slightly reduced, and ZWI was up-regulated moderately; on the contrary, the transcriptional level of GL3 was increased dramatically (Fig. 7b). These results demonstrated that overexpression of TMM took a

123

Plant Cell Rep Fig. 5 tmm alleles displayed different sensitivity to ABA. a Seedling growth treated with 0.5 lM ABA. The scale bar represented 1.5 cm. b ABAinhibited seed germination. Bars represented SE, n = 3. The superscript letters represent statistical significance at p \ 0.05 (Duncan test), and values with the same letter are not statistically different. c Seed dormancy in different genotypes plants. The scale bar represented 2 mm. d ABAinduced stomatal closure. Bars represented SE, n = 90. Asterisks indicate statistically significant differences (p \ 0.05; Student’s t test) e Percentage of water loss. Bars represented SE, n = 15

considerable role in the trichome morphogenesis by regulating trichome formation and its branch pattern.

Discussion New tmm alleles harbored mutational sites different from each other TMM encodes a LRR-containing receptor-like protein of 496 amino acids, which consists of the N-terminal non-LRR

123

region (NNL), C-terminal non-LRR region (CNL), a transmembrane domain and ten LRRs (Nadeau and Sack 2002b). As a functional conservative region, LRRs of TMM contained 237 amino acids from positions 160–397 (http://www. uniprot.org/). In our study, three new tmm alleles containing different mutational sites were identified. Similar to tmm-1, nonsense mutation could result in premature termination of translation and produce a truncated version of TMM protein deletion of two LRR repeats in tmm-4 (Fig. 2a), since the mutational sites of them are so close (Nadeau and Sack 2002b). Nevertheless, nonsense mutation happened in tmm-5

Plant Cell Rep

Fig. 6 Expression levels of ABA-inducible genes in tmm alleles. The inset graph in PP2C shows expression in mock-treated seedlings. Transcript levels of detected genes were normalized to those of

ACTIN, and the relative level of transcript in mock-treated wild-type seedlings was set to 1

would produce a loss-of-function TMM protein only harboring the NNL region (Fig. 2a). Unlike other tmm mutants, missense mutation in tmm-6 could lead to a polar, neutral amino acid (Gly) replaced by a nonpolar, basic amino acid (Arg), which possibly interferes with protein fold and further higher order structure (Fig. 2a). In addition, mutational site in tmm-6 locates in NNL region without significant function reported up to date, which might distinguish tmm-6 from other tmm mutants.

that the clusters occasionally appeared and the number of stomata in cluster was fewer in tmm-6 (Figs. 3, 4). The formation of clusters in tmm-6 was similar to that in stomatal density and distribution 1 (sdd1), which only had a minor fraction of stomata in clusters (Berger and Altmann 2000). This characteristic revealed that tmm-6 appeared to have an intermediate phenotype between typical tmms and the wild type, suggesting tmm-6 was a weak allele of tmm. Increased stomatal density was another characteristic in tmm mutants (Geisler et al. 1998; Yang and Sack 1995). Statistical analysis showed that stomatal density both on cotyledon and rosette leaves appeared to be higher in tmm alleles than that in wild type during the whole developmental stage (Fig. 3). This indicated that more entry and spacing divisions happened and led to cell increase which could enter stomatal development pathway in tmm mutants compared to wild type during leaf expansion. Through successive observation of stomata development, we found that, in tmm-4 and tmm-5, the orientation of spacing and amplifying divisions was abnormal, and entry divisions usually happened in cells adjacent to two or more stomata or their precursors, which were coincident with tmm-1 as

Reduction of entry divisions led to weak stomatal phenotype in tmm-6 In classical mutant tmm-1, stomata were often formed between two or more already existing stomata or precursors, and all these stomata connected together and composed a stomatal cluster usually with large size (Fig. 8) (Geisler et al. 1998; Yang and Sack 1995). But in tmm-6, the cells next to two stomata or their precursors seldom formed stomata, and the chance of large-size clusters formation was rare (Fig. 8). Therefore, the most striking feature distinguishing tmm-6 from its allelic mutants was

123

Plant Cell Rep

Fig. 7 Reduction of leaf trichome number in TMM overexpression plant. a Phenotype of leaf trichome at vegetative stage of plant. b Expression levels of marker genes regulating trichome initiation and branching in TMM overexpression lines. Transcript levels of detected genes were normalized to those of ACTIN, and the relative level of transcript in wild-type seedlings was set to 1

previous reports (Geisler et al. 2000; Yang and Sack 1995). Further investigation showed that, although entry divisions seldom happened in the cells adjacent to two or more stomata or their precursors, the stomatal density in tmm-6 was similar to other tmms, indicating that the number of entry and spacing divisions also increased during leaves development in tmm-6. Meanwhile, the increased stomatal density and the decreased cluster density revealed that extra formed stomata partially kept correct orientation, however, random distribution of stomata was reduced in tmm-6. As a weak tmm mutant due to the mild phenotype of stomata cluster, tmm-6 could be a better material or model to exploit deeply the molecular mechanism of entry division and stomata formation. TMM is involved in ABA signaling transduction ABA plays a considerable role in various aspects of plant growth and development, including embryo maturation, seed dormancy, germination and post-germinative growth (Finkelstein et al. 2002; Leung and Giraudat 1998). It is also involved in the signaling regulation to adapt to stress environments by control of stomatal aperture and expression of stress-responsive genes (Himmelbach et al. 2003). ABA could rapidly induce the expression of stress-responsive genes such as RD29A, MYB2, PP2C and ABI5, in which ABI5 plays a dominant suppression role in seed germination and

123

seedling development (Dai et al. 2013; Lopez-Molina et al. 2002). A genomic study about RLPs in Arabidopsis indicated that both the typical mutant tmm-1 and a null mutant Atrlp17-1 showed poorer growth and slower chlorosis in response to exogenous ABA (Wang et al. 2008), which hinted that ABA sensitivity might be a potential factor for TMM in control of stomatal distribution. However, in our study, TMM seemed to be a positive regulator in tmm-4 due to the defective ABA response to seed dormancy, but showed negative regulation in seed germination and seedling growth because of the hypersensitivity of tmm-5 and tmm-6. And ABA possibly induced stomatal closure and environmental pressure response independent of TMM in mature plant (Fig. 5). In accordance with the speculation, the expression of ABA-inducible genes was up-regulated in response to ABA in all tmm alleles, however, compared to other three genes, the transcriptional level of ABI5 in tmm-4 was slightly lower than in other tmms both in normal growth and ABA treatment seedlings (Fig. 6), which was consistent with the repression role of ABI5 and the defect phenotype of tmm-4 in seed dormancy (Dai et al. 2013). Although tmm-5 and tmm-6 were hypersensitive to ABA in seed germination, the expressions of three ABA-inducible genes in them were generally comparable with the wild type regardless of ABA treatment, perhaps hinting that some alternative ABAresponsive genes potentially regulated by TMM contributed to the hypersensitivity of tmm-5 and tmm-6. All these results suggested that TMM might perform regulation with different directions in ABA-induced physiological processes, which were dependent on developmental stages of plant. It was a similar situation for TMM as a negative or positive regulator of entry into the stomatal pathway, with the direction determined by organ and location (Geisler et al. 2000). However, our study also indicated that the function of TMM in ABA response was independent on that in stomatal patterning regulation. A complex network of signaling transduction pathway is used for achieving ABA functions (Wang and Zhang 2008). Though several genes, such as FLOWERING TIME CONTROL PROTEIN A (FCA) (Razem et al. 2006), ABAR/CHLH(Shen et al. 2006), GPCR-type G proteins 1 (GTG1) and GTG2 (Pandey et al. 2009) have been reported to encode ABA receptors, more proteins may participate in these pathways as receptors or signal transducers as well. TMM belongs to LRR-receptor-like proteins family (Nadeau and Sack 2002b; Wang et al. 2008), and it probably interacted with multiple receptor-like kinases (RLKs) in various signaling pathways to achieve its different regulations of ABA response. This possibility is supported by the functions of ER-family RLKs, which can compose a receptor complex with TMM to achieve the function of extracellular signal transduction to manipulate the stomatal distribution (Shpak et al. 2005; Torii et al. 1996), and also can compose with other partners to regulate plant transpiration efficiency,

Plant Cell Rep Table 2 Leaf trichome number and branch number Growth stages

Vegetative growth

Genotypes

WT

27.2 ± 4.3ab a

Branches (br)/trichome (%) 1 br

2 br

0d

12.2 ± 3.1d

85.3 ± 2.8a

2.6 ± 1.3a

d

d

a

0.8 ± 0.5a

79.2 ± 2.7a

1.6 ± 1.0a

80.7 ± 4.0

a

2.4 ± 1.6a

81.7 ± 2.0

a

2.1 ± 1.0a

37.8 ± 8.0

b

0a

S4

33.3 ± 3.9

0

tmm-4

24.0 ± 1.3b

0d

tmm-5 tmm-6 WT (empty vector) TMM-OX lines T2-1

Reproductive growth

Trichomes per leaf

ab

28.5 ± 1.8

ab

27.7 ± 2.7

ab

30.7 ± 2.8

0

d

0

d

16.7 ± 2.2

3 br

19.3 ± 3.4cd

16.2 ± 5.9

c

16.9 ± 2.8

d

16.2 ± 1.5

d

46.0 ± 4.3

a

82.5 ± 2.0

4 br

7.2 ± 2.5c

60.3 ± 8.6a

36.0 ± 5.6ab

5.6 ± 5.6d

0a

T2-2

14.3 ± 2.2c

54.8 ± 7.9a

30.0 ± 5.8bc

15.2 ± 3.4cd

0a

T2-3

c

b

39.8 ± 3.4

ab

14.9 ± 0.5

c

15.0 ± 1.4

c

11.5 ± 0.6

c

WT S4

15.5 ± 2.3

39.8 ± 3.2 a

167.2 ± 10.3

a

159.5 ± 12.5 a

6.0 ± 0.7

c

5.3 ± 0.5

c c

tmm-4

148.3 ± 6.7

6.3 ± 0.4

tmm-5

141.3 ± 15.8a

11.2 ± 2.2c

tmm-6 WT (empty vector)

a

168.5 ± 9.7

a

157.3 ± 7.8

6.9 ± 2.4

c

1.5 ± 0.6

c

17.9 ± 1.9c 18.0 ± 1.0

c

15.4 ± 1.2

c

20.4 ± 3.3

c

0a

76.0 ± 0.9

ab

3.0 ± 0.7a

77.1 ± 1.0

ab

2.7 ± 0.6a

79.1 ± 0.5

a

3.1 ± 0.5a

69.7 ± 3.3b

1.2 ± 0.4b

73.3 ± 2.6

ab

0.3 ± 0.2b

79.8 ± 1.1

a

3.3 ± 0.7a

TMM-OX lines T2-1

13.7 ± 3.1b

33.1 ± 7.2ab

52.4 ± 6.5a

14.5 ± 5.3c

0b

T2-2

20.3 ± 4.6b

24.5 ± 4.8b

54.1 ± 5.6a

21.4 ± 2.2c

0b

T2-3

b

a

b

c

0b

19.5 ± 1.8

38.0 ± 5.8

40.0 ± 6.3

21.9 ± 2.1

Data, including S.E., were obtained from at least six of the third leaves of 3-week-old plants and 6-week-old plants, respectively. ‘‘T2-1’’ represented ‘‘one line of second generation lines from wild type transformed with 1300-TMM construct’’ and so on. The superscript letters represent statistical significance at p \ 0.05 (Duncan test), and values with the same letter are not statistically different

organ size and shape (Masle et al. 2005; Shpak et al. 2004). A reported LRR-RLK named RPK1 has been demonstrated to function as an important positive regulator in early ABA signal transduction (Osakabe et al. 2005). Possibly, TMM interacted with RPK1 to positively regulate seed dormancy in early stage of development. Meanwhile, the mode of action of TMM affecting ABA signaling probably was correlated with different domains of the protein, for the tmm alleles displayed different sensitivity to ABA. On the other hand, similar to the function mode of the LRR-containing protein COI1, TMM may be implicated in stomata closure by regulating certain anion channels activity to respond to ABA induction (Munemasa et al. 2007; Xie et al. 1998). TMM affects trichome development Trichomes are normally present on the leaves, stems and sepals of Arabidopsis and protect plant against pests, pathogens and herbivores. In our study, a novel feature was identified that TMM could affect the development of trichomes. The number of trichomes on leaves was significantly decreased in TMM overexpression lines, and many of the formative trichomes had abnormal branches (Table 2; Fig. 7), which partially mimicked the mutants with fewer or

loss of trichomes such as gl and ttg1 (Hulskamp et al. 1994; Koornneef 1981), and mutants with reduced branches, such as sti, an, sta, zwi and frcs (Folkers et al. 1997; Luo and Oppenheimer 1999). The expression patterns of trichome formation genes GL1 and GL3, and branching genes ZWI and KIS were basically consistent in different TMM-OX lines, suggesting that overexpression of TMM potentially regulated their expression at transcriptional level, and up-regulated TMM level could play a negative role in trichome initiation and branching. Moreover, the reduction level of trichome was more significant in reproductive other than vegetative stage, which indicated that TMM might make more important contribution in later stage of trichomes development. However, the number of trichomes and branches was at normal levels in tmm-4, tmm-5 and tmm-6, which suggested that single gene mutation of TMM was not enough to influence the development of trichomes. Although more than 50 genes, excluding LRR proteins, have been isolated to control trichome development (Marks et al. 2009), more new genes or novel functions of known genes might be proved to involve in this process. Perhaps there exist some cooperators of TMM, which could function redundantly to regulate the generation of trichomes and formation of branches. In summary, we provided further evidences and materials for

123

Plant Cell Rep

Fig. 8 Sketch map of dental resin sequence showing placement and division of stomata in leaves. In tmm mutants, the orientation of spacing and amplifying divisions was interfered, and stomata and their precursors often contacted with each other. In tmm-6, entry

divisions occasionally appeared in cells adjacent to two stomata or their precursors. New produced meristemoids which contacted with each other or previous existed stomata were indicated by arrows

supplement and demonstration of TMM functions in the study.

Bergmann DC, Lukowitz W, Somerville CR (2004) Stomatal development and pattern controlled by a MAPKK kinase. Science 304:1494–1497 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 Dai M, Xue Q, McCray T, Margavage K, Chen F, Lee JH, Nezames CD, Guo L, Terzaghi W, Wan J, Deng XW, Wang H (2013) The PP6 phosphatase regulates ABI5 phosphorylation and abscisic acid signaling in Arabidopsis. Plant Cell 25:517–534 Daszkowska-Golec A, Szarejko I (2013) Open or close the gatestomata action under the control of phytohormones in drought stress conditions. Front Plant Sci 4:138 Finkelstein RR, Gampala SSL, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell 14:S15–S45 Folkers U, Berger J, Hulskamp M (1997) Cell morphogenesis of trichomes in Arabidopsis: differential control of primary and secondary branching by branch initiation regulators and cell growth. Development 124:3779–3786 Geisler M, Yang M, Sack FD (1998) Divergent regulation of stomatal initiation and patterning in organ and suborgan regions of the Arabidopsis mutants too many mouths and four lips. Planta 205:522–530 Geisler M, Nadeau J, Sack FD (2000) Oriented asymmetric divisions that generate the stomatal spacing pattern in Arabidopsis are disrupted by the too many mouths mutation. Plant Cell 12:2075–2086

Acknowledgments We thank Prof. F. Sack (University of British Columbia) for useful discussion and gifts of tmm-1 and TMMpro:: TMM-GFP; Dr. Pengfei Jia for taking the confocal and DIC images. This work is supported by the National Natural Science Foundation of China (NSFC) (Grant NO. 30300029, 30670124, 31070247, 91017002 and 31271460), the National Basic Research Program of China (Grant NO. 2009CB941500), and the program for New Century Excellent Talents of the Ministry of Education (Grant NO. NCET-060897).

References Balkunde R, Pesch M, Hulskamp M (2010) Trichome patterning in Arabidopsis thaliana from genetic to molecular models. Curr Top Dev Biol 91:299–321 Berger D, Altmann T (2000) A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes Dev 14:1119–1131 Bergmann DC, Sack FD (2007) Stomatal development. Annu Rev Plant Biol 58:163–181

123

Plant Cell Rep Hara K, Kajita R, Torii KU, Bergmann DC, Kakimoto T (2007) The secretory peptide gene EPF1 enforces the stomatal one-cellspacing rule. Genes Dev 21:1720–1725 Himmelbach A, Yang Y, Grill E (2003) Relay and control of abscisic acid signaling. Curr Opin Plant Biol 6:470–479 Hulskamp M, Misra S, Jurgens G (1994) Genetic dissection of trichome cell development in Arabidopsis. Cell 76:555–566 Hou S, Baker A, Webb A, Jia J, WJ L (2006) Segregation and identification of IAA6-knocked out mutant of Arabidopsis. In: Xu ZH, Li JY, Pua EC, Xue YB (eds) 11th IAPTC&B Congress Abstracts. Kluwer Academic Publishers, Boston p 64 Koornneef M (1981) The complex syndrome of ttg mutants. Arabidopsis Info Serv 18:45–51 Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49:199–222 Liu A, Gao F, Kanno Y, Jordan MC, Kamiya Y, Seo M, Ayele BT (2013) Regulation of wheat seed dormancy by after-ripening is mediated by specific transcriptional switches that induce changes in seed hormone metabolism and signaling. PLoS ONE 8:e56570 Lopez-Molina L, Mongrand S, McLachlin DT, Chait BT, Chua NH (2002) ABI5 acts downstream of ABI3 to execute an ABAdependent growth arrest during germination. Plant J 32:317–328 Luo DL, Oppenheimer DG (1999) Genetic control of trichome branch number in Arabidopsis: the roles of the FURCA loci. Development 126:5547–5557 Marks MD, Wenger JP, Gilding E, Jilk R, Dixon RA (2009) Transcriptome analysis of Arabidopsis wild-type and gl3-sst sim trichomes identifies four additional genes required for trichome development. Mol Plant 2:803–822 Masle J, Gilmore SR, Farquhar GD (2005) The ERECTA gene regulates plant transpiration efficiency in Arabidopsis. Nature 436:866–870 Munemasa S, Oda K, Watanabe-Sugimoto M, Nakamura Y, Shimoishi Y, Murata Y (2007) The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production. Plant Physiol 143:1398–1407 Nadeau JA, Sack FD (2002a) Stomatal development in Arabidopsis. The Arabidopsis Book Nadeau JA, Sack FD (2002b) Control of stomatal distribution on the Arabidopsis leaf surface. Science 296:1697–1700 Ni BR, Bradford KJ (1993) Germination and dormancy of abscisic acid- and gibberellin-deficient mutant tomato (Lycopersicon esculentum) seeds (sensitivity of germination to abscisic acid, gibberellin, and water potential). Plant Physiol 101:607–617 Ni M, Cui D, Einstein J, Narasimhulu S, Vergara CE, Gelvin SB (1995) Strength and tissue-specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J 7:661–676 Osakabe Y, Maruyama K, Seki M, Satou M, Shinozaki K, Yamaguchi-Shinozaki K (2005) Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis. Plant Cell 17:1105–1119 Pandey S, Assmann SM (2004) The Arabidopsis putative G proteincoupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell 16:1616–1632

Pandey S, Nelson DC, Assmann SM (2009) Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell 136:136–148 Parcy F, Valon C, Kohara A, Misera S, Giraudat J (1997) The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development. Plant Cell 9:1265–1277 Pei ZM, Kuchitsu K, Ward JM, Schwarz M, Schroeder JI (1997) Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants. Plant Cell 9:409–423 Pillitteri LJ, Sloan DB, Bogenschutz NL, Torii KU (2007) Termination of asymmetric cell division and differentiation of stomata. Nature 445:501–505 Razem FA, El-Kereamy A, Abrams SR, Hill RD (2006) The RNAbinding protein FCA is an abscisic acid receptor. Nature 439:290–294 Sachs T (1991) Pattern formation in plant tissue. Cambridge University Press, New York Shen YY, Wang XF, Wu FQ, Du SY, Cao Z, Shang Y, Wang XL, Peng CC, Yu XC, Zhu SY, Fan RC, Xu YH, Zhang DP (2006) The Mg-chelatase H subunit is an abscisic acid receptor. Nature 443:823–826 Shpak ED, Berthiaume CT, Hill EJ, Torii KU (2004) Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation. Development 131:1491–1501 Shpak ED, McAbee JM, Pillitteri LJ, Torii KU (2005) Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309:290–293 Shu K, Zhang H, Wang S, Chen M, Wu Y, Tang S, Liu C, Feng Y, Cao X, Xie Q (2013) ABI4 regulates primary seed dormancy by regulating the biogenesis of abscisic acid and gibberellins in Arabidopsis. PLoS Genet 9:e1003577 Telfer A, Bollman KM, Poethig RS (1997) Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Development 124:645–654 Torii KU, Mitsukawa N, Oosumi T, Matsuura Y, Yokoyama R, Whittier RF, Komeda Y (1996) The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats. Plant Cell 8:735–746 Wang XF, Zhang DP (2008) Abscisic acid receptors: multiple signalperception sites. Ann Bot 101:311–317 Wang G, Ellendorff U, Kemp B, Mansfield JW, Forsyth A, Mitchell K, Bastas K, Liu CM, Woods-Tor A, Zipfel C, de Wit PJ, Jones JD, Tor M, Thomma BP (2008) A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis. Plant Physiol 147:503–517 Wang Z, Cao H, Sun Y, Li X, Chen F, Carles A, Li Y, Ding M, Zhang C, Deng X, Soppe WJ, Liu YX (2013) Arabidopsis paired amphipathic helix proteins SNL1 and SNL2 redundantly regulate primary seed dormancy via abscisic acid-ethylene antagonism mediated by histone deacetylation. Plant Cell 25:149–166 Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280:1091–1094 Yang M, Sack FD (1995) The too many mouths and four lips mutations affect stomatal production in Arabidopsis. Plant Cell 7:2227–2239

123