Cytokinin biosynthesis

South African Journal of Botany 109 (2017) 96–111

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Cytokinin biosynthesis ISOPENTENYLTRANSFERASE genes are differentially expressed during phyllomorph development in the acaulescent Streptocarpus rexii (Gesneriaceae) Y.-Y. Chen a,b,c,1, K. Nishii b,d,⁎,1, A. Spada e, C.-N. Wang a, H. Sakakibara f, M. Kojima f, F. Wright g, K. MacKenzie g, M. Möller b a

Institute of Ecology and Evolutionary Biology, Department of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, UK c University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK d Tokyo Gakugei University, 4-1-1 Nukuikitamachi, Koganei, Tokyo 184-8501, Japan e Dipartimento di Scienze Agrarie e Ambientali, Milan University, Via Celoria 2, 20133 Milan, Italy f RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan g Biomathematics and Statistics Scotland, JCMB, The King's Buildings, Peter Guthrie Tait Road, Edinburgh EH9 3FD, Scotland, UK b

a r t i c l e

i n f o

Article history: Received 17 June 2016 Received in revised form 6 October 2016 Accepted 13 December 2016 Available online xxxx Edited by K Doležal Keywords: Streptocarpus Cytokinin Isopentenyltransferase Leaf Meristem

a b s t r a c t The enzyme ISOPENTENYLTRANSFERASE (IPT) is responsible for the rate limiting step of cytokinin biosynthesis, an important plant hormone with key roles in meristem maintenance and organ development. In this study, we isolated IPT genes from the acaulescent Streptocarpus rexii, a plant that shows an unorthodox development starting with post-germination anisocotyly, in which cytokinins play an integral role. Three adenosine phosphate-IPTs and two tRNA-IPTs were isolated from S. rexii. Their expression levels and patterns in different tissues were compared by means of realtime-PCR and mRNA in-situ hybridization. We found that each SrIPT had a distinctive expression pattern. Interestingly, in vegetative tissues as well as in meristems only the adenosine phosphate-IPT SrIPT5 and the tRNA-IPT SrIPT9 were found. In addition, they were differentially affected by external hormone application, suggesting their different regulation and expression during meristem formation and maintenance and lamina growth. Our results indicate that SrIPTs are involved in shaping the architecture of S. rexii, working differentially and redundantly, and show that differentially expressed IPT genes regulate plant form. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Cytokinins are important plant hormones and play key roles in plant development and maintenance (Werner et al., 2001, 2003; Osugi and

Abbreviations: BAP, 6-Benzylaminopurine; cZ, cis-Zeatin; cZR, cZ riboside; cZRPs, cZR 5′-phosphates; DAU, days after cotyledon unfolding; DIG, digoxygenin; DMAPP, dimethylallyl diphosphate; DMSO, dimethylsulfoxide; DZ, dihydrozeatin; DZR, DZ riboside; DZRPs, DZR 5′-phosphates; GA, gibberellic acid; GA2ox, GA2-oxidase; GA20ox, GA20-oxidase; iP, N6-(Δ2-isopentenyl)adenine; iPR, iP riboside; iPRPs, iPR 5′-phosphates; IPT, isopentenyltransferase; KNOX1, class 1 KNOX genes; MS medium, Murashige and Skoog medium; NAA, 1-Naphthaleneacetic acid; RAM, root apical meristem; SAM, shoot apical meristem; STM, SHOOTMERISTEMLESS; tZ, trans-Zeatin; tZR, tZ riboside; tZRPs, tZR 5′-phosphates. ⁎ Corresponding author at: Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, UK. E-mail addresses: [email protected] (Y.-Y. Chen), [email protected], [email protected] (K. Nishii), [email protected] (A. Spada), [email protected] (C.-N. Wang), [email protected] (H. Sakakibara), [email protected] (M. Kojima), [email protected] (F. Wright), [email protected] (K. MacKenzie), [email protected] (M. Möller). 1 Both authors contributed equally: KN, YY.

http://dx.doi.org/10.1016/j.sajb.2016.12.010 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

Sakakibara, 2015), sink-source balances (Werner et al., 2001; Takei et al., 2001a; Werner et al., 2003), senescence (Gan and Amasino, 1995) and cell division (Riou-Khamlichi et al., 1999). Cytokinins are also important for maintaining the shoot apical meristem (SAM), the source of stem tissue and lateral organs (Werner et al., 2003; Miyawaki et al., 2006). In Streptocarpus rexii, a plant with a unique morphology, cytokinins have important roles in shaping its architecture, since external cytokinin applications severely altered its development and morphology. This acaulescent species lacks a conventional SAM and develops an irregular rosette composed of meristem-bearing leaves termed phyllomorphs (Jong, 1970; Hilliard and Burtt, 1971; Jong and Burtt, 1975; Jong, 1978). Just after germination, both cotyledons are equal in size as in typical dicots (Fig. 1a). However, a SAM is not established between the cotyledons and soon after they unfold, the two cotyledons develop unequally establishing anisocotyly (Jong, 1970; Hilliard and Burtt, 1971; Jong and Burtt, 1975; Nishii and Nagata, 2007; Fig. 1b,c). The larger cotyledon, the macrocotyledon, continuously expands in size while the smaller cotyledon, the microcotyledon, stops any further development and eventually withers away. The macrocotyledon develops into the first phyllomorph (cotyledonary phyllomorph)

Y.-Y. Chen et al. / South African Journal of Botany 109 (2017) 96–111

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Fig. 1. The unique development of the rosulate Streptocarpus rexii. a Seedling with fully unfolded cotyledons representing ′day 1 after cotyledon unfolding′ (1 DAU). b 10 DAU, beginning of the anisocotylous phase. c 30 DAU, strongly anisocotylous seedling. Bars: 0.5 mm. d Anisocotylous seedling with large cotyledonary phyllomorph (macrocotyledon) and microcotyledon (arrowhead). e Plant with several additional phyllomorphs, formed in numbered succession. f Inflorescence initiated at the base of the phyllomorph in the reproductive stage. Bars; 1 cm. g Schematic illustration of a phyllomorph (modified from Jong and Burtt, 1975). The macrocotyledon retains the basal meristem and the groove meristem, and grows to become the cotyledonary phyllomorph. BM: basal meristem, GM: groove meristem, Mc: macrocotyledon, mic: microcotyledon, cp: cotyledonary phyllomorph.

(Fig. 1). Phyllomorphs consist of a lamina and petiolode, a stem-like petiole (Jong, 1970; Hilliard and Burtt, 1971; Jong and Burtt, 1975; Fig. 1g) and their development is governed by three meristems, the basal meristem at the proximal end of the lamina for lamina growth, the petiolode meristem for petiolode and midrib extension and thickening, and the groove meristem located at the juxtaposition between the lamina and petiolode that forms new phyllomorph primordia (Fig. 1g), the reiteration of this process results in a false rosette (Fig. 1e, f). Previous studies (Rosenblum and Basile, 1984; Nishii et al., 2004; Mantegazza et al., 2009) demonstrated that the synthetic cytokinin 6-benzylaminopurine (BAP) causes both cotyledons to develop into cotyledonary phyllomorphs creating macrocotyledonary isocotyly in several Streptocarpus species (i.e. S. prolixus, S. wendlandii and S. rexii), suggesting a pivotal role for cytokinin in the establishment of anisocotyly. The enzyme ISOPENTENYLTRANSFERASE (IPT), is responsible for the rate limiting step of cytokinin biosynthesis (Kakimoto, 2001; Takei et al., 2001b). IPTs transfer the isoprenoid group from isoprenyl donor dimethylallyl diphosphate (DMAPP) to the N6-position of adenosine, forming isopentenyladenosine, which is a precursor for cytokinin biosynthesis (Sakakibara, 2006). There are two different forms of IPTs in plants, adenosine phosphate-IPTs and tRNA-IPTs. Adenosine phosphate-IPTs in plants use primarily ATP, ADP as substrates and are responsible for the synthesis of N6-(Δ2-isopentenyl)adenine (iP) or trans-Zeatin (tZ) type cytokinins. tRNA-IPTs, on the other hand, catalyse the addition of prenyl-moiety to a tRNA-bound adenine nucleotide and contributes to the formation of certain cytokinins, mainly cisZeatin (cZ) in Arabidopsis (Miyawaki et al., 2006; Sakakibara, 2006). Seven adenosine phosphate-IPTs and two tRNA-IPTs (Kakimoto, 2001; Takei et al., 2001b; Golovko et al., 2002; Miyawaki et al., 2004) are known to belong to the multigene ISOPENTENYLTRANSFERASE (IPT)

family in Arabidopsis thaliana. Each AtIPT gene showed a specific expression patterns and different hormonal feedback regulations (Miyawaki et al., 2004). At the same time, AtIPTs work highly redundantly, since single or double IPT gene mutants did not show phenotypes, only multiple (triple or quadruple) mutants did (Miyawaki et al., 2006). Although there is a high degree of redundancy, some IPT genes have specific roles in meristems, with some under the control of class 1 KNOX genes (KNOX1). KNOX1 genes are responsible for the formation and maintenance of the SAM (Hake et al., 2004) and these induced upregulation of AtIPT7 but not of other AtIPTs (Yanai et al., 2005). The present study investigates the role of the cytokinin biosynthesis genes ISOPENTENYLTRANSFERASE in the morphogenesis of Streptocarpus, with particular focus on the meristems. We isolated IPT genes from the rosulate S. rexii and characterized their expression patterns during plant growth with special emphasis on macrocotyledon and phyllomorph development, in an attempt to find specific IPT genes linked to meristem formation and maintenance. The results are compared with those obtained for model plants, particularly A. thaliana, and the role of cytokinin and IPTs in the phyllomorph morphogenesis in S. rexii discussed. 2. Materials and methods 2.1. Plant material Seeds of Streptocarpus rexii (originally collected from Tsitsikamma, Cape Province, South Africa, and subsequently grown at the Royal Botanic Garden Edinburgh, RBGE, accession number 20030814) were cultivated at 22 °C–24 °C under 16 h of light and 8 h of darkness, and a relative humidity of 80%, on sterilized soil (Potgrond H, KlasmannDeilmann GmbH, Germany). The plants were fertilized once a week

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with HYPONeX #4 (N–P–K = 6.5–6–19; 1:1000 w/v in tap water; HYPONeX Corporation, OH, USA). The pot-grown plants were used for harvesting tissue for the RT-PCR, realtime-PCR and in-situ hybridization experiments. For cytokinin treatments, seeds were sterilized and sown in Petri dishes on 30% strengths of Murashige and Skoog (MS) medium solidified with 0.8% agarose as described in Nishii et al. (2004). 2.2. Cytokinin treatments The cytokinins 6-Benzylaminopurine (BAP, EC 605–343-5) and trans-Zeatin (tZ, EC 214–927-5) were used (Sigma-Aldrich, St Louis, MO, USA). The chemicals were diluted in dimethylsulfoxide (DMSO) to a concentration of 10−3 M, and added to MS medium before solidification at 1000 times dilution for a final concentration of 10−6 M. In controls, the same amount of DMSO was added. Seedlings germinating (defined here as cotyledon unfolding) on MS agarose medium without hormones were moved to MS medium containing hormones. The seedlings were cultivated as before and their morphology observed 30 days after cotyledon unfolding (DAU).

shared an isopentenylpyrophosphate transferase domain (IPPT: Pfam accession number PF01715). Thus, the IPPT domains were aligned using the HMMER v3.0 package (Eddy, 2008). The alignment was trimmed using BMGE (Criscuolo and Gribaldo, 2010), and model selection was carried out using TOPALi v2.5 (Milne et al., 2009) and the WAG model selected under the Akaike information criterion (AIC). The model was used to estimate a PhyML Maximum likelihood (ML) tree calculated in TOPALi. ML bootstrap support values were estimated with 1000 replicates under the same settings. The tree was visualized in FigTree v1.4.1 (http://tree.bio.ed.ac.uk/software/figtree/). 2.7. Intron search The presence of introns was examined by comparing PCR amplified product using complementary DNA (cDNA) and gDNA as templates. Genomic DNA and total RNA was extracted and cDNA synthesized as described above. Their intron positions were drawn with GSDS v2.0 (Hu et al., 2015). Primers used for intron isolation are listed in Table A1.

2.3. Morphological observations

2.8. Gene expression analyses by realtime-PCR

Digital images of seedlings were taken under a stereomicroscope DP71 (Olympus, Tokyo, Japan) and the length of cotyledons measured using ImageJ v1.44× (Schneider et al., 2012). For observations under a scanning electron microscope, samples fixed in FAA (5% acetic acid, 5% formaldehyde, in 50% ethanol) were dehydrated using an ethanol series and immersed in isoamyl acetate. Following drying and ion-sputtering, the samples were observed under an S-2250 N scanning electron microscope (Hitachi, Tokyo, Japan) at the University of Tokyo (Tokyo, Japan).

To compare the levels of expression of the IPT genes, realtime-PCR with the absolute copy number measurement method was employed (see Whelan et al., 2003). Briefly, plasmids containing sequences of each IPT gene isolated here and 18S ribosomal RNA (18S rRNA) as reference gene were constructed using the pGEM-T Easy vector system (Promega, Fitchburg, WI, USA), and all plasmid insertions were checked by sequencing. The molecular weight and the predicted copy numbers of the plasmids were calculated (see Table A2). The concentration of plasmids was measured with NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Realtime-PCRs (primers listed in Table A1) were performed with a dilution series of plasmids (25 × 107 ~ 25 × 103 copies) as controls (Table A2), on a Bio-Rad CFX384 realtime-PCR machine (BioRad Laboratories, Hercules, CA, USA) as described previously (Nishii et al., 2010). The results were analysed in REST (Pfaffl et al., 2002). For all realtime-PCR analyses, each sample was run in triplicates and each experiment was conducted at least two times for confirmation of the results. Realtime-PCR were performed with RNA extracted from; 1) seedlings 35 DAU, excluding the lower parts of the hypocotyls and roots, 2) macrocotyledons excised from 3 months old plants, 3) lamina and 4) midrib of young phyllomorph (5 cm in length), 5) lamina and 6) midrib of a fully expanded phyllomorph (20 cm in length), 7) flower buds of 0.5–1.0 cm length, 8) fully opened flowers 9) developing fruits of 4–10 cm length, 10) peduncles, 11) mature phyllomorph with inflorescences removed, 12) roots of a mature flowering plant (see Fig. A1). To determine the localization of the expression of the IPT genes in the phyllomorph, RNA was extracted from the proximal and distal region of young (5 cm lamina length) and fully expanded (20 cm lamina length) phyllomorphs. Gene expression levels of SrIPT5 and SrIPT9 were examined using plasmids as control and normalized between samples using 18S rRNA as reference.

2.4. Isolation of IPT genes from S. rexii Five IPT-like partial sequences, named SrIPT1, SrIPT2, SrIPT3, SrIPT5, and SrIPT9, were retrieved from the S. rexii transcriptome database (Chiara et al., 2013). Total RNA was extracted from an entire plant using Trizol reagent (Invitrogen, Waltham, MA, USA) and cDNA was synthesized using Superscript III (Invitrogen) as described before (Nishii et al., 2014). 3′RACE PCRs were carried out to obtain the 3′region of SrIPT1, SrIPT2, SrIPT3, SrIPT5, and SrIPT9 as described in Mantegazza et al. (2007). Genomic DNA (gDNA) was extracted using a CTAB method (Doyle and Doyle, 1987). Inverse PCR (Ochman et al., 1988) was carried out for isolating the 5′region of SrIPT1 from gDNA digested with MboI (New England Biolabs, Ipswich, MA, USA), and TAIL-PCR (Liu and Whittier, 1995) for SrIPT3. The primers used are listed in Table A1. The amplified products were sent for sequencing to Genomic BioSci & Tech (Taipei, Taiwan). The full sequence of five SrIPTs were confirmed by PCR and sequencing from cDNA. 2.5. Homology search The obtained SrIPT sequences were compared with AtIPT genes from A. thaliana, retrieved from GenBank. The deduced amino acid sequences were aligned using MAFFT v7 (Katoh and Standley, 2013). Sequence identity (number of identical nucleotides per alignment length) and amino acid similarity were analysed in SIAS (http://imed.med.ucm.es/ Tools/sias.html). 2.6. Phylogenetic analysis of IPT genes A phylogenetic analysis was conducted to assess the relationships between the IPT genes of S. rexii, A. thaliana, rice, and maize. Bacteria, yeast, human, and fruit fly dimethyltransferase genes were also included in the analyses as outgroup. Pfam searches (Finn et al., 2014) showed that the deduced amino acid sequences of the IPT genes

2.9. Hormone treatment Seedlings were grown on soil for 35 DAU as described above and then transferred to Petri dishes and grown on for 7 days on filter paper soaked with 10−4 M 1-naphthaleneacetic acid (NAA), 10−5 M 6-benzylaminopurine (BAP), or 10−4 M gibberellic acid (GA3) (SigmaAldrich, Missouri, USA). The hormone concentrations were chosen based on a previous study (Nishii et al., 2004). NAA, BAP, and GA3 1000× stock solutions were prepared in DMSO and as control seedlings were treated with DMSO without hormones. RNA was extracted from entire seedlings excluding the lower part of the hypocotyls and roots. Relative gene expression was calculated against the expression level of control samples using 18S rRNA as reference.


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primers used are listed in Table A1. Digoxygenin (DIG)-labelled RNA probes were synthesized using a DIG RNA labelling kit (Roche, Basel, Switzerland) following the manufacturer's protocol. Three months old seedlings were fixed in a solution of 4% paraformaldehyde, 10% DMSO, 0.5% glutaraldehyde in phosphate buffered saline, pH 7.0. The samples were passed through an ethanol series and finally ethanol was replaced by xylene prior to paraffin embedding. The embedded samples were sectioned at 10 μm thickness and hybridized with RNA probes as previously described (Mantegazza et al., 2007). The signal was detected as purple-blue colour with beige-brown as background. Sense transcribed probes were hybridized as negative control. 2.11. Measurement of cytokinins

Fig. 2. Length of cotyledons 30 days after cotyledon unfolding (DAU). Control seedlings show anisocotyly, whereas BAP and tZ treated seedlings do not show anisocotyly and both cotyledons are almost equal in size. Dotted line indicates a 1:1 ratio.

Seedlings were grown on 30% strength MS medium solidified with 0.8% agarose as described before (Nishii et al., 2004). 35 DAU seedlings were excised into root tissue, microcotyledon, and into equal-sized proximal and distal regions of the macrocotyledon. The material collected separately was immediately frozen into liquid nitrogen. 100 mg of tissue were used for each measurement. Extraction and determination of the cytokinins was performed as previously described (Kojima et al., 2009), based on biological triplicates.

2.10. Gene expression analyses by RNA in-situ hybridization

3. Results

RNA in-situ hybridization was carried out as described in Mantegazza et al. (2007) to examine the expression patterns of SrIPT5 and SrIPT9. The partial sequence of SrIPT5 (amino acid: 11–115), and SrIPT9 (amino acid: 344–449) were PCR amplified from cDNA and inserted into pGEM-T Easy vector system (Promega) as templates. The

3.1. Exogenous cytokinin treatment effects on seedling development

a

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In Streptocarpus rexii, both cotyledons were equal in size at 1 DAU (Fig. 1a). Anisocotyly became noticeable around 10 DAU (Fig. 1b), and the seedling were strongly anisocotyl at 30 DAU (Fig. 1c). Exogenous

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Fig. 3. SEM micrographs of cytokinin treated seedlings 30 days after cotyledon unfolding (DAU). a, d, g Seedling treated with 10−6 M tZ. b, e, h Seedling treated with 10−6 M BAP. c, f, i Seedling without hormone treatment. d, e, f Magnified view of the proximal part of cotyledons. g, h, i Magnified view of the distal part of cotyledons. In S. rexii, small meristematic cells are observed in the proximal region of the macrocotyledon (f, arrows), but larger jigsaw puzzle shaped cells are present in the differentiated distal parts of the cotyledonary lamina (i), and throughout the microcotyledon (f, asterisk). Cytokinin (tZ and BAP) treated seedlings show two macrocotyledons (a, b), and small cells in the proximal region of the lamina of both cotyledons (d, e), resembling the macrocotyledon in the control seedling (c, f, i).

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donor) binding motifs characteristic to adenosine phosphate-IPTs (Fig. 4; Chu et al., 2010). The tRNA-IPTs, SrIPT2 and SrIPT9, retained the ATP-binding P-loop domain (Fig. 5). SrIPT9 additionally possessed the motifs for tRNA and phosphate group recognition and binding. However, in SrIPT2, a premature stop codon was present in the amino acid sequence at position 234 (Fig. A2), truncating the ORF before the functional domain (Fig. 5; Soderberg and Poulter, 2001; Zhou and Huang, 2008; Chimnaronk et al., 2009). Thus, SrIPT2 might not be properly functional and it might represent a pseudogene (Fig. A2). On the basis of those findings, SrIPT2 was excluded from further analysis. Introns were only found in SrIPT2 and SrIPT9, but not in the other three SrIPTs. The exon-intron structures of SrIPT2 and SrIPT9 were very similar to those of AtIPT2 and AtIPT9 respectively, although SrIPT2 only retained the first seven exons (Fig. 5b).

application of BAP and tZ resulted in two similarly enlarged cotyledons at 35 DAU (Figs. 2, 3). Both cotyledons showed small cells in the proximal region (Fig. 3d, e) and larger ones in the distal region of the lamina (Fig. 3g, h). These resembled the macrocotyledon in control seedlings (Fig. 3f, i), and confirmed that the basal meristems in both cotyledons were active in the hormone treated seedlings. 3.2. Isolation of genes from the IPT multigene family from S. rexii Three adenosine phosphate-IPT genes and two tRNA-IPT genes were isolated from S. rexii (Figs. 4, 5). The adenosine phosphate-IPTs were assigned SrIPT1, SrIPT3, and SrIPT5, and the tRNA-IPTs SrIPT2 and SrIPT9. The nucleotide sequence identity and amino acid similarity compared between the SrIPTs and their closest ortholog of AtIPT genes isolated from the model plant Arabidopsis thaliana (Miyawaki et al., 2006; Table A3) indicated that SrIPT1 showed the highest identity and similarity with AtIPT1, SrIPT2 with AtIPT2, both SrIPT3 and SrIPT5 with AtIPT5, and SrIPT9 with AtIPT9 (Table A3). The phylogenetic analysis of the SrIPTs also showed that SrIPT1, SrIPT3, and SrIPT5 belong to the adenosine phosphate-IPT clade, and SrIPT2 and SrIPT9 are tRNA-IPT genes, with the first falling in the AtIPT2-type tRNA-IPT clade, and the latter in the AtIPT9-type tRNA-IPT clade respectively (Fig. 6). The deduced amino acid sequences of the adenosine phosphate-IPTs, SrIPT1, SrIPT3, and SrIPT5 showed the ATP-binding P-loop domain ([G or A]-X4-G-K-[S or T]) near the N-terminal region (Fig. 4). They also retained the conserved adenine, ribose and DMAPP (prenyl group

3.3. Tissue-specific differential expression between SrIPT genes The levels of expression of SrIPT1, SrIPT3, SrIPT5, and SrIPT9 in various tissues were examined by realtime-PCR (Fig. 7). SrIPT1 expression was found in the roots and reproductive organs, and was relatively high in the flower bud and open flower. SrIPT3 expression was found almost exclusively in roots, but low in other tissues examined (Fig. 7). SrIPT5 and SrIPT9 were expressed in all vegetative and reproductive tissues examined, including the root (Fig. 7). SrIPT5 expression was high in open flowers, peduncle, flowering phyllomorph and root. SrIPT9 expression P-loop

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.

.

.

.

160 | .

- A RG E L T - E HG E L T - E - ADLT - E AG E V T - TYEDF T - Q DG E L T - E AG N L T - DD SELT D S HP E F T - E - SDF S - D - EDF T

.

.

.

|

.

.

.

.

70 |

.

.

.

.

.

.

|

.

.

.

.

170 | .

.

.

.

|

.

.

.

.

80 |

.

.

.

.

|

.

.

.

.

90 |

.

.

R R - Q - - - R H R K E K L L V L MG RME Q S R S R N R K D K V V V I L G - M L T L N P Y G P K D K V V V I MG - - - - - - - - K C N D KM V V I MG - M V D V P F F R R K D K V V F V MG C MG H A G R K N I K D K V V L I T G F - - - - - - L H P K E K V I F V MG V C M E Q - - - S Y KQ K V V V I MG - - - - - - - - - - - - K I I V I MG - - I L Q - - - G Q K E K V V V V MG - - - - - - RQ R R K D K V V V V I G

.

|

.

.

.

.

180 | .

.

P A D F R S L AG K A V S E I T G R R K L P AG E F R S A A S N V V K E I T S RQ K V P A A N Y C HM A N L S I E S V L N R G K L P A A E F R VMA A E A I S E I T Q R K K L P A E D F Q R E A I R A V E S I VQ R D R V P P AE F R S L AT L S I S KL I S S KKL P A TQ Y S R L A SQ A I S KL S A N N KL P T S E F R S L A S R S I S E I T A RG N L P P S D F R R SG A A V I SQ I V S R R R I P AADF RA S A S S AVR S I HL RKKL P V D D F V Y H A S L A A D R I T R RG R L P

.

.

.

V L VG G I I AG G I I VG G I L AG G I I AG G I V VG G I V AG G I I AG G F I VG G V I AG G I I VG G

.

.


N N N N N N N N N N N

|

.

190 | .


.

.

.

|

A TG A TG A TG A TG A TG T TG A TG A TG P TG A TG A TG

.

.

|

F I HAL F VHAL YVEAL Y I HAL Y I EAL F NHAL Y I EAL F I HAL F I YAL F IQAL YVKAL

.

.

TG AG TG SG TG TG SG SG CG AG TG

.

.

.

.

100 |

K K K K K K K K K K K


R R R S R R R C K R R

.

.

200 |

LVDRF L AQ R F VDDKE LAKSY VNDCV LAERF VNHS S LVDRF L AKKY I DRDF VNDDV

†† .

.

.

|

.

.

.

.

240 | .

.

.

.

|

.

.

.

.

250 | .

.

.

.

|

.

.

.

T D Y L A K R V D DML E L G MF D E Y E Y L L R R V D E MM D S G M F E E HG F V S E R V D KM V E S G M V E E F E Y L S L R L D L MM K S G M F E E H S F V S E R V D KM V D MG L V D E F Q H L C N R V DQ M I E SG L V E Q N S F V S K R V D R MM E A G L L E E F E Y L S K R V D Q MM E S G M F E E NQ H L I K R V D DML D SG MF E E H S F V S D R V D RMV E RG MV K E H S F V S K R V D RMV D A R L V D E

330 | .

340 | .

430 | .

.

- - - - - - - - - - - - - - - - V SL S S L L S F T K R R R K HQ P L V S S I SP TLDF P P ARFG P N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - H S S L L P T VT T KFG S P RL VT T - Q P I L CF KNKL S KVN - - VN S T S P R L R L P P P R S V V P - - MT T D S A T T S A T L R R HM - - - - - - A R P T L R I P G N A I AQ - - - - - - - - - - - - - - - - - - - - - - - - -

.

.

.

|

A K RQ A K RQ A C RQ T K DQ A C RQ V K KQ A C RQ A K KQ A K RQ A C RQ A C RQ

.

.

.

IGK VKK REK I TK LQ K KE K L KK I ER VG K VEK VKN

.

I I I I I I I I I I I

* † † .

.

.

.

.

.

440 | .

.

.

.

|

.

.

.

.

350 | .

.

.

.

|

.

KG - A G W D L R R L D A T K D - AG WE I E R V D A T R K V K KW S I Q R V D A T R N - AG WD I K K V D A T Y KQ W K W N M H R V D A T I R - G G WE I K R L D A T H K KW KM S M H R V D A T K S - S G WD I Q R L D A T R D A AG WE L K R V D S T R N A KG WD L H R L D A T E G L L E R RMH R L D A T

.

.

.

.

L * -

S -

|

.

.

.

.

450 | .

.

|

.

.

LRL EML ERL NKL QRL MKL QRL MKL LRL MR L LRL

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I A S R R P L V E A S T A V A A AME R E - - - - - - - - - - - - - - - - - - - - - - - - - - - AN I LLP E I SAVP P LP AAVAA I SR * - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L K N D D V E H C L A A S YG G G SG S R A H NM I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V A YG G S A V S V E A P L MA A AG * - - - - - - - T P MF S P P V A S I F L K S N A V A T A T R * - - - -

|

.

40 |

- - - - - SF S SHS - - - - - - - - - - NFQG N - - - - - - - - - - - - - - - SF SNNK - - - - - - - - - - -

L WV D V S V K V L I WV D V S E T V L L WV D V A L P V L I W I D V DQ S V L L WV D V S R P V L L WV D V L E P V L I WV D V S L P V L L WV D V S V S V L I WV D V S S P V L L W V D V S MQ T L L WV D V L M P I L

E D P G R D R V R RG A F E E A V R A I K E N T C H L KM I KW D A L R K A A Y D K A V D D I K R N T W T L - - F L NVE DRE E L L S KVL E E I KRNT F E L D M D KW D P M R K E A Y E K A V R A I K E N T F Q L - - NYP AE T T E RL L E T A I E K I KE NT CL L D KG I W D L A R K A A Y E E T V KG M K E R T C R L - - L V D R A T K S KM L D V A V K N I K K N T E I L KM S E W D Q A R KG A Y D E A V Q E I K E N T W R L E S R V H E M E KQ A A Y E E A V R A I K E N T C Q L - - S S D E E T R V R V L A E A I D R I KM N T S K L - - HE E HE S R V K I L E E A I EQ I K S N T C I L

|

.

*

.

- ELRYDCCF - DLRYECCF R - SRYDCCF - E L KYDCCF R - LRYNCCF - DL RYKCC I - - NNYDCCF - - LRYECCF KE L RYRCCF - - - RYDCCF R - N KYE CCF

320 | .

30 |

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CKNHNT - - - - - - - - - - -

**† .

EG S H S V V SG S C - L I - - - - F - D H - KG - S - - - - - - PG S SL ST - - - - - - - - S SET S DF HSDVD - - - - - - - - - - - - 310 | .

.

20 |

- - - - TTTTTTN - - SDRF TTTTTT SP - - M K I S M A M C KQ P - - - - - - - - - - - - - - - - K P CMT A L RQ V I Q - - QQ LMT L L S P P - - - K F S I S S L KQ V - - - QNLT STF V SP - - - KRL S VF P T T P HF - - Q I S L S M C KQ A - - DQ L I L S - - - - - - -

|

.

.

.

.

260 | .

.

.

.

|

.

.

.

.

270 | .

.

.

.

L AE F Y S - P E DE DHDE L S R F Y D - P V K SG L E VREF F D - F SN SDY S I AE F HR - S KKAP KE VRR I F D - P S S SDY S L A E L Y D - P V V D SG R VRE VF N - P KAN - Y S L AG F Y D - P R Y SG S A L AE YF L HPQ S NP V - VRDLF D - PQ ADY S - V KE F F N - P SG D Y T - -

.

360 | .

.

.

.

|

.

.

.

.

370 | .

E S F R A AMT S D S - - - A S F K A V MM K S S S - - P VF T KRR S - - - - - - A S F R E A I R A A KEG EG EVF LRR - - - - - - - - A A I MA E L NQ S T A K - G E VF L KR - - N - - - - - P SFG - - - - R S S - - - A A L L A A MG - - - - - - DVF RK- - S - - - - - - DAF L KL - - - - - - - - -

- - - - RCL - - - - - - - - - - - - - - - - -

.

.

- - VA - - - - - - - - -

.

.

.

|

D -

S -

.

|

.

.

.

.

280 | .

A T R TG - TRFG - - R - G - - P LG - - - AG - - R LG - - - VG I R A HG - S K CG - - - SG - - - RG

.

.

.

.

.

.

.

L RKA I RKA I KKA I WK A I RRA VRKT I RRA I HKT L KKA I RRA I RRA

380 | .

|

.

I I I I I I I I I I I

G G G G G G G G G G G

.

.

VP VP FP VQ VP VE VP I P VP VP VP

.

†** .

.

.

|

.

.

.

290 | .

.

.

.

EF DRYF E F DG Y F EF DRF F EF DDYL ELDEF L EF DRYF E L HE YL EF DRYF E FQ KYF ELDEF F EMHE Y F

.

390 | .

.

.

- G E K C T E I W E KQ V L E P S V K I V - E K KW R E N W E E Q V L E P S V K I V - KM D A N V A W E R L V A G P S T D T V V A E MQ R K I W N K E V L E P C V K I V R - G E E A D E AWD N S V A H P S A L A V E G K NG R E I WE K H I V D E S V E I V - V E E Q D E AWE N L V A R P S E R I V - - - - - R E I WD N T V L D E S I K V V - G R R A T E I WE RQ V L E P S V K I V - G K D V E A AWE NM V AG R S A A I V - G D E A DQ AWE R S V A K P S T K I V -

.

S -

|

.

.

.

.

300 |

E KF RPG KE YP - P R N EQ - KM Y K - W R S EMR RVYP - K RNE S - SLYP - P ERYD - RTE S - RTEP F -

|

.

- - - HL - - - - - - - -

.

.

400 |

- - - DQ - - - - - - - -

P -

I -

.

460 |

* -

Fig. 4. Alignment of adenosine phosphate-IPTs. Open box: ATP-binding P-loop domain, asterisks (*): adenine and ribose binding site, dagger (†): phosphate group binding site. Domain information obtained from HlAIPT (Chu et al., 2010).


101

a

P-loop .

.

.

.

.

|

.

.

.

10 |

ScMOD5 AtIPT2 AtIPT9 SrIPT2 SrIPT9

M L KG P L KG C L MMM L N P S N G G M - V I G SG V - M- DVDP DADE M - I G G L R VG G


S D S MQ A D A MQ A D S VQ A D S MQ A D S VQ

.

.


DP - - - Q DMV V E QTEY - T KP DCE E R DG - -

S L -


L I F S F


KL ML EF - YF


S S T

.

.

.

.

.

|

.

.

.

.

.

.

.

.

.

.

.

.

|

|

.

.

.

.

.

.

.

|

.

EDC I EEA I YAF L - - - YTF L

.

.

|

.

.

.

.

S

P

L

|

.

.

.

T T S T S

.

.

20 |

- - - - RL

P

F

.

.

.

NKHP NKVT AKP S NKI P AKP S

.

.

.

|

.

.

.

.

.

|

.

.

.

.

30 |

- - - - - - - - AAE

P

L

L

L

F F

L L

T T

.

|

.

.

.

.

.

120 | .

.

.

.

LQ E R EG I P V D E Q KG V P D S DRKVVP V Q E R KG V P I CERREVP

* .

.

.

310 | .

.

220 | .

.

.

.

|

.

.

.

- - RT - R -

130 | .

.

.

.

|

- - - - CYL - - - - -

.

.

|

.

.

40 |

.

- - - - R LQ - - - - Q

P P

P S

S I

.

.

.

.

.

.

140 | .

.

.

.

- - - - LVL - - A SF

.

.

.

H H VMN H V DW S E E Y Y S H R F H H L L G T V S S DME F T A R D F H H L I D I L H P SQ D Y S VG Q F HHL LG T I S P D I E F T A KE F H H L I D I L H P S E D Y S VG Q F

.

.

.

.

.

.

.

.

|

.

.

.

.

320 | .

.

.

.

|

.

.

- - R L D D R V D DML E R - - G D R Y V E Q R V D AMV D A - - G Y R S I D F R C E DML S G P NG - - - - - - - - - - - - - - - - Y R S I D F R C E DML L - DG G

410 | .

† .

.

E RMK T R T DRVKL NT NKFQ T A S - - - - - - S E FQ KA S

.

|

P I I DVL D VG DLL D IG

210 | .

.

.

.

.

230 | .

.

.

.

|

.

.

.

.

240 | .

.

.

.

- - - - - - D V I Y N T L V K C D P D I - A T K Y H P N D Y R R VQ RML V F G R D D L S HG Y E L L K E L D P V A A N R I H P N N H R K I N Q Y L - - - - - NWD A A V E L V V N AG D P K A S S L P R N DWY R L R R S L E P KG A A C D Y T H A R L E N L D P V S A N R I H P N D H R K I R Q Y L - - - - - DWD A A VQ L V V K AG D P G VQ S L A A N DWY R L R R R L

Y S KP E P L FQ CMD A E T A V L F L S SP RVAL YLVLE * - - F L S T HRL DL

.

.

- - I - - - N - LRV

110 | .

VYKD I I Y SG L V Y KG L V YQ G L V Y RG L

**** .

.

510 | .

.

|

.

.

.

.

* * .

.

420 | .

RQ Y A K RQ R R L L R RQ R N F A K RQ - - - - - - R N F A K RQ

.

|

.

.

.

.

.

.

.

|

- - - - KRRV S - M T WF - - - - - L T WF

520 | .

.

.

.

|

.

.

.

.

- - RLE RCE - - RNE

.

KG E T T M K K L D D W T H Y T C N V C R N A D G - - - - - - - E R D L WT Q Y V C E A CG N K - KNR - - - - RE D - - - - - - - - - C S S V - - - - - - - - - - - - - - - - - - - - - - - - - K N R H F I T R E D - - - - - - - - - CG S I - -

.

.

330 | .

A LQ E LLDE VL SE - - - I LTE

.

.

430 | .

530 | .

.

.

.

- - - - - - RRRF - - - HRR I

|

.

.

.

.

.

.

|

.

.

.

.

.

.

.

.

.

K KM L - - - - - - - - - - - - -

I -

|

.

.

* .

.

KNVVA I G - - - I L RG - - - - - - - - - - - - - - - - - - -

.

|

340 | .

.

.

.

Y E Y Y SQ N KF YKPG - ADYT L DLG L L P N S - - - - - - - - L D LG LMP N S

.

.

.

.

440 | .

P -

.

D -

I -

540 | .

.

.

.

KG - - - - -

.

.

50 |

.

.

.

150 | .

.

E T E CMN A I RDF TVP L I Y D DG RQ A T RE HA I P L I Y E D A RQ I T

|

.

E I SL E I NL E I

.

.

|

.

TP RG NP - N S

.

|

.

D N -

I I -

|

.

250 | .

Y Y KTG H A S RG L K S TG Y E S RG L K S SG

.

.

.

350 | .

.

.

.

YLL HY I - - L - - - - I

.

.

.

450 | .

.

.

- - AC - VC

.

ED EE KD DE EE

|

I I I I I

.

|

S S S S S

.

.

|

KKP VLP SPP VLP A SP

EQ C E NG L RQ S I G ATRA I G - - - - - ATRA I G

.

.

- - - - - - - - CAATT - - - - S RHCT

** .

I KQ L VYD I A RWL - - - AQ WL

- - V KW I T VFGWP M Y HW - - - - - P I YQ W -

.

|

.

.

|

V WQ VRE Y RQ - - Y RQ

.

S S

.

.

.

.

- - VP - S -

L -

.

.

.

.

.

|

550 | .

.

.

.

|

.

.

.

.

.

160 | .

260 | .

ETFN K L YQ G SFR I P KVFQG AF P LP

.

.

.

.

.

.

|

.

.

.

.

70 |

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

170 | .

.

.

S S

F F

.

.

460 | .

K C F

560 | .

RQ A D F E R HKN SQ A * - - - - - - - - * - - - - -

.

.

.

|

I I I I I

|

.

.

AG MG SG MG SG

.

.

.

.

.

|

.

.

.

.

.

.

.

S I I

.

.

|

.

.

.

.

270 | .

.

.

370 | .

.

.

.

.

.

|

.

.

.

.

|

.

T -

S -

L -

S -

.

.

|

.

470 | .

.

- S Q WD T N A S Q R A I A E E S WN AQ V V K P A S E Y D A Y E S E A - - EMV E - - - - - - - - - - - - - C D A Y H VQ N - - G N L S

.

.

570 | .

T E -

VE AE - - - -

.

.

|

.

.

.

- - - - - - - - - - DADDF L - - - - - EV - EVL

- HL - - - -

KW K I N K K E T Y K N R E VQ - - - - - - - - - - - - - - - - - - - - - -

.

.

.

VG SG AG SG AG

180 | .

.

.

K K K K K

.

.

|

.

SQ L S KL SRL S KL SRL

.

|

.

.

.

.

90 |

S IQL AVDL AME L A I DL SLEL

.

.

.

.

.

.

.

AQ K F A S HF AKRL A S HF AKRL

190 | .

.

.

.

.

.

100 |

NG E P VE NG E P I E NG E

V I I I I

I I I I I

N N S N S

.

.

.

200 |

|

|

.

.

.

T H Y Y L Q T L F N K R V D T K S S E R K L T R KQ L D I L E S T T HYY I Q AVV S KF L L DDAAE DT E E CC ADVA S VVD T G L Y L RWF M YG K P D V P K P S P E V I A E A H DM L VG F T N Y Y I Q A L V SQ Y L L N D F A D D L E A N C S L D L P G D T T G L Y L RWF I YG K P N V P K A S P E I A S E V H S E L S G L

- - - - - - - - - - - - RVNLVAP - - - - - - KE K SG S S

|

80 |

T TG P TG P TG ATG P TG

* * .

- - YD - YN

360 | .

|

I V VG G V L VG G I V TGG V I VG G I V SGG

V I G F K E F L P WL T G K F E D F L K I H L S E T C AG AME Y L L Q C R R Y E G E - - - - - - - - - - - - - - AMD Y L L A C R E Q G SW -

.

.

- - - - - - - NM S K K V I V - - E G E KM K K K A K V V V NG N K K K K S E K E K V I V - - F T P N P N S K P K I VM S H A Q T E KG H R Q K V I V

DAT - - - DL - - DAT - - - EY I L S N A S KP L D S I LQ - - - - - - - - - - D A AQ P ME N V L S

E K YWK I H L G S R R H K S N L K R N T R H E W E H H KQ G R T H R - - - K R T T - L EWI R S EG C K S E A S C V E S A I - - - - - - - - - - - - - - - - - - - - - L E WV K K - - T Q Q G Q A T V P A V L

.

H R RG K I P V SRNHI P L N RG R V P WSRNCL P L S KG R V P

* * .

60 |

.

.

* VN - - - -

.

* -

.

.

.

280 | .

- - - - E DG - - EMN

.

.

.

S S

S S

380 | .

.

.

.

.

.

- - - - - - - N D D K VMK E - - - - - - - - - - - - - - - - - - - - - -

.

.

I I I I

S I P P

.

480 | .

NDF RCF E SL - - R SL

.

|

.

- - - KT AE AD I S N TME TDEL

.

.

|

.

- NL - - - -

|

.

.

.

.

290 | .

.

.

.

EQ K I T L KF N WG C I N A S IQN I ETDL V C Y T AWL L HD S K S KD L

.

.

- RK - - - -

.

.

.

I -

.

390 | .

L -

.

.

- NF - - - -

P -

490 | .

.

.

.

|

.

.

.

.

300 |

DT L F LW RF DYCL DYDF LC CKF I VP DF DF I C

|

.

.

.

.

400 |

- TDDNTV KDDKL R I - - - S SPR - - - - - - - - - S S SR

.

|

.

I S N R P I KQ E R A P K A L L E T E - T E SG R D P T SG RM S K D V R D S R E A S E L - - - - - - - - - - - - - - I MK K D V S N H R E VQ D L

.

.

.

500 |

EELL KS I KG Y R - - - KAYR

b

Fig. 5. tRNA-IPTs. a. Alignment. Open box: ATP-binding P-loop domain, asterisks (*): tRNA-binding sites, dagger (†): phosphate group binding site, grey line: predicted invading ɑ14 helix domain. Domain information obtained from bacteria and yeast IPTs (e.g. MOD5; Zhou and Huang, 2008). SrIPT2 shows a premature stop codon (grey arrowhead) and lacks the functional domains, and thus might be a pseudogene. AtIPT: Arabidopsis thaliana IPT, SrIPT: Streptocarpus rexii IPT, ScMOD5: Saccharomyces cerevisiae. b. Exon-intron structure of tRNA-IPTs in S. rexii and A. thaliana.

was lower in the midrib of fully expanded phyllomorphs, the capsule and roots compared to the relatively high level in other tissues (Fig. 7). 3.4. SrIPT5 and SrIPT9 expression pattern in the phyllomorph In order to characterize the role of SrIPT5 and SrIPT9 in meristem formation and maintenance their expression was further characterized. In young 5 cm long phyllomorphs, the SrIPT5 and SrIPT9 expression levels were very similar between the proximal and distal tissue (Fig. 8). In 20 cm long fully expanded phyllomorphs, the expression of SrIPT5 was higher in the proximal tissue and very low in the distal tissue, whereas SrIPT9 was expressed in both the proximal and distal tissue, with higher expression in the distal part (Fig. 8). 3.5. The groove meristem harbours SrIPT expression Three months old macrocotyledons (cotyledonary phyllomorphs) showed a strongly focused expression of SrIPT5 on the petiolode at the base of the lamina where the groove meristem is located (Fig. 9a, b). Expression was observed in the subepidermal layer and beneath the groove meristem in an inverted triangular shape over 3–4 cell layers deep, but it was excluded from the epidermal layer. SrIPT9 showed a similar but more diffused expression pattern (Fig. 9c, d). 3.6. Phytohormone treatment affected the expression level of SrIPTs The effects of the external hormone treatment on the expression level of SrIPT5 and SrIPT9 in seedlings showed that NAA strongly

reduced the expression of both SrIPT5 (P b 0.05) and SrIPT9 (P b 0.01) (Fig. 10). On the other hand, BAP and GA3 differentially affected SrIPT5 and SrIPT9: BAP only mildly reduced the expression, though without statistical significance, of SrIPT5 (P = 0.19) and SrIPT9 (P = 0.14). GA3 reduced the expression of SrIPT9 (P b 0.05), but not of SrIPT5. 3.7. Cytokinin measurement in anisocotylous seedling Cytokinins measured in 35 DAU anisocotylous seedlings of S. rexii showed that the concentration of each cytokinin was similar between the proximal- and the distal region of the macrocotyledon, and also between the proximal region of the macrocotyledon and the microcotyledon, except for minor differences of iP and iPR (Table 1). Overall, iP-type cytokinins were slightly more abundant than tZ-type cytokinins. cZ was not detected in the macro- or the microcotyledons or the root, though cZR and cZRPs were found. DZ, DZR, DZRPs were also not detected in any tissue analysed (Table 1). 4. Discussion 4.1. Cytokinins are able to impair anisocotyly In this study we show that the naturally occurring cytokinin, trans-Zeatin (tZ), induced the development of two macrocotyledons. This result is fully congruent with previous studies where synthetic cytokinins (e.g. BAP) resulted in the formation of two macrocotyledons (Rosenblum and Basile, 1984; Nishii et al., 2004; Mantegazza et al., 2009).

102


100

99

100

MIAA_RHIRD MIAA_ECOLI MIAA_PSEPU ZmIPT10 OsIPT10 AtIPT9 SlIPT6 85 SrIPT9 tit1_SCHPO 99 MOD5_YEAST DmtRdim TRIT1_HUMAN 74 100 Trit1_MOUSE ZmIPT1 98 OsIPT9 SrIPT2 95 95 SlIPT5 55 AtIPT2

I

99

91

II

OsIPT6 OsIPT7 99 ZmIPT8 95 OsIPT8 52 ZmIPT3b 99 ZmIPT3 96 SrIPT1 97 SlIPT1 54 SlIPT2 86 AtIPT6 AtIPT8 96 AtIPT4 70 AtIPT1 AtIPT3 SlIPT4 SrIPT3 88 SlIPT3 83 SrIPT5 AtIPT7 52 AtIPT5 ZmIPT7 99 OsIPT3 ZmIPT2 90 OsIPT2 100 56 OsIPT1 ZmIPT6 90 98 ZmIPT5 99 OsIPT4 OsIPT5 ZmIPT9 82 99 ZmIPT4

III

0.5 Fig. 6. Maximum likelihood tree of deduced amino acid sequences of IPT genes isolated from S. rexii. SrIPT2 and SrIPT9 belong to the AtIPT2- and AtIPT9-type tRNA-IPT clades respectively. SrIPT1 is closely related to adenosine phosphate-IPT AtIPT1, AtIPT4, AtIPT6, and AtIPT8, and SrIPT3 and SrIPT5 to AtIPT3, AtIPT5, AtIPT7. Bootstrap values (N50%) of 1000 replicates are shown along the branches. Clade I: AtIPT9 type tRNA-IPT; Clade II: AtIPT2 type tRNA-IPT; Clade III: adenosine phosphate-IPT. Accession numbers: Arabidopsis thaliana: AtIPT1 (AB062607), AtIPT2 (AB062609), AtIPT3 (AB062610), AtIPT4 (AB062611), AtIPT5 (AB062608), AtIPT6 (AB062612), AtIPT7 (AB062613), AtIPT8 (AB062614), ATIPT9 (AB062615); Solanum lycopersicum: SlIPT1 (AB690812), SlIPT2 (AB690813), SlIPT3 (AK329766), SlIPT5 (NM_001257987), SlIPT6 (AK324787); Oryza sativa: OsIPT1 (AB239797), OsIPT2 (AB239798), OsIPT3 (AB239799), OsIPT4 (AB239800), OsIPT5 (AB239801), OsIPT6 (AB239803), OsIPT7 (AB239804), OsIPT8 (AB853903), OsIPT9 (AB239806), OsIPT10 (AB239807); Zea mays: ZmIPT1 (AC216794), ZmIPT2 (AC210040), ZmIPT3 (AC209075), ZmIPT3b (AC192238), ZmIPT4 (AC194228), ZmIPT5 (AC202968), ZmIPT6 (AC206179), ZmIPT7 (AC183318), ZmIPT8 (AC203907), ZmIPT9 (AC209018), ZmIPT10 (AC208425); Pseudomonas putida MIAA_PSEPU (O30762); Escherchia coli MIAA_ECOLI (WP_032208303); Agrobacterium tumefaciens complex MIAA_RHIAD (WP_010972016); Saccharomyces cerevisiae: MOD5_YEAST (NM_001183693); Schizosaccharomyces pombe tit1_SCHPO (NM_593436); Drosophila melanogaster DmtRdim (ACV91664); Mus musculus Trit1_MOUSE (AAH51040); Homo sapiens TRIT1_HUMAN (NP_060116.2).

Continuous cytokinin treatment is not necessary for inducing and maintaining two macrocotyledons, but the developmental stage at which seedlings are treated appears to be crucial, since the BAP

treatment for three days at the isocotylous stage (Nishii et al., 2004) or single treatment during germination (Rosenblum and Basile, 1984) was sufficient to induce two macrocotyledons. Moreover, Tsukaya


103

Fig. 7. Realtime-PCR expression patterns of SrIPTs in the vegetative and reproductive phase in various organs, including roots. Absolute gene copy numbers were calculated from the plasmid controls. 1, seedlings 35 days after cotyledon unfolding (DAU); 2, macrocotyledons of 3 months old plants; 3, lamina and 4, midrib of young phyllomorph (5 cm in length); 5, lamina and 6, midrib of a fully expanded phyllomorph (20 cm in length); 7, flower buds; 8, open flowers; 9, developing fruits; 10, peduncles; 11, mature phyllomorph with inflorescences removed; 12, roots.

(1997) demonstrated that in Monophyllaea, another anisocotylous genus in Gesneriaceae, the two cotyledons are physiologically equivalent during germination and both are able to become macrocotyledons. Their fate is determined during early development

stages and later on becomes irreversible. We suggest that cytokinin treatment during the anisocotyly determination phase leads to an abolishment of the suppression of the microcotyledon by the macrocotyledon.

Fig. 8. Distribution of SrIPT5 (a) and SrIPT9 (b) in the phyllomorph. RNA was extracted separately from proximal and distal parts of young phyllomorphs (5 cm in length) and fully expanded phyllomorphs (20 cm in length) respectively. Plasmids containing each IPTs were used as respective controls, and the expression level of 18S rRNA was used to normalize the result between samples. SrIPT5 expression was localized in the proximal region of developed, but not in young phyllomorphs. SrIPT9 expression was observed in both, proximal and distal parts, but was higher in distal parts in fully expanded phyllomorphs.

104


a

IPT5

b

IPT5

c

IPT9

d

IPT9

f

IPT9 sense

e

IPT5 sense

Fig. 9. In-situ hybridization expression patterns of SrIPT5 and SrIPT9 in 3 months old cotyledonary phyllomorphs. a, c. Transverse section through the proximal part of the phyllomorph showing the groove meristem. b, d. Magnified view of a, c. a–b. SrIPT5. c–d. SrIPT9. e, f. Negative controls hybridized with sense transcribed probes. SrIPT5 expression was strongly detected in internal layers of the groove meristem, and SrIPT9 follow this pattern. Bars: 100 μm.

Fig. 10. Effect of hormone treatments on SrIPT5 (a), SrIPT9 (b) expression levels. Seedlings 35 days after cotyledon unfolding (DAU) were treated with 10−5 M NAA, 10−4 M BAP, 10−5 M GA3, and DMSO for control respectively, for 7 days. The expression levels of above-ground parts were compared between control and hormone treated seedlings. NAA treatment inhibited the expression level of both of SrIPT5 and SrIPT9, whereas GA3 only inhibited the expression level of SrIPT9. BAP did not have an effect on the expression levels. *:0.01 b P b 0.05,**: P b 0.01.

11.07 ± 0.92 9.96 ± 1.35 9.64 ± 0.77 8.29 ± 0.44 P = 0.741 P = 0.109 0.25 0.24 0.35 0.26 P= P= 0.56 0.38 0.39 0.39 P= P= 0.19 0.14 0.07 0.22 P= P= N.D. N.D. N.D. N.D. – – Statistics

Root pMac vs dMac pMac vs mic

Distal part Proximal part

* 0.001 b P b 0.05; ** 0.0001 b P b 0.001. †pMac: Macrocotyledon proximal part, dMac: Macrocotyledon distal part, mic: microcotyledon, tZ: trans-zeatin; tZR: tZ riboside, tZRPs: tZR 5′-phosphates, cZ: cis-zeatin, cZR: cZ riboside, cZRPs: cZR 5′phosphates, DZ: dihydrozeatin, DZR: DZ riboside, DZRPs: DZR 5′-phosphates, iP: N6-(Δ2-isopentenyl)adenine, iPR: iP riboside, iPRPs: iPR 5′-phosphates.

DZRPs

N.D. N.D. N.D. N.D. – – N.D. N.D. N.D. N.D. – –

DZR DZ

N.D. N.D. N.D. N.D. – – ± 0.18 ± 0.22 ± 0.08 ± 0.15 0.180 0.061

cZRPs cZR cZ

± 0.18 ± 0.21 ± 0.10 ± 0.11 0.238 0.072 0.54 0.44 0.25 0.72 P= P=

tZRPs

± 0.04 ± 0.03 ± 0.02 ± 0.01 0.333 0.254 0.08 0.07 0.05 0.12 P= P=

tZR

± 0.09 ± 0.01 ± 0.02 ± 0.05 0.395 0.190 tZ

0.18 0.11 0.09 0.19 P= P= Microcotyledon Macrocotyledon Cytokinin concentration

Table 1 Measurement of cytokinin molecules in cotyledon tissue of Streptocarpus rexii. Cytokinin concentration as pmol g−1 FW (n = 3).

± 0.07 ± 0.05 ± 0.02 ± 0.01 0.101 0.056

0.46 0.38 0.16 0.61 P= P=

iP

± 0.08 ± 0.04 ± 0.02 ± 0.12 0.338 0.022⁎

iPR

± 0.02 ± 0.01 ± 0.03 ± 0.06 0.005⁎ 0.012⁎

iPRPs


105

4.2. Tissue-specific expression of SrIPT genes The cytokinin biosynthesis gene tRNA-IPT SrIPT2 isolated from S. rexii lacked the functional domain and may be a pseudogene. This is not unusual, and IPT pseudogenes have been found in other plants, including A. thaliana (Miyawaki et al., 2004), rice (Sakamoto et al., 2006), and tomato (Matsuo et al., 2012). Since redundancy has been shown among IPT genes, tRNA-IPT SrIPT9, might compensate for a lacking functional SrIPT2 (see Miyawaki et al., 2006), which might be reflected in the relatively high expression level of SrIPT9 (Fig. 7), though there is no direct evidence at present. The SrIPT gene expression patterns resemble those of A. thaliana, where IPTs show differential expression patterns (Miyawaki et al., 2004). SrIPT1 was primarily detected in floral organs, SrIPT3 mainly in roots, even if low expression was detected in seedlings with the lower part of hypocotyls and roots excluded. This is likely due to the ability of hypocotyls to develop adventitious roots. SrIPT5 and SrIPT9 were detected ubiquitously. These expression patterns are comparable to the orthologous AtIPTs. SrIPT1 grouped with AtIPT1, AtIPT4, AtIPT6, AtIPT8 and shares a similar transcription profile in reproductive organs, though AtIPT1 is also expressed in vegetative tissues and roots. SrIPT3 is expressed in roots and SrIPT5 ubiquitously. Both fell in the clade including AtIPT3, AtIPT5 and AtIPT7 (Fig. 6): AtIPT3 is expressed in the phloem, AtIPT5 present in the root apical meristem (RAM), stems, and fruit abscission zones, and AtIPT7 in trichomes of young leaves and the endodermis of the root elongation zone (Miyawaki et al., 2004). The ubiquitously expressed tRNA-IPT SrIPT9 clustered with AtIPT9 which has a similar ubiquitous expression (Miyawaki et al., 2004). The similarities of the IPT gene expressions of A. thaliana and S. rexii, including redundancies and some conservation of function, may suggest a promoter sequence similarity and conserved regulation mechanisms. This could be due to their shared evolutionary pathway of IPT gene duplication. 4.3. Role of cytokinin on S. rexii meristem activity In A. thaliana, the regulation of the SAM is tightly linked with cytokinins since IPT gene mutants have a reduced size of the SAM (Miyawaki et al., 2006). Moreover, the IPT genes in A. thaliana play different roles in meristem and organ formation. For instance, AtIPT7 is strongly expressed when the KNOX1 gene SHOOTMERISTEMLESS is overexpressed (Yanai et al., 2005). On the other hand, the nuclear protein AS1-AS2 complex and its modifiers, interact negatively with KNOX1 (Guo et al., 2008), down-regulating the expression of AtIPT3, but not AtIPT5 or AtIPT7 (Takahashi et al., 2013). Streptocarpus rexii has an unusual plant architecture without a typical SAM but possesses intercalary meristems in the proximal region of the phyllomorph. Two SrIPTs are expressed in the phyllomorph, SrIPT5 and SrIPT9. However, it cannot be ruled out that other SrIPT genes are indirectly involved, since cytokinin can be translocated (Kudo et al., 2010). High cytokinin concentrations are important for meristem maintenance (Shani et al., 2006; Veit, 2009; Murray et al., 2012) and it could be hypothesised that higher levels of cytokinins are established in the proximal region of the S. rexii phyllomorph where the meristems are located and lower levels in the distal region. However, we could not find such a cytokinin gradient (Table 1), which is in accordance with a previous study on mature Streptocarpus phyllomorphs (Van Staden, 1973). Since cytokinin levels and signalling respond to environmental factors (Takei et al., 2004; Schäfer et al., 2015) and have physiological roles, such as maintaining chlorophyll (Talla et al., 2016), not all cytokinins in the phyllomorphs might be directly linked to meristem formation and its maintenance. Although we found cytokinins throughout the phyllomorph, the location of maximum cytokinin biosynthesis is important for meristem localization and development (Jasinski et al., 2005). In the phyllomorph, SrIPT5 and IPT9 are expressed strongly in the groove meristem (Fig. 9a–d), suggesting that they are involved in maintaining high levels of cytokinin in the groove meristem. However, some indirect evidence indicates that

106

Y.-Y. Chen et al. / South African Journal of Botany 109 (2017) 96–111 Table 2 Summary of hormonal responses of IPTs in Streptocarpus rexii and diverse model plants.

Species

Genes

Chemical

Material

Result

Reference

Streptocarpus rexii

SrIPT5

A

NAA

Seedling

Down

This study

Streptocarpus rexii

SrIPT5

CK

BAP

Seedling

Down*

This study

Streptocarpus rexii

SrIPT5

GA

GA3

Seedling

No change

This study

Streptocarpus rexii

SrIPT9

A

NAA

Seedling

Down

This study

Streptocarpus rexii

SrIPT9

CK

BAP

Seedling

Down*

This study

Streptocarpus rexii

SrIPT9

GA

GA3

Seedling

Down

This study

Arabidopsis thaliana

AtIPT1

A

IBA

Root

No change

Miyawaki et al., 2004


AtIPT1

CK

BAP

Root

Down



AtIPT1

CK

BA

Seedling

No change

Brenner et al. 2005


AtIPT2

A

IBA

Root

No change



AtIPT2

CK

BAP

Root

No change



AtIPT3

A

IBA

Root

No change



AtIPT3

CK

BAP

Root

Down



AtIPT3

CK

BA

Seedling

No change

Brenner et al. 2005


AtIPT5

A

IBA

Root

Up



AtIPT5

CK

BAP

Root

Down



AtIPT7

A

IBA

Root

Up



AtIPT7

CK

BAP

Root

Down



AtIPT7

CK

BA

Seedling

No change

Brenner et al. 2005


ATIPT8

CK

BA

Seedling

No change

Brenner et al. 2005


AtIPT9

A

IBA

Root

No change



AtIPT9

CK

BAP

Root

No change



AtIPT9

CK

BA

Seedling

Down

Brenner et al. 2005

A

NAA

Root

No change

Ando et al. 2005

CK

BAP

Root

Up

Ando et al. 2005

BrIPT1 Brassica rapa (BrIPT1-2) BrIPT1 Brassica rapa (BrIPT1-2) Brassica rapa

BrIPT2

CK

BAP

Leaf

Up*

Liu et al. 2013

Brassica rapa

BrIPT3-1

CK

BAP

Leaf

Down

Liu et al. 2013

Brassica rapa

BrIPT3

A

NAA

Root

Down

Ando et al. 2005

CK

BAP

Root

Down

Ando et al. 2005

(BrIPT3-2) BrIPT3 Brassica rapa (BrIPT3-2) Brassica rapa

BrIPT3-2

CK

BAP

Leaf

Down

Liu et al. 2013

Brassica rapa

BrIPT5-1

CK

BAP

Leaf

No change

Liu et al. 2013 (continued on next page)


107

Table 2 (continued)

Species

Genes

Chemical

Material

Result

Reference

BrIPT5 Brassica rapa

A

NAA

Root

Down

Ando et al. 2005

CK

BAP

Root

Down

Ando et al. 2005

A

NAA

Root

Down

Ando et al. 2005

CK

BAP

Root

Down

Ando et al. 2005

(BrIPT5-2) BrIPT5 Brassica rapa (BrIPT5-2) BrIPT7 Brassica rapa (BrIPT7-1) BrIPT7 Brassica rapa (BrIPT7-1) Brassica rapa

BrIPT7-1

CK

BAP

Leaf

Down

Liu et al. 2013

Brassica rapa

BrIPT8-2

CK

BAP

Leaf

Down

Liu et al. 2013

Brassica rapa

BrIPT9-1

CK

BAP

Leaf

No change

Liu et al. 2013

Brassica rapa

BrIPT9-2

CK

BAP

Leaf

Up*

Liu et al. 2013

Pisum sativum

PsIPT1

A

IAA

Stem

Down

Tanaka et al., 2006

Pisum sativum

PsIPT1

A

IAA

Root

Down

Tanaka et al., 2006

Pisum sativum

PsIPT2

A

IAA

Stem

Down

Tanaka et al., 2006

Pisum sativum

PsIPT2

A

IAA

Root

Down

Tanaka et al., 2006

Glycine max

GmIPT1

A

NAA

Leaf

Up*

Ye et al., 2006

Glycine max

GmIPT1

GA

GA

Leaf

Up

Ye et al., 2006

Up: up-regulated, Down: down-regulated, Up*: very mildly up-regulated and/or no statistic significance, Down*: very mildly down-regulated and/or no statistical significance. A: auxin, CK: cytokinin, GA: gibberellin.

SrIPT5 is the major IPT acting in the meristem: i) in A. thaliana, the adenosine phosphate-IPT mutant, atipt1 3 5 7, shows a reduced SAM activity which was not observed in the atipt2 9 tRNA-IPT mutant (Miyawaki et al., 2006); ii) a major cytokinin molecule for regulating shoot morphogenesis is tZ, a downstream product of adenosine phosphate-IPT (Kiba et al., 2013); iii) SrIPT5 belongs to the same group as AtIPT3 and AtIPT7, that have been shown to interact with meristem regulating genes (Yanai et al., 2005; Takahashi et al., 2013); iv) SrIPT5, but not SrIPT9, is stable under GA3 treatment (Fig. 10). Previous work has shown that GA3 treatments suppressed the basal meristem but not the groove meristem in S. rexii (Mantegazza et al., 2009; Nishii et al., 2014). These aspects suggest that the adenosine phosphate-IPT SrIPT5 might have major roles in the groove meristem in S. rexii. Further studies are needed to reveal its role and signalling in this species. On the other hand, tRNA-IPT SrIPT9 might have different roles in the lamina of the phyllomorph. In A. thaliana, the tRNA-IPT double mutant (atipt2 9) was reported to be often chlorotic (Miyawaki et al., 2006). We found that GA treatments induce chlorosis in S. rexii, in the distal region of the phyllomorph (Fig. A3), and also inhibited the expression of SrIPT9. In a previous study on the phyllomorphic Streptocarpus molweniensis, the levels of cytokinin in the distal part of the lamina increase in summer and decrease in autumn, which was suggested to be linked to the formation of abscission zones and the senescence of the distal part of the lamina (Van Staden, 1973). This suggests that SrIPT9 might be involved in preventing chlorosis of the distal part of the phyllomorph, perhaps through the known function of cytokinin to induce chlorophyll production (Fletcher and McCullagh, 1971; Talla et al., 2016). Interestingly, we detected both iP-type cytokinins and tZ-type cytokinins in S. rexii seedlings, but no tRNA-IPT-type cZ, although it is found in A. thaliana and rice (Sakakibara, 2006). On the other hand, the

precursors of cZ (cZR and cZRMP) were found in S. rexii. Thus, SrIPT9, a tRNA-IPT which is involved in the synthesis of cZ-type cytokinins, might produce as final product cZR, a weakly active cytokinin form. A possible explanation is that, the catalysation of cZR → cZ is very low in S. rexii, or cZ is metabolized rapidly. DZ, DZR and DZRMP were also not detected in S. rexii seedlings, and the steps catalysed by zeatin reductase (Martin et al., 1989) might be low in activity here. 4.4. Phytohormone regulate the expression of SrIPT We found that the expression levels of SrIPT5 and SrIPT9 in S. rexii seedlings were differently affected by external hormone treatments. Such differential effects were observed in previous work on IPT genes, although with sometimes opposing responses (summarized in Table 2); i.e. in A. thaliana, auxin does not affect AtIPT1, AtIPT2, AtIPT3, AtIPT8, and AtIPT9, but up-regulate AtIPT5 and AtIPT7 (Miyawaki et al., 2004; Brenner et al., 2005). In S. rexii, auxin reduced SrIPT5 and SrIPT9. It is clear that the responses to hormones have diversified between species. For instance, in Brassica rapa, closely related to A. thaliana, auxin inhibited the expression of BrIPT3–2, BrIPT5–2, and BrIPT7–1 (Ando et al., 2005; Table 2). It was shown that hormones regulate IPT's expression through ciselements: IAA inhibited the expression of PsIPTs, interacting with the IPT promoter in Pisum sativum (Tanaka et al., 2006; Table 2), and GA interacted with the IPT promoter in Glycine max (Ye et al., 2006; Table 2). It appears that during gene duplication and differentiation processes, cis-elements of IPT genes diversified and established unique regulatory networks for each copy that can be individual for each species. Such a flexible system might be necessary to coordinate the different events occurring at the same time in different plant organs and tissues, where cytokinin concentration has to be finely tuned.

108


4.5. Conclusion In this study, we isolated cytokinin biosynthesis IPT genes from S. rexii. Each SrIPT paralog had unique expression patterns, and among these, SrIPT1 was expressed in flower buds and flowers, SrIPT3 predominantly in roots, and SrIPT5 and SrIPT9 in the groove meristem and throughout the phyllomorph. Comparisons with IPTs characterized in model plants allowed us to hypothesise that adenosine phosphate-IPT SrIPT5 is the cytokinin biosynthesis candidate gene for meristem formation and maintenance in S. rexii. The tRNA-IPT SrIPT9 might be involved in different aspect of plant growth such as the prevention of lamina senescence in S. rexii. The differentiation of the cytokinin biosynthesis gene paralogs during evolution and their fine tuning with different cis-elements might have contributed to the diversification of morphological traits such as the unique morphology in S. rexii. Funding This work was supported by a Taiwan-Italy Scientific Research Cooperation grant from the National Science Council in Taiwan (NSC, Taiwan)

and National Research Council in Italy (CNR, Italy) [grant Number 99– 2923-B-002-007-MY2]; the NSC funding [grant number NSC 101– 2811-B-002-150]; the Ministry of Science and Technology, Taiwan [grant number 102–2621-B-002-002]; the Sibbald Trust at Royal Botanic Garden Edinburgh (RBGE, UK); and the JSPS KAKENHI [grant number 15K18593]. Acknowledgement We thank M. Kawaguchi (National Institute of Basic Biology, Japan) and G.-J. Wu (South China Botanic Garden, China) for helpful comments and technical supports at the initial stage of this work, T. Suzaki (Tsukuba University, Japan) for supporting SEM work. We thank K.-J. Tang and Y.-Y. Gao in TechComm at National Taiwan University (NTU) for technical support and enabling access to realtime-PCR facilities, and Yen-Wei Chou and Meng-Jung Ho (NTU) for technical support and helpful comments on this work. We thank the Science Division of RBGE for supporting this work. RBGE is supported by the Rural and Environment Science and Analytical Services division (RESAS) in the Scottish Government.

Appendix A

b

c

d

e

f

g

h

i

a

Fig. A1. Materials used for expression analysis of SrIPTs, with dashed lines marking the cutting sites. Arrows indicate the samples used. a. Seedlings 35 days after cotyledon unfolding (DAU). b. Macrocotyledon of 3 months old seedling. c. Young phyllomorph 5 cm in length. d. Fully expanded phyllomorph 20 cm in length. e. Peduncle. f. Flower bud. g. Fully open flowers. h. Flowering phyllomorph (inflorescences were removed prior to RNA extraction). i. Developing fruit. Bars =1 cm.


109

Fig. A2. DNA sequence and its deduced amino acid sequence of SrIPT2.

a

b

Fig. A3. GA3 treatment induces chlorosis in Streptocarpus rexii seedlings. Seedlings were grown on soil for 3 months and moved to filter paper containing DMSO or GA3. a. Control seedlings treated with DMSO. b. Seedlings treated with 3 × 10−3 M GA3 for 1 h. Bars = 1 cm.

110


Table A1 List of primers used in this study. Method

Gene

3′RACE

Table A3 (continued) S. rexii IPT

Forward/reverse Primer sequence (5′-3′)

SrIPT1 SrIPT2 SrIPT3 SrIPT5 SrIPT9 Adaptor Inverse PCR SrIPT1 SrIPT1 TAIL PCR Adaptor SrIPT3 SrIPT3 SrIPT3 Intron SrIPT2 isolation SrIPT2 SrIPT2 SrIPT2 SrIPT9 SrIPT9 SrIPT9 SrIPT9 SrIPT9 SrIPT9 Realtime PCR SrIPT1 SrIPT1 SrIPT3 SrIPT3 SrIPT5 SrIPT5 SrIPT9 SrIPT9 18S rRNA 18S rRNA ISH probes SrIPT5 SrIPT5 SrIPT9 SrIPT9

Forward Forward Forward Forward Forward Reverse Forward Reverse Forward Reverse Reverse Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

TGGACTCCGCCTTCAGTAAG GAGTGCCGCATCATCTGCTTGG GAGTGCCGCATCATCTGCTTGG GTGGAATCCGGCGTGCTATC CAGAGAACAAGGGAGCTGGA GACTCGAGTCGACATCG TGGACTCCGCCTTCAGTAAG TCTGGGTTGTATTTCTTCGCCA AGWGNAGWANCAWAGG CCTCCACGGAAACTGCGCTGCCACCGTAA ACGTCGGTGGCGTCGAGGCGGTGTAGGT AGCGGCGGAGAAGTCGGATTCGGGGTCA CCGGATGCAGACGAGAATTT CGCTGAAACTGGGTCAAGAT CCACTTCCCCATCGAAATTA AGCTTCGCATCTTCCTCAAA CATGCTCAAACGGAGAAAGG GGGTAATGGAAAGGCTGATG AGCAGGTGATCCAGGTGTTC TCCAGCTCCCTTGTTCTCTG CAGAGAACAAGGGAGCTGGA GCAAGTGCCAGAACTTCTTTACC CAGTCGAACCCTGTCAGCAAA GGACCCGAGATTCGCCATTAT CCGCCAGGTGGAGAAGATTAT TCGACATCCTTGCCGCTTT GCTTTCCTCAAACTTGGCGAC GCGAGAACATTGGTGTGGTGT GAGGCACAGTGGCTTCTTGA CCCTTGTTCTCTGCACGCTA TGACGGAGAATTAGGGTTCGA GGATGTGGTAGCCGTTTCTCA CGGAAAGACAAGGTGGTTGT CATAGGAATTCGACCCTCCA CAGAGAACAAGGGAGCTGGA ATGCTGCCACAGTCCTCTCT

Table A2 Partial sequences of SrIPT genes were inserted into pGEMt-easy vectors (Promega) and molecular weights calculated for the estimation of copy numbers per weight. Plasmids were used as controls in realtime-PCR, and thus the expression levels are comparable between SrIPT genes. Gene

Plasmid Molecular Weight per Copy number Plasmid (ng) for size (bp) weight (Da) plasmid (ng) per ng 25 × 107 copies

SrIPT1 SrIPT3 SrIPT5 SrIPT9 18S rRNA

3323 3180 3327 3624 3073

2,193,180 2,098,800 2,195,820 2,391,840 2,028,180

3.64 3.48 3.64 3.97 3.26

× × × × ×

10−9 10−9 10−9 10−9 10−9

274,578,466 286,925,862 274,248,345 251,772,694 306,679,792

0.91 0.87 0.91 0.99 0.82

Table A3 Comparison of deduced amino acid sequences between Streptocarpus rexii IPT genes (SrIPT) and Arabidopsis thaliana IPT genes (AtIPT), using SIAS (http://imed.med.ucm.es/ Tools/sias.html). AtIPT genes showing the highest value of identity and similarity are highlighted in bold. S. rexii IPT

A. thaliana IPT

Identity

Similarity

SrIPT1

AtIPT1 AtIPT2 AtIPT3 AtIPT4 AtIPT5 AtIPT6 AtIPT7 AtIPT8 AtIPT9 AtIPT1 AtIPT2 AtIPT3 AtIPT4 AtIPT5

51.00% 34.29% 38.98% 48.00% 45.45% 44.34% 40.00% 44.10% 31.03% 35.89% 53.84% 34.61% 35.04% 37.13%

59.34% 34.37% 45.47% 48.95% 46.73% 58.82% 46.41% 56.14% 30.51% 34.45% 36.97% 34.48% 32.94% 36.71%

SrIPT2

SrIPT3

SrIPT5

SrIPT9

A. thaliana IPT

Identity

Similarity

AtIPT6 AtIPT7 AtIPT8 AtIPT9 AtIPT1 AtIPT2 AtIPT3 AtIPT4 AtIPT5 AtIPT6 AtIPT7 AtIPT8 AtIPT9 AtIPT1 AtIPT2 AtIPT3 AtIPT4 AtIPT5 AtIPT6 AtIPT7 AtIPT8 AtIPT9 AtIPT1 AtIPT2 AtIPT3 AtIPT4 AtIPT5 AtIPT6 AtIPT7 AtIPT8 AtIPT9

33.05% 36.32% 36.32% 29.05% 48.57% 38.92% 50.31% 42.22% 57.91% 42.40% 49.36% 47.16% 30.37% 44.07% 38.48% 51.97% 36.84% 57.23% 40.13% 51.64% 41.77% 24.01% 26.89% 25.91% 27.67% 29.30% 27.48% 26.66% 27.79% 27.67% 58.83%

33.13% 34.13% 34.82% 22.19% 50.26% 36.98% 59.58% 48.76% 65.19% 49.03% 57.48% 54.87% 30.90% 45.95% 36.78% 56.03% 48.50% 64.04% 48.18% 59.88% 47.50% 27.58% 32.98% 36.61% 26.08% 30.10% 31.14% 25.63% 29.62% 26.08% 66.45%

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