www.sciencemag.org/cgi/content/full/323/5920/1485/DCl Hams Supporting Online Material for Paternal Control of Embryonic Patterning in Arabidopsis thaliana Martin Bayer, Tal Nawy, Carmela Giglione, Mary Galli, Thierry Meinnel, Wolfgang Lukowitz* *To whom correspondence should be addressed. E-mail: lukowitz@uga.edu Published 13 March 2009, Science 323, 1485 (2009) DOI: 10.1126/science.1167784 This PDF file includes: Materials and Methods Figs. S1 and S2 Tables S1 to S3 References and Notes M. Bayer, T. Nawy, C. Giglione, M. Galli, T. Meinnel, W. Lukowitz Paternal control of embryonic patterning in Arabidopsis thaliana. Supplementary Online Material Materials and Methods Supplementary References Figures S1, S2 Tables S1-3 Materials and methods Plant stocks and growth conditions. Plants for phenotypic analysis were grown in walk-in chambers under constant illumination (65 ^mol/m2/s) at approximately 21°C and 60% relative humidity on commercial potting mix (RediEarth, Sun Gro Horticulture) containing systemic insecticide (Marathon 1% G, Olympic Horticultural Products) and slow-release fertilizer (19/12/6 Osmocote, Scotts Micacle-Gro Co.). In the greenhouse, where culture conditions were more variable, the phenotype of ssp embryos was occasionally skewed toward the weaker or stronger side of the spectrum reported in Fig. 1, suggesting an influence of environmental factors. For crosses, the stamens of designated female partners were manually removed prior to pollen dehiscence, and the pistils pollinated two days after emasculation. The ssp-1 allele was induced in the Landsberg erecta (Ler) accession by chemical mutagenesis (S1) and harbors a premature stop codon at amino acid position 141 (C-to-T nucleotide substitution at position +689 of the open reading frame). Additional alleles were found in sequenced-tagged insertion libraries: ssp-2, generated by T-DNA transformation in the Columbia (Col) accession, harbors an insertion in intron 2 (SALK_051462) (S2); ssp-3 and ssp-4, generated by transposon mutagenesis of Ler, harbor insertions after amino acid position 383 (exon 8) and amino acid position 425 (exon 9), respectively (GT11708, ET11486) (S3). Other mutants and marker lines have been described: yda-1 and hyperactive YDA lines (Leraccession) (S1), AtSUC3-GFP (C24 accession) (S4), met1 (also termed ddm2-1; Col accession) (S5), ddm1-2 (Col accession) (S6), cmt3 (SALK_148381; Col accession) (S7), drm2 (SALK_150863; Col accession) (S8), rdr1-1, rdr2-1 (SAIL_672F11, and SAIL_1277H08, respectively; Col accession) (S9), rdr6-15 (Col accession) (S10), cdc2a (Col accession) (S11), fie (Ler accession) (S12). Map-based cloning. The ssp-1 mutation maps to an approximately 4cM interval on the top arm of chromosome II, defined by the PCR-based markers S499 and T320 (table S2). 97 recombination events within this interval were collected from a mapping population of about 2400 F2 plants as described (S13). Fine-mapping with internal markers (table S2) positioned ssp-1 between SNP8866 (7 recombination events) and S622 (4 recombination events), a 71kbp segment containing 15 predicted genes (At2g17040 to At2g17170). SNP1809 co-segregated with the mutation. Sequence analysis of candidate genes surrounding SNP1809 revealed a single base pair substitution in At1g17090 (see above). A PCR-based marker for this substitution (table S2) also co-segregated with ssp-1 in the mapping population. 1116 F2 plants were 1 Bayer et al., Paternal control of embryonic patterning, SOM analyzed, of which 306 were wild-type, 550 heterozygous, and 260 mutant, revealing normal transmission of the ssp-1 allele through the haploid gametophytic generation. T-DNA constructs. A 9.8kbp Smal/EcoRI fragment covering the SSP locus was isolated from the BAC clone F6P23 and transferred to a modified pCAMBIA3300 binary vector (S14). To facilitate genotyping of transgenic plants without interference from SSP transgenes, intron 3 of the SSP gene was eliminated by overlapping extension PCR (S15; primer pair I3-F/R, table S3). This construct served as a basis for all further manipulations. For tagging of the SSP protein, Apal/Stul restriction sites were inserted into two low-complexity regions N- and C-terminal of the kinase domain by overlap extension PCR (primers SSPKT-F/R and SSPMT-F/R, table S3). These sites were then used to insert the coding sequence of YFP (mCitrine variant, described in ref. S16; amplified by primer pair YANS-F&R, table S3). Point mutations in the myristoylation/palmitoylation motif and the ATP binding pocket were introduced by overlap extension PCR with the following primers (table S3): G2-F/R (G2A substitution), C34-F/R (C3,4S substitution), and K78-F/R (K78R substitution). Truncated variants of SSP, harboring premature stops at amino acid position 301 and 434, were generated by inserting a synthetic linker with stop codons in all reading frames (5'-CTAGTCTAGACTAG-3') into a blunted Ndel site and an Mscl site, respectively. Constructs directing ectopic expression of SSP were generated by fusing a CaMV 35S enhancer and transcriptional start site to the coding sequence of YFP-tagged as well as G2A and C3,4S variants of SSP, utilizing an MspA1l site located in the 5' untranslated region of the gene. A genomic DNA fragment extending from the translational start site of SSP to 6.4kbp upstream was PCR amplified using Phusion DNA polymerase (New England Biolabs; primer pair SSP5'-F&R; table S3) and inserted into a plasmid for recombination-based cloning (pDONR221; lnvitrogen). This fragment, containing the presumptive SSP promoter as well as the 5' untranslated region of the SSP transcript, was then joined with the coding sequence of a nuclear localized triple YFP reporter gene (Venus variant, described in ref. S17) in a modified pGreen binary vector (S18) by multi-site recombination (Gateway, lnvitrogen). RT-PCR and cDNA synthesis. RNA was extracted using the Spectrum Plant Total RNA kit (Sigma). 1 total RNA was used as a template for cDNA synthesis primed with oligo dT (annealing at 30 min at 50°C, reverse transcription for 30 min at 60°C with Thermoscript, lnvitrogen). SSP cDNAs were amplified from aliquots of the reaction with the primer pair SSP-27-F and SSP+939-R (30 cycles, annealing at 62oC; table S3). YFP and actin cDNAs were amplified with the primer pairs YFP-F/R and Actin-F/R (same conditions; table S3). A complete SSP coding sequence was amplified from a cDNA sample of mature pollen with the primer pair SSP-27-F and SSP+2215-R (40 cycles, annealing at 62oC; table S3) and subcloned into pGEM-T (Promega). Sequencing of individual plasmids revealed the existence of two splice variants. MspA1l/SphI restriction sites were utilized to replace the genomic coding sequence with the corresponding sequences of both cDNA variants in T-DNA constructs. 2 Bayer et al., Paternal control of embryonic patterning, SOM To enable discrimination between the paternal and maternal contribution to SSP expression, flowers of the Wassilewskija (Ws) accession were hand-pollinated with pollen of Col plants. Approximately 2000 immature seed from 70 crosses were dissected 24 hours after pollination and collected in RNAlater solution (Applied Biosystems). RNA extraction and cDNA synthesis were as above, except reverse transcription was primed with random hexamer primers. Nested PCR was performed using the primer pair SSP+204-F/SSP-2215R (30 cycles, annealing at 62oC; table S3). 1 [J of the primary PCR product was used as template to amplify a 273 bp fragment of the SSP cDNAs with the primer pair cSSP-dCAPS-F& and -R (30 cycles, annealing at 62oC; table S3). The resulting secondary PCR product was incubated with Bcll. Col-derived cDNA species remained uncut, whereas species derived from Ws were cleaved (245bp and 28bp fragments). This procedure maps the single nucleotide polymorphism SGCSNP1810 (www.arabidopsis.org), which was originally found in the Ler but is also present in the Ws accession. In situ hybridization. Pollen was germinated for 4h on microscope slides as described (S19) and fixed over night by adding 4% formaldehyde directly to the medium. In situ hybridization was performed on whole-mount germinated pollen. Siliques were fixed over night at 4oC (4% formaldehyde in PBS) and processed for sectioning and hybridization as described (S20). Digoxigenin-labeled RNA-probes were generated by in vitro transcription with T7 polymerase (Stratagene) from purified PCR-generated templates covering non-conserved sequences up- and downstream of the kinase domain (primer pairs SSP+32-F and SSP+409T7-R, SSP+859-F and SSP+1345T7-R, respectively, for SSP; PIN7-F and PIN7-T7-R for PIN7; table S3). The SSP probes were used individually as well as combined to increase signal intensity. Pollen nuclei were counter-stained with DAPI (1mg/l). Size measurements of embryonic cells. Whole-mount immature seed were dissected from siliques, cleared in modified Hoyer's solution (70% w/v chloralhydrate, 4% w/v glycerol, 5% w/v gum arabic) and examined by DIC microscopy (Leica DMRB compound scope equipped with Qimaging MicroPublisher 5.0RTV digital camera). Measurements were taken from digital images and calibrated by comparison to a stage micrometer. Fluorescence and confocal laser scanning microscopy. Embryos, immature seed, leaves, and roots were mounted in water containing 10uM propidium iodide as a counter stain and imaged with a confocal laser-scanning microscope. Mature pollen was harvested in 12% sucrose and germinated on glass slides for about 4 hours (S19). Nuclei were stained with DAPI (1mg/l) in germination medium, and the pollen tubes imaged with a fluorescence compound scope (Zeiss Axioplan2 microscope equipped with a Zeiss Axiocam HRm camera). Assay for N-myristoylation. Recombinant AtNMT1 with an N-terminal 10xHis tag was expressed from pET16b (Novagen) in bacteria and purified as described (S21). Myristoylation activity was assayed indirectly, by coupling the reaction to pyruvate dehydrogenase and measuring the produced NADH (S22). Absorbance at 340nm was monitored over time using a spectrophotometer equipped with a temperature control unit (Ultrospec-4000, AP Biotech). The reaction mixtures contained 50mM Tris at pH8.0, 3 Bayer et al., Paternal control of embryonic patterning, SOM 1mM MgCl2, 0.193mM EGTA, 0.32mM DTT, 0.2mM thiamine pyrophosphate, 2mM pyruvate, 0.1mg/ml bovine serum albumin, 0.1% Triton X-100, 5-1000uM peptide, 2.5mM NAD+, 0.125U/ml porcine heart pyruvate dehydrogenase (0.33U/mg) and were incubated at 30oC. Peptides were custom-synthesized (Genscript Corporation; >95% purity) and dissolved in water at a final concentration of 4mM. The value for kcat/Km was obtained from the experimental data by iterative non-linear least square fits of the Michaelis-Menten equation as described (S23). Genetic analysis of methylation effects. Pollen of ssp-1 plants was crossed to pistils of DNA methylation-deficient mutants to test whether expression of the maternal SSP allele can be restored by abolishing maintenance methylation (met1 and ddm1) (524) or small RNA-mediated de novo methylation (cmt3, drm2, and rdr1;-2;-6 triple mutants) (S24). All embryos from these crosses showed an ssp phenotype. Generation and analysis of fie seed generated by cdc2a pollen. Seed with unfertilized endosperm was generated using cdc2a and fie mutant strains as described (525) . Briefly, loss of CDC2a affects cell division in the male gametophyte, resulting in pollen with a single sperm. This sperm fertilizes the egg cell. As the central cell remains unfertilized, a normal, triploid endosperm cannot develop to sustain zygotic development (S11). Second mutations in FERTILIZATION-INDEPENDENT SEED (FIS)-class Polycomb-group genes suppress this zygotic arrest by triggering the formation of a diploid endosperm. While this endosperm lacks any paternal contribution, is able to support essentially normal growth of the embryo (S25). Double heterozygous cdc2a+/-;fie+/- plants were identified by PCR (S11, S25) and allowed to self-fertilize. 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(A) Top panel: RT-PCR with SSP-specific primers from total RNA of whole seedlings (1), rosette leaves (2), stems (3), whole inflorescences (4), and mature pollen (5); arrowhead in (5) points to a double band representing two SSP splice forms; lanes marked with a minus sign show control reactions without reverse transcriptase. Bottom panel: sample of total RNA used for RT-PCR reactions stained with ethidium bromide. Pollen-specific expression of SSP is in coincordance with microarray data in the public domain (S26). (B) Top panel: RT-PCR with YFP-specific primers from total RNA prepared from pollen of transgenic ssp-1 plants harboring a functional YFP-tagged SSP variant (lane 1), ssp-1 plants, transgenic Col wild type plants harboring the SSP::3xVenus reporter construct (3), and Col plants (4); RNA from transgenic seed expressing an inactive YFP-tagged SSP variant from a strong, broadly active promoter shown as positive control (lane 5); arrowhead marks position of YFP-specific band; although the assay was not meant to be quantitative, a stronger signal was consistently obtained with the triple-YFP transgene; lanes marked with a minus sign show reactions without reverse transcriptase. Bottom panel: RT-PCR with actin-specific primers from the same RNA samples. (C-F) Fluorescence microscopy of germinated pollen harboring a functional YFP-tagged SSP variant (C), and a reporter construct based on the promoter of the GEX2 gene (S27) (E); overlays with DIC images of pollen tubes shown in (D, F); inlet: detail of two sperm cells in close proximity, boxed in (E); note the absence of specific fluorescence in (C), although SSP and GEX2 show similar patterns of RNA expression and report similar normalized expression levels by microarray profiling (S26); scale bar 20|am. Fig. S2. Presence of SSP transcripts in the zygote and endosperm. In situ hybridization with SSP-specific probes to histological sections of seed containing zygotes (A, B) and 1 -cell embryos (C, D); arrows point to weak signals in the zygote and the micropylar region of the endosperm; note that signals persist beyond the first division in the endosperm but not in the embryo; nonspecific staining was independently assessed using control probes directed against PIN7 mRNA, preferentially accumulating in the basal cell after division of the zygote (E, arrow) (S28) and ML1 mRNA, preferentially accumulating in the apical cell (F, arrow) (ref. S29); scale bar 20|am. Classes of suspensor phenotypes Exaggerated Normal Short Rudimentary Cone-shaped Unrecognizable Self-fertilized ssp-1/ssp-1 Self-fertilized yda-1/+ Self-fertilized ssp-1/ssp-1; yda-1/+ 0 0 0 344 (60%) 0 0 75 (16%) 67 (12%) 4 (1%) 246 (53%) 25 (4%) 145 (47%) 114 (25%) 20 (4%) 99 (32%) 30 (6%) 116 (20%) 60 (19%) Self-fertilized D-YDA/+; yda-1/yda-1 Self-fertilized D-YDA/+; ssp-1/ssp-1 242 (45%) 305 (60%) 142 (27%) 94 (18%) 149 (28%) 113 (22%) Tab. S1. Genetic interactions of SSP and YDA. Frequency of suspensor phenotypes observed in the progeny of self-fertilized plants of the listed genotype; "exaggerated" suspensors were longer than wild type, contained more cells, and were often associated with aberrant development of the proembryo (see ref. S1 for images). Fig. 2 shows representative examples of the "short" (I), "rudimentary" (H), "cone-shaped" (G), and "unrecognizable" (F) suspensor classes; for the analysis of plants containing hyperactive YDA variants, the phenotypic classes of "short" to "unrecognizable" suspensors combined in one; hyperactive YDA variants are associated with a number of dominant effects that eventually become lethal, and can only be propagated in the heterozygous state. Marker Position Primer pair Comments 5499 (CEREON449449) S643 (CEREON459643) SNP8866 (SGCSNP8866) SNP1809 (SGCSNP1809) S622 (CEREON444622) 5500 (CEREON459500) T650 (CEREON458650) T320 (CEREON460320) ssp-1 (EMS-induced allele) At2g16400/10 CATTGCTTGACCGAGCAGAG GTGAGAATAGAAGTGCGTTGG At2g16780/90 CCTCCTTACCTTCATCTGCC GCTGTTTCGAAACAGTAAAACG AtAt2g17036/40 TTTGGCCTCCACCTCC CAAGGTGACTGTATTTCACTGG At2g17090 CAAACAAGCCTCTCCTAGTGG GCCTCTCAATGTTTGAAGtACGAGG At2g17170/80 GAAGAAACACCATAATCCATGC GAG G AAG AG C ATAAC AT AGC C At2g17230/40 GTTCGTAATCCTTCCATGTCC GTCTAACAAATTTGCTTCCAGC At2g17660 GAAACGAGTCCATTTCGTAGG CCTTCCAGCTTCATTCGACG At2g18380/90 GTGACATTGTCACTCACGTGG CAAAGATGTCTTCAGTGTCTGG At2g17090 TTAG AG AC C AC AC G AG AAGG C TAACATGGCTTGGTCTGATCC Length polymorphism Col: ~200bp; Ler>Col Length polymorphism Co/:~180bp; Ler>Col Length polymorphism Col: ~610bp; Ler>Col dCAPS marker, creates polymorphic Box\ site Col: ~240bp+20bp; Ler. ~260bp Polymorphic C/al site: Col: ~490bp+280bp; Ler. ~770bp Length polymorphism Col: ~350bp; Ler