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1 Supplemental Methods Isolation and mapping of SPCH An EMS-mutagenized population of tmm-1 (Col);E1728 (an enhancer trap GFP marking guard cells) was created. M2 seeds were collected from M1 s planted in pools of 10 to aid in the recovery of lethal mutations through sibling selection. From a screen of 50,000 M2 seedlings, two alleles of SPCH were identified. Lines were backcrossed twice to the parental unmutagenized line to remove extraneous background mutations, to ensure that a single locus was responsible for the mutant phenotype and to determine the dominant/recessive nature of the mutant allele. A mapping population was generated by outcrossing to Ler. An initial screen of 145 homozygous mutant F 2 plants identified linkage to a 330 Kb region on chromosome 5 markers cer and cer recombinants between these markers were identified from a population of 720 F 2 individuals and were used to narrow the region to 37 Kb cer and cer (TAIR). Candidate genes in the region were PCR amplified and sequenced. spch-1 and spch-2 exhibited G A mutations in the coding region of At5g A 6787 bp genomic spch fragment was generated by HindIII digestion of BAC K19E1 and was cloned into pcambia3300 and transformed into Col. Basta resistant T2 transformants were crossed with spch-1/+. Homozygous mutant F2 plants were confirmed by PCR genotyping and tested for restoration of normal stomatal densities. Total RNA from homozygous spch mutant plants (4 alleles) was collected from whole 2 week old seedlings. 100ng of RNA was used in PCR reactions. The SPCH, ACTIN, MUTE and FAMA cdnas were amplified for 35, 25, 37 and 27 cycles respectively for 15 sec at 95, 30 sec at 52 and 1 min at 68 using primers in table at end of methods. Phenotypic analysis For qualitative and quantitative examination of epidermal phenotypes, tissues were cleared in 70% ethanol and then overnight in Hoyer s solution 16. Ten plants each of spch-2;tmm-1, tmm-1, spch-2, and Col were examined for stomatal density in expanded cotyledons, pedicles and siliques. Cotyledon stomata were counted 10 days post germination in a 200X field between the midvein and margin halfway between petiole and tip. The number of stomata in contact as well as the number of stomata within one cell of another stomatal lineage cell were quantified in Col and spch-2 to assay the effects of the spch-2 mutation on spacing divisions. Counts of pedicle and silique stomata were made from epidermal peels extending the entire length of the organ. Pedicels were used to quantify the number of amplifying divisions because the sister cells of meristemoids are most unambiguously identified in this tissue 2.Stomatal density was determined as the number of stomata/area or the number of stomatal units (all stomata in contact are considered a single unit/area. Stomatal index was calculated as the number of stomata/total number of epidermal cells. Values are reported as the mean for all 10 plants +- the standard error. Following a test of data normality by normal probability plots, two-tailed t tests were carried out between spch-2 and the relevant background genotype using R statistical computing software ( and an alpha=0.05. P values were as reported (Fig S1). Confocal analysis of early divisions was aided by introgression of the plasma membrane marker Q8 17 into wt, spch-1 and spch-2 backgrounds. Embryos were isolated from mixed-stage spch- 1/+ and spch-3/+ siliques and scored in bulk (n= 50) for morphological defects. Root tips of 7- dpg homozygous spch-1 seedlings were counterstained for 1-2 minutes in 100ug/ml propidium iodide (molecular probes) and assayed by confocal microscopy (n=10). 1

2 Plant materials. Markers, mutants and previously published transgenic lines are as follows: Enhancer trap line E1728, Poethig lab, (enhancertraps.penn); Plasma membrane marker Q8 17 FAMA T-DNA insertion allele (SALK_100073) ABRC stock center; tmm-1 18, yoday295 3, FAMA Estpro::FAMA 10. Genotypes were confirmed with PCR using previously described primers or those indicated in table at end of methods. Col (0) was used as the wild type in all studies. T- DNA inserts were confirmed using primers designed by isect tools (salk.signal.edu). Plants were grown initially on half strength MS agar plates in a Percival incubator with 24 hour light for 7 days then transferred to soil in a 22C growth chamber with 16hr light/8 hour dark cycles. Double mutants between spch and other stomatal pathway genes were constructed by crossing and were obtained in the F2 progeny and confirmed by PCR-based genotyping using previously described primers or those indicated in table at end of methods. DNA manipulations. Plant binary vectors based on Gateway cloning technology (Invitrogen) were used for most manipulations. Descriptions of the vectors are found in 19 and 20. cdna clones of SPCH (pyat5g53210) and MUTE (pyat3g06120) were obtained from the ABRC and served as the templates for the creation of rescuing, and over-expression constructs using primers indicated in table at end of methods. Reporters for SPCH and MUTE were made by PCR amplification of 2.5KB and 1.7KB, respectively of DNA 5 of the translational start site. Transcriptional fusions for SPCH were made to GUS (pmdc163) 19, GFP (pmdc107) 19 and nucgfp (pbggn) 20. Transcriptional fusions for MUTE were made to GFP (pmdc107). A translational fusion for SPCH was made by cloning the 2.5KB promoter into a Not1 site 5 of the SPCH cdna in pentr/topo and recombining this vector into pmdc107. Promoter swap experiments used the 2.5KB SPCH promoter cloned into a Not1 site 5 of MUTE (see above) and FAMA 10 cdnas in pentr/topo. Translational SPCH fusions and promoter-swap constructs were transformed into spch-3/+. Individual T1 lines were allowed to set seed and lines segregating the T-DNA insertion for spch- 3 were characterized for epidermal phenotypes in the T2 and T3 generations. Overexpression of SPCH was achieved through the use of the native promoter (in a SPCH+ background), an estrogen-inducible promoter (pmdc7) and the CaMV35S promoter (pmdc32). 35S driven expression of SPCH often caused a recapitulation of the loss of function phenotype. This observation suggested that MUTE could also be silenced by incorporating the cdna into a plasmid containing the 35S promoter (pmdc32). 7/18 lines showed reduced MUTE RNA levels and co-suppression phenotypes. Lines were tested for RNA expression using primers in table at end of methods. Plants were stably transformed using Agrobacterium-mediated transformation (strain GV3101) with standard protocols. Transgenic lines were selected on 1/2MS medium containing mg/l Hygromycin or Basta as appropriate. 2

3 Primers used in this study Purpose Primer name Sequence SPCH promoter cloning pro f CACCAGATCATCACTGCGATAAGG pro r GCGGCCGCGTGATTAGAGATATATCCT SPCH homologue (MUTE) promoter 06120_pro f2 CACCGCGGCCGCGTTGGTTTGGTTTTCGACCC 06120_pro r2 GCGGCCGCCAAGATTCTCTTCTGGAGTTCC SPCH cdna gatestart CACCATGCAGGAGATAATACCGGA bhlh rev AGAAAGTGAGTACGTACTGC MUTE cdna MUTEgs f CACCATGTCTCACATCGCTGTTGA MUTEr GGATCCTTAATTGGTAGAGACGATCAC Sequencing primers SPCHseqfor AGACAAAACAACGAACGAGG SPCHseqrev AGAAAGTGAGTACGTACTGC SPCHseqint TAAAAAAAGGGGGACCAAGC RT-PCR primers SPCHrtPCRf AAAATCGGCTTTGGCTGATGTGAAG SPCHrtPCRr AGAAAGTGAGTACGTACTGC MUTErtPCRf CATCAAAAGGGGAGATCAAG MUTErtPCRr CAGAGATGATCTTTACGAGC FAMArtPCRf GCTCGAGCAACTCCTACAATG FAMArtPCRr GGAACTTGCTATGTCTTCTGC ActinF GGCGATGAAGCTCAATCCAAACG ActinR GGTCACGACCAGCAAGATCAAGACG genotyping primers SAIL_36_B06L GAAAAACCTAGATCCTCCCCC SAIL_36_B06R TCCTATGATCGATGCTTGGTC spch2dcaps for AAAATCGGCTTTGGCTGATGTGAAG spch2dcaps rev TTCGTCGACGGTGTTAATATTAACC Salk078595L TATGAGGGACTCGCATTCATC Salk078595R AAAACAAATTCGTTTGCTCCC spch1dcapsf AAAGAACAATTATCAATGTATACTTCTC spch1dcapsr TAATTCAGCAAACATTCTGCTAGAT 3

4 MacAlister et al., Supplemental Figures Genotype mean +/-SE Organ tmm-1 tmm-1;spch-2 P-value Stomatal index Abaxial cots / / e-4 Adaxial cots / / e-6 Col 0 spch-2 P-value Pedicels / / e-3 Siliques / / a Adaxial cots / / e-14 tmm-1 tmm-1;spch-2 Stomata/mm 2 Abaxial cots / / e-9 Adaxial cots / / e-5 Stomatal Units/mm 2 Abaxial cots / / e-9 Adaxial cots / / e-5 Col 0 spch-2 #SLGC/stomata Pedicels / / e-9 stomata within 1 cell of other stoma/total stomata Adaxial cots Col 0 spch / / e-5 Figure S1 changes in stomatal density and index in spch-2 organs. Stomatal density was determined as the number of stomata/area or the number of stomatal units (all stomata in contact are considered a single unit/area). Stomatal index was calculated as the number of stomata/total number of epidermal cells. Values are reported as the mean for all 10 plants +- the standard error. Following a test of data normality by normal probability plots, two-tailed t tests were carried out between spch-2 and the relevant background genotype using R statistical computing software ( and an alpha=0.05. a not significant 4

5 Figure S2 SPCH is not required for asymmetric cell divisions outside of the stomatal lineage. a-b, Confocal images of propidium iodide-stained wild type (a) and spch-1 (b) 7-dpg roots. White arrows indicate cortex/endodermis division. c-f, DIC images of cleared globular (cd) and heart stage (e-f) embryos of wildtype (c,e) and representative embryos from spch-1 segregating siliques (d,f). No obvious defects in morphology or early cleavage planes were seen, and suspensors appeared normal in 60 embryos (4 siliques) from a spch-1/+ plant. 5

6 SPCH MQEIIP MUTE FAMA MDKDYSAPNFLGESSGGNDDNSSGMIDYMFNRNLQQQQKQSMPQQQQHQLSPSGFGATPF 60 SPCH ---DFLEECEFVDT SLAGDD LFAILESLEGAGEISPT 40 MUTE FAMA DKMNFSDVMQFADFGSKLALNQTRNQDDQETGIDPVYFLKFPVLNDKIEDHNQTQHLMPS 120 SPCH AASTPKDGTTSS------KELVKDQDYENSSPKRKKQRLETRKEEDEEEEDGDGEAEEDN 94 MUTE FAMA HQTSQEGGECGGNIGNVFLEEKEDQDDDNDNNSVQLRFIGGEEEDRENKNVTKKEVKSKR 180 SPCH KQD GQQKMSHVTVERNRRKQMNEHLTVLRSLMPCFYVKRGDQASIIGGVVE 145 MUTE MSHIAVERNRRRQMNEHLKSLRSLTPCFYIKRGDQASIIGGVIE 44 FAMA KRARTSKTSEEVESQRMTHIAVERNRRKQMNEHLRVLRSLMPGSYVQRGDQASIIGGAIE 240 *:*::******:****** **** * *::**********.:* SPCH YISELQQVLQSLEAKKQRKTYAEVLSPRVVPSPRPSPPVLSPRKPPLSPRINHHQIHHHL 205 MUTE FIKELQQLVQVLESKKRRKT----LN RPS FAMA FVRELEQLLQCLESQKRRRILG ETG :: **:*::* **::*:*:... SPCH LLPPISPRTPQPTSPYRAIPPQLPLIPQPPLRSYSSLASCSSLGDPPPYSPASSSSSPSV 265 MUTE --FPYDHQTIEP SSLGAATTRVPFSR-----I 94 FAMA --RDMTTTTTSSSSP ITTVANQAQPLIITG----NV 295 *.. :::.. : : spch-2 M SPCH SSNHESSVINELVANSKSALADVEVKFSGANVLLKTVSHKIPGQVMKIIAALEDLALEIL 325 MUTE ENVMTTSTFKEVGACCNSPHANVEAKISGSNVVLRVVSRRIVGQLVKIISVLEKLSFQVL 154 FAMA TELEGGGGLREETAENKSCLADVEVKLLGFDAMIKILSRRRPGQLIKTIAALEDLHLSIL :.* * :* *:**.*: * :.::: :*:: **::* *:.**.* :.:* spch-1 stop SPCH QVNINTVDETMLNSFTIKIGIECQLSAEELAQQIQQTFC MUTE HLNISSMEETVLYFFVVKIGLECHLSLEELTLEVQKSFVSDEVIVSTN FAMA HTNITTMEQTVLYSFNVKITSETRFTAEDIASSIQQIFSFIHANTNISGSSNLGNIVFT 414 : **.::::*:* * :** * ::: *:::.:*: * Figure S3 SPCH protein domains and alleles and alignment of SPCH and homologues. ClustalW alignment of SPCH, MUTE and FAMA protein sequences. Identical residues among all three marked with *, conservative substitutions with :, and semi-conservative changes with.. The putative nuclear localization sequence is shown in green, the bhlh domain is shown in blue, and the basic region is additionally indicated by bold type. Amino acid changes in the EMSinduced mutations in spch-1 and spch-2 are noted in red. 6

7 m, graph of microarray expression profiles max expression roots hypocotly veg rosette leaf 4 leaf 12 leaf 7 proximal leaf 7distal stem SAM (veg) SAM (floral) flower stage 15 silique stage 5 mature pollen 7 FAMA MUTE SPCH

8 Figure S4 Additional SPCH expression pattern data a-c, Confocal images of SPCHpro::SPCH-GFP expression (green) with propidium iodide counterstaining to visualize cell outlines (red). White arrows indicate small dividing cells (a) Example of epidermis in SPCHpro::SPCH-GFP line that overproduces stomata. Note extra GFP-positive small cells, but no GFP expression in morphologically distinct GCs. (b) SPCH- GFP expression in 2-dpg cotyledon (c) Absence of SPCH-GFP in roots. d-e, Confocal images of transcriptional reporter SPCHpro::nucGFP in (d) stomatal lineage cells and (e) cells dividing next to stomatal lineage cells. f-h, brightfield images of SPCHpro::GUS expression. (f) Expression in expanded cotyledons is restricted to the stomatal lineage, (g-h) overstained samples indicate that highest expression is in young dividing tissues and (h) expression is negligible in the root. i-k, Closeup of cluster in tmm-1; SPCHpro::SPCH hypocotyl showing that the green cells are E1728-expressing GCs. (j) and (k) are the single PI and GFP channels corresponding to the merged image in i. l, Phenotypic consequence of MUTE expression under the control of the SPCH promoter in wild type plants. Bracket indicates a small cluster of ectopic guard cells and the black arrow indicates a normal stoma. m, Expression data for MUTE, SPCH and FAMA as determined from tissue-specific microarray experiments 21. The X-axis indicates tissue type consistent with designations in ntools ( 22. The maximum expression for the gene in the entire suite of microarray experiments is indicated under max expression. 8

9 Figure S5 Model of stomatal development. The three stomatal bhlh proteins have distinct and successive expression patterns and each is required for progression through a different stage in stomatal development. Expression of SPCH (blue) in young epidermal cells allows these cells to make asymmetric cell divisions. SPCH expression stays high in small cells with no morphological evidence of differentiation. In meristemoids, SPCH expression is downregulated and MUTE (red) expression commences. MUTE is required to limit the number of rounds of meristemoid division. Reduction of SPCH or MUTE activity has opposite effects on amplifying divisions consistent with a model that SPCH may promote divisions and that MUTE is required to stop divisions and promote differentiation into GMCs. After differentiation of meristemoids into GMCs, expression of MUTE diminishes and FAMA (pink) expression begins. FAMA is required both to limit cell divisions and to promote guard cell fate during the final stage in stomatal formation. FAMA acts contemporaneously with two MYB-type transcription factors, FLP and MYB88, but it is likely that these proteins work independently 9,10. In addition to these transcription factors, stomatal development is influenced by several genes encoding putative signaling components. Phenotypic studies and genetic evidence suggest that TMM, the ER-family and YODA act as positive (arrows) and negative (T-lines) regulators of stomatal development at several stages including (1) entry into the pathway, (2) amplifying divisions and (3) the orientation of spacing divisions 2, 3, 4. Several of these events overlap with events controlled by the stomatal bhlhs, but it remains to be tested whether SPCH, MUTE or FAMA is a direct target downstream of TMM or the ER-family and YODA kinases. 9

10 Supplementary notes: 16. Liu, C. M. & Meinke, D. W. The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development. Plant J. 16, (1998). 17. Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S. & Somerville, C. R. Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl. Acad. Sci. U. S. A. 97, (2000). 18. Yang, M. & Sack, F. D. The too many mouths and four lips mutations affect stomatal production in Arabidopsis. Plant Cell 7, (1995). 19. Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, (2003). 20. Kubo, M. et al. Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 19, (2005). 21. Schmid, M. et al. A gene expression map of Arabidopsis thaliana development. Nat. Genet. 37, (2005). 22. Toufighi, K., Brady, S. M., Austin, R., Ly, E. & Provart, N. J. The Botany Array Resource: e-northerns, Expression Angling, and promoter analyses. Plant J. 43, (2005). 10