The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis
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- Schwarz, S., Grande, A.V., Bujdoso, N. et al. Plant Mol Biol (2008) 67: 183. doi:10.1007/s11103-008-9310-z
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Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested.
KeywordsArabidopsisJuvenile phasemiRNAPhase changeSBP-box genesShoot maturation
Days after sowing
Quantitative real-time PCR
Shoot apical meristem
SQUAMOSA PROMOTER BINDING PROTEIN
SQUAMOSA PROMOTER BINDING PROTEIN-LIKE
During maturation, plants pass through several developmentally distinct growth phases in which the shoot gradually gains reproductive competence (Poethig 1990). After the transition from embryonic to postembryonic growth, plants undergo at least two further phase transitions, the vegetative as well as the reproductive phase change. During vegetative growth, rosette leaves are initiated at the flanks of the shoot apical meristem (SAM) with a certain frequency, referred to as plastochron (Erickson and Michelini 1957). After going through the reproductive phase transition, also known as the floral transition, the SAM starts to initiate floral buds instead of leaves. In Arabidopsis, as in many other plants showing day length dependent flowering, the floral transition is preceded by a transition from juvenile to adult growth. This switch, known as the vegetative phase change, is physiologically defined as achieving competence to respond to photoperiodic induction of flowering (Poethig 1990).
The transition from juvenile to adult growth is gradual and rather subtle but generally can be followed by several morphological markers. In Arabidopsis, for example, leaves produced in the juvenile phase have long petioles, are small, round and lack abaxial trichomes. In contrast, short petioles, elliptical anatomy and the development of trichomes on the abaxial side represent adult traits (Telfer et al. 1997).
Regulation of these developmental transitions is largely dependent on (changes in) environmental cues such as day length, light intensity and temperature, as well as on endogenous factors such as the plant hormone gibberellin (Telfer et al. 1997). Whereas the molecular genetic mechanisms underlying the floral transition are already worked out in increasing detail (Komeda 2004), it has only been recently that we begin to understand the molecular genetic basis of the vegetative phase change. Most genes suggested to play a role in promoting the latter phase change have been identified by the analysis of mutants showing a precocious onset of adult traits and intriguingly, link vegetative phase change to RNA silencing pathways. These genes include the Arabidopsis ortholog of exportin 5/MSN5, HASTY (HST; Telfer and Poethig 1998; Bollman et al. 2003), the zinc-finger-domain protein encoding locus SERRATE (SE; Clarke et al. 1999) and ZIPPY (ZIP), an AGO-family member (Hunter et al. 2003). More recently, screens for mutations with zip-like phenotypes resulted in alleles of SUPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT POLYMERASE6 (RDR6), both genes required for posttranscriptional gene silencing (PTGS) and acting in the same pathways as ZIP and HST (Peragine et al. 2004). Furthermore, a precocious vegetative phase change has also been found in dicer-like 4 (dcl4) mutants (Gasciolli et al. 2005; Xie et al. 2005; Yoshikawa et al. 2005).
One explanation for the observed effects could be that target genes of this silencing pathway play a positive role on the vegetative phase change and their down-regulation consequently promotes juvenility. Hence, mutations in genes involved in this silencing pathway, as the ones described above, cause an accelerated vegetative development.
In line with this idea, members of the plant specific SBP-box gene transcription factor family have been implicated in promoting vegetative and floral phase transitions. In particular, overexpression of the Arabidopsis SBP-box gene SPL3 leads to early flowering and a significant earlier appearance of abaxial trichomes on the rosette leaves (Cardon et al. 1997; Wu and Poethig 2006). Interestingly, together with 10 of 16 other family members, SPL3 expression is post-transcriptionally controlled by miR156 and probably also by the very closely related miR157 (Rhoades et al. 2002; Schwab et al. 2005; Wu and Poethig 2006; Gandikota et al. 2007). Consistent with its role of down-regulating SPL3 and related SPL target-genes, constitutive overexpression of miR156 encoding loci has been shown to cause the production of a significantly larger number of leaves with juvenile characteristics and a delay in flowering (Schwab et al. 2005; Wu and Poethig 2006).
Although the available data clearly point to a regulatory role for the miRNA regulated SPL genes in the temporal development of the Arabidopsis shoot, the contribution of the single genes to the described phenotypes remains to be determined. Therefore, we identified and isolated mutant alleles for single SPL genes. In comparison to other miR156 targeted SPL genes, available expression data (AtGenExpress; Schmid et al. 2005) show SPL9 and SPL15 to be already quite active in the vegetative shoot apex. Accordingly, their mutant phenotypes were found to affect vegetative development. Here we report the mutant analysis of SPL9 and SPL15, two likely paralogous members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor family (Cardon et al. 1999), and discuss their redundant regulatory role on the vegetative phase change and the temporal initiation of rosette leaves.
Materials and methods
Plant material and plant growth conditions
All of the genetic stocks described in the paper were in Columbia background. The T-DNA insertion lines SALK_006573 (spl9-2), SALK_074426 (spl15-1) and SALK_138712 (spl15-2) were obtained at the Nottingham Arabidopsis Stock Centre (NASC). The T-DNA insertion lines GABI-Kat 544F04 (spl9-3) and WiscDsLox 457 (spl15-3) were obtained from GABI-Kat and the Arabidopsis Biological Research Centre (ABRC), respectively. Insertion mutant information for NASC- or ARBC-lines was obtained from the SIGNAL website at http://signal.salk.edu. Plants homozygous for the T-DNA insertions were identified by PCR using T-DNA left border- and gene-specific primers. T-DNA specific left border primers for SALK, GABI-Kat and WiscDsLox T-DNAs were 5′-GCGTGGACCGCTTGCTGCAACT-3′, 5′-ATATTGACCATCATACTCATTGC-3′ and 5′-TGGCAGGATATATTGTGGTGTAAACA-3′, respectively. In combination with the respective left border primer we used the following gene-specific primers: 5′-GCTATGGCTTAAGCCTTAAGTTAAAAGG-3′ for SALK_006573, 5′-CGTAGCTGTCGTGGACTAGTGTCAATC-3′ for SALK_074426 and SALK_138712, 5′-AACCTCTGTTCGATACCAGCCACAG-3′ for GABI-Kat 544F04 and 5′-AGCCATTGTAACCTTATCGGAGAATGAG-3′ for WiscDsLox 457. The stable En-1 insertion mutant 5ABA33-H1 (spl9-1) was obtained from the ZIGIA-population (Unte 2001). Plants homozygous for a four base pair insertion in the first exon of SPL9 caused by the excision of the En-1 transposon were backcrossed with wild type twice to obtain plants exclusively containing the four base-pair insertion without any further transposon contamination. In order to identify plants containing the mutation we used the following primer combination: 5′-AGTAAGAGGAAACCACCATGGAGATGG-3′ (forward) and 5′-AACCTTCCACTTGGCACCTTGGTATA-3′ (reverse, recognises the insertion).
All plants were grown in plastic trays or pots filled with ready-to-use commercial, pre-fertilized soil mixture (Type ED73, Werkverband eV, Sinntal-Jossa, Germany). For stratification, seeds were kept on moist paper at 4°C in the dark for 4–5 days before transferring to soil (i.e. “sowing”) in growth chambers at 22°C, 50% relative humidity. Germination and cultivation of the plants in long-day conditions (16 h light, 8 h dark) were either under approx. 70 μE/cm2/s (LD1) or 175 μE/cm2/s (LD2) light provided by fluorescent tubes (L58W/840 and L58W/25 Osram, Munich, Germany). Plants in short-day (SD) conditions (8 h light, 16 h dark) were cultivated under approx. 450 μE/cm2/s light. To determine sensitivity to photoperiodic induction of flowering, stratified seeds were germinated in a modified SD with a 9 h light period. The developing plants were kept in these conditions for 21 days before they were transferred to similar growth chambers with continuous light provided by Osram HQIT 400 W lamps. Batches of plants were returned to the modified SD conditions after 1, 3 and 5 days.
Flowering time was measured as the time between sowing and anthesis (opening of first flower). Bolting time was recorded when the main inflorescence had reached a height of 0.5 cm. Inflorescence height was measured between the rosette and the first flower of the main inflorescence of plants with their first siliques fully ripened. In order to count the number of site shoots, all site shoots longer than 0.5 cm were scored. Abaxial trichomes were scored using a Leica MZFLIII stereomicroscope (Wetzlar, Germany). An estimation of the rosette leaf initiation rate (L/D day−1) was obtained by dividing the number (L) of rosette leaves having reached at least 0.5 cm in length through the number of days (D) between sowing and determination. Note that this value reflects but does not equalize (average) plastochron as it should be corrected for the true start of initiation of the first leaf as well as the time needed for the first adult leaf to reach a size of 0.5 cm.
Apical regions were isolated from plants grown in SD for 41 days by trimming with a razor blade. The tissue was fixed in 4% formaldehyde/0.1 M PO4, pH 7.0 for 48 h and embedded in paraffin using a Leica ASP300 tissue processor (Wetzlar, Germany). Embedded apices were cut into 8 μm thin cross sections using a Jung Autocut 2055 and photographed using a Zeiss Axiophot microscope (Göttingen, Germany) equipped with a KY-F5U 28CCD camera (JVC, Yokohama, Japan). The first cross section in which the apex was visible plus two successive sections were used to determine the diameter of the apical region. From these three measured values the average was taken for comparison. In addition, cross sectional area and circularity factor (=4π A P−2, A is area, P is perimeter) of the leaf primordia, outlined by hand on the photographs, were determined as well. The measurements were performed with help of the program ImageJ 1.35s (Wayne Rasband, National Institutes of Health, USA).
Col-0, the spl9 spl15 double mutant and the 35S::MIR156b overexpressor were grown in LD1 conditions. Immediately after germination, half of the plants were treated by spraying 100 μM GA3, 0.02% Tween 20 and this was repeated twice per week until they started flowering. The other half of the plants was similarly treated with 0.02% Tween 20.
Multiple alignments of amino acid sequences were generated by the program ClustalW of the MacVector 7.2.2 software package (Accelrys Ltd., Cambridge, UK) using the BLOSUM 30 matrix with an open gap penalty of 10 and an extend gap penalty of 0.05. Only the SBP-domain was used for the phylogenetic reconstruction. The tree was constructed using the neighbour-joining algorithm of the MacVector 7.2.2 software package.
Quantitative real-time PCR analysis
To perform quantitative RT-PCR (using the iQ5 real-time PCR detection system, Bio-Rad, Munich, Germany) apical regions were collected (roots and as much of the leaves as possible were removed using tweezers) of plants cultivated 5, 9, 13, 27 and 32 days after sowing in LD1 conditions. Total RNA was extracted using the RNeasy plant mini kit (Qiagen, Hilden, Germany), including an on-column DNase digestion. First-strand cDNA was synthesized using SuperScript III RNase H reverse transcriptase (Invitrogen) starting with 2 μg of total RNA primed with an oligo(T)12–18 primer (Gibco BRL, Karlsruhe, Germany). SPL9-specific primers, 5′-AGAACATTGGATACAACAGTGATGAGG-3′ (forward) and 5′-GTTTGAGTCGCCAATTCCCTTGTAGC-3′ (reverse) as well as SPL15-specific primers, 5′-TTGGGAGATCCTACTGCGTGGTCAACC-3′ (forward) and 5′-AGCCATTGTAACCTTATCGGAGAATGAG-3′ (reverse), were designed to generate a PCR product of 171 and 300 bp, respectively. Based on the analysis of Czechowski et al. (2005) PP2A expression was used as reference for transcript normalization with the primer pair 5′-TAACGTGGCCAAAATGATGC-3′ (forward) and 5′-GTTCTCCACAACCGCTTGGT-3′ (reverse). The PCR efficiencies for SPL9, SPL15 and PP2A primers were determined to be 96, 95.5 and 96%, respectively. Quantifications, in triplicate, were performed using the Brilliant SYBRGreen QPCR kit (Stratagene, La Jolla, CA, USA), according to the manufacturer’s protocol, in a final volume of 25 μl. PCR was carried out in 250 μl optical reaction vials (Stratagene) heated for 10 min at 95°C to hot-start the Taq polymerase, followed by 40 cycles of denaturation (30 s at 95°C), annealing (30 s at 58°C) and extension (30 s at 72°C).
Semi-quantitative RT-PCR analysis
Total RNA was extracted from seedlings of the Col-0 wild-type, mutant- and transgenic lines using the RNeasy plant mini kits (Qiagen). RT-PCR with equal amounts of RNA was performed using the one-step RT-PCR kit (Qiagen). SPL9 knockout lines were identified using the following primer pair: 5′-GGTCGGGTCAGTCGGGTCAGATACC-3′ (forward) and 5′-ACTGGCCGCCTCATCACTCTTGTATCC-3′ (reverse). SPL9 mRNA is expected to yield a 415 bp fragment whereas genomic SPL9 DNA is expected to yield a 1,138 bp fragment. SPL15 knockout lines were identified using the following primer pair: 5′-AGAAGCAAGAACCGGGTCAATACC-3′ (forward) and 5′-AGCCATTGTAACCTTATCGGAGAATGAG-3′ (reverse). SPL15 mRNA is expected to yield a 666 bp fragment whereas genomic SPL15 DNA is expected to yield a 1,004 bp fragment. RT-PCR of the loading control (RAN3; At5g55190) was performed with the primer pair 5′-ACCAGCAAACCGTGGATTACCCTAGC-3′ (forward) and 5′-ATTCCACAAAGTGAAGATTAGCGTCC-3′ (reverse) to yield a fragment of 531 bp when derived from RAN3 mRNA (genomic RAN3 is expected to yield a 1,314 bp fragment).
Graphical representations of numerical data were generated with the Microsoft Excel program (Microsoft Germany, Munich) and statistical tests were performed using the Student’s t-Test within this program. P-values lower than 0.05 were considered to be statistically relevant and the data involved to represent significant differences.
Remaining techniques and methods
Standard molecular biology techniques were performed as described by Sambrook et al. (1989). Graphical plots and digital photographic images were cropped and assembled using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
Molecular characterization of the Arabidopsis SBP-box genes SPL9 and SPL15
Also three independent T-DNA insertion lines for SPL15 could be obtained and confirmed (see “Materials and methods”; Fig. 1a). Two alleles designated as spl15-1 and spl15-2 were identified within the SALK collection and one, spl15-3, within the WiscDsLox T-DNA collection.
According to data available from the AtGenExpress micro-array database (Schmid et al. 2005), both SPL9 and SPL15 transcript levels increase during development and are preferentially found in the shoot apical region and in young flowers. We confirmed this temporal expression pattern with the help of qRT-PCR (Fig. 1b). In LD1 growing conditions (see “Materials and methods”), SPL9 and SPL15 transcript levels remain comparable during the first 2–3 weeks. Thereafter, the expression level of SPL9 starts to increase followed by that of SPL15. Around 32 days after sowing (DAS), at about the time Col-0 plants have undergone their reproductive phase transition, SPL9 transcript levels have become approximately two and a half times higher in comparison to SPL15 and six times in comparison to day 5. Arabidopsis lines carrying as a transgene a genomic fragment encompassing the locus for SPL15 and with a GUS reporter gene inserted downstream of the ATG start codon, confirmed the predominantly apical expression of SPL15 (Supplementary Fig. 1).
RT-PCR performed on mRNA isolated from whole seedling plants homozygous for any of the three SPL9 or SPL15 mutant alleles (see “Materials and methods”) did not result in the detection of RNA derived of the respective genes (Fig. 1c). This strongly suggests that all mutant alleles isolated represent functional null-alleles. Accordingly, plants homozygous for any of the three spl9 mutant alleles showed highly identical phenotypes, as did all three homozygous spl15 mutants (see phenotypic analysis below; Supplementary Fig. 2). Allelic tests confirmed that the observed phenotypes are indeed due to mutation in either SPL9 or SPL15, respectively (data not shown).
Phenotypic analysis of spl9 and spl15 mutants
For a phenotypic analysis, we compared spl9 and spl15 single mutants, spl9spl15 double mutants to Col-0 wild type as well as to an 35S::MIR156b transgenic line (kindly provided by D. Weigel and R. Schwab).
Phenotypic evaluation of spl9 and spl15 mutant alleles in comparison to Col wt and a 35S::MIR156b transgene under LD conditions
Infloresc. heightb (cm)
Also with respect to the development of side shoots, the spl9spl15 double mutant differed more from wild type than the single mutants. In fact, spl9-1 and spl15-1 single mutants were found not to differ significantly from Col-0 plants that had formed, on average, 0.9 ± 0.6 side shoots of at least 0.5 cm in length by the time that the first siliques ripened. With an average of 2.1 ± 1.1 side shoots, the spl9spl15 double mutant did significantly differ from wild type as did the 35S::MIR156b transgenic line with, on average, 4.1 ± 0.8 side shoots. Taken together, the phenotypic data of the spl9spl15 double mutant clearly suggests a redundant function of SPL9 and SPL15 in shoot development and in the maintenance of apical dominance.
MiR156 is assumed to target, besides SPL9 and SPL15, exclusively other SPL genes (Rhoades et al. 2002). These too were shown to be down regulated in MIR156b over-expressing plants (Schwab et al. 2005). As in comparison to the spl9 spl15 double mutant the MIR156b over-expressor displays an even more severe aberrant phenotype, it can also be deduced that in addition to SPL9 and SPL15, other miR156-controlled SPL genes act redundantly to control shoot development and apical dominance.
In addition to the number of leaves formed before the appearance of the first flowers, we also determined for the same plants the time they needed to bolt as well as to anthesis (Table 1). On average, the spl9 and spl15 single mutants behaved similar to wild type but, as expected based on the data of Schwab et al. (2005), the 35S::MIR156b line bolted and flowered somewhat later. The spl9 spl15 double mutants showed an intermediate behaviour. Whereas for the single mutants the few more leaves formed may be accounted for by the slight delay in the transition to flowering, this delay is unlikely to explain the increased rosette leaf number of the spl9 spl15 double mutant. In line with the observation of Schwab and co-workers (2005) who reported a leaf-initiation rate per day in SD of 2.2 vs. 1.4 for the MIR156b overexpressor and the wild type, respectively, this is probably best explained by assuming a shortened plastochron during vegetative growth.
After sectioning the paraffin embedded material, a small but not significant difference in average SAM-diameter of the spl9spl15 double mutant and Col-0 wild type could be observed (Fig. 4b, e–f). However, with an average diameter of 104 μm, the MIR156b overexpressor showed also a slight but yet significant (P < 0.05) decrease in its SAM size compared to Col-0 (Fig. 4b, e, g). Furthermore, both the spl9spl15 double mutant and the MIR156b overexpressor exhibited the same phyllotaxy as wild type with rosette leaves initiated either clock- or anticlockwise with an angle of divergence of about 137.5° between successive leaves and forming a spiral lattice with a parastichy pair (3,5) (Fig. 4e–g). From these observations, it is concluded that the observed shorter plastochron is neither the result nor the cause of an altered phyllotaxy in the spl9spl15 double mutant or the MIR156b overexpressor. The shortened plastochron, however, seems to correlate with a reduced SAM size.
As obvious from cross sections shown in Fig. 4e, g, the young leaves of the MIR156b overexpressor appear more roundish in shape in comparison to wild type leaves at similar positions. In particular, the vacuolated cells surrounding their midveins seem larger and the developing laminas reduced, i.e. represented by less small cytoplasm rich cells along their lateral margins. In addition, the stipules of the MIR156b overexpressor seem to be more prominent. In these aspects of leaf development, the spl9spl15 double mutant seems to behave intermediate (Fig. 4e).
To quantify the difference in shape and size of the leaf primordia, we determined their circularity and cross sectional area (see “Materials and methods”) starting from the first leaf cross section found to be separated from the apical meristem. To reduce effects due to imperfect cross sectioning, i.e. not absolute perpendicular to the longitudinal axes, as well as in correlating sequentially numbered primordia of different sections, we averaged the values obtained over 10 successive primordia. As shown in Fig. 4c, circularity of the youngest 10 leaf primordia was highly similar between the different genotypes. Circularity of subsequent older primordia decreased in all, however, more rapidly in wild type such that, on average, leaf 10–20 did differ significantly between the genotypes. Interestingly, it cannot be excluded that this difference is a direct consequence of a shortened plastochron in the mutant lines. In particular as the leaf initiation rate of the MIR156b overexpressor line lies roughly one and a half times above that of wild type. Accordingly, and with respect to absolute age, leaf 11–20 of the MIR156b overexpressor may be more comparable to leaves 6–15 of wild type and to which indeed no significant difference in circularity was found. However, in cross sectional area these young leaves differed significantly. On average, 6.5 ± 2.4 × 103 μm2 for wild-type leaves 6–15 and 26.7 ± 16.2 × 103 μm2 for leaf 11–20 of the MIR156b overexpressor.
It is known that the shape and other characteristics of newly formed leaves progressively change in correlation with the vegetative phase transition (Telfer et al. 1997). Furthermore, likely due to a changed plastochron, the correlation between leaf number and flowering seems to differ for the mutants and wild type. Therefore, we further investigated the possibility that the observed differences correlate with relative altered timing of the vegetative phase transition.
Functional analysis of SPL9 and SPL15 during the vegetative phase transition
In order to determine the timing of the vegetative phase change we used the absence or presence of abaxial trichomes on rosette leaves as a morphological marker for leaves formed during the juvenile or adult growth phase, respectively (Telfer et al. 1997). On average, in our LD2 growing conditions, the first abaxial trichomes developed on rosette leaf number six of Col-0 wild-type plants (Table 1). The spl9spl15 double mutants displayed its first abaxial trichomes on leaf number twelve and the 35S::MIR156b overexpressor on leaf number 16. Although less than the spl9spl15 double mutant, the respective single mutants also developed significantly more juvenile leaves than wild type (Table 1).
Photoperiodic floral induction in wild type and spl mutants
Percentage of plantsa induced after treatment with continuous light for
The role of gibberellin in the function of SPL9 and SPL15
Transgenic plants constitutively over-expressing the plant specific miRNA156 have been described for Arabidopsis (Schwab et al. 2005; Wu and Poethig 2006) and rice (Xie et al. 2006). Recently, overexpression of a miR156 encoding locus has also been shown to be the cause of the natural maize mutant Corngrass1 phenotype (Chuck et al. 2007). Interestingly, in all three different species, overexpression of this well conserved miRNA (Axtell and Bartel 2005; Arazi et al. 2005) causes a similar phenotype suggesting an evolutionary conserved role for the function of miR156 and its SPL target genes. Generally, in comparison to the respective wild type, miR156 over-expressing plants are smaller, flower later, tend to lose apical dominance and initiate more leaves with a shorter plastochron. MiR156 targets eleven SBP-box genes in Arabidopsis but the results presented here clearly show that already simultaneous silencing of the two likely paralogous target genes, SPL9 and SPL15, well approximate the miR156 over-expressing phenotype regarding the traits mentioned above. SPL9 and SPL15 thus act as important and functionally redundant transcription factors regulating diverse processes in shoot maturation and most likely in combination with other miR156 regulated SPL genes. In agreement with this latter statement is our observation that in addition to spl9-1 and spl15-1, mutation of a third miR156 controlled gene, SPL2 (At5g43270; T-DNA insertion line SALK_022235), results in triple mutant plants showing an even better approximation to the MIR156b overexpressor phenotype (Supplementary Fig. 2; despite the absence of detectable SPL2 transcript, single homozygous spl2-1 mutant plants lack an obvious mutant phenotype, data not shown). In greenhouse LD conditions we found the triple mutant to have produced on average 17.3 ± 2.1 (n = 16) rosette leaves in comparison to 15.6 ± 2.6 for the spl9 spl15 double mutant. Col-0 wild-type and the MIR156b overexpressor plants grown in parallel produced 12.9 ± 1.7 and 22.5 ± 3.5 rosette leaves before flowering, respectively.
SPL9 and SPL15 positively regulate the juvenile-to-adult growth phase transition
It became clear with the detailed analysis of Arabidopsis MIR156b overexpressors by Wu and Poethig (2006) as well as with the description of the Corngrass1 mutant in maize by Chuck et al. (2007), that one of the major phenotypic alterations in miR156 over-expressing plants is an extended juvenile growth phase. This suggests that one of the important functions of miR156 targeted SBP-box genes is to promote the vegetative phase change. In agreement with this observation, Wu and Poethig (2006) showed that overexpression of the miRNA156 regulated gene SPL3 and its likely paralogs leads to a greatly shortened juvenile phase in Arabidopsis. Based on morphological markers (abaxial trichomes) and physiological parameters (response to photo-inductive stimulus) we found that the spl9spl15 double mutant exhibit a delayed vegetative phase transition and, therefore, conclude that both genes are very likely involved in the positive regulation of this developmental process in a redundant fashion. Most likely, because of this redundancy, photoperiodic induction of the single spl9 and spl15 mutants is not much affected. However, the effect on the appearance of abaxial trichomes as a marker for the juvenile-to-adult phase transition appears to be stronger in the spl9 mutant in comparison to spl15. This may be due to the fact that in shoot apical development expression of SPL9 starts to increase before that of SPL15 (Fig. 1b).
As SPL9 and SPL15 promote the juvenile-to-adult growth phase transition and thus competence to respond to photoperiodic induction of flowering, it is interesting to note that both SPL9 and SPL15 themselves are strongly upregulated in the shoot apex upon such induction (Schmid et al. 2003). An additional role for these genes in establishing inflorescence or floral meristem identity may thus be suggested.
SPL9 and SPL15 negatively regulate leaf initiation rate
Our data on the leaf initiation rate suggest that SPL9 and SPL15 act negatively on leaf initiation. Silencing of both genes leads to a shorter plastochron. Other miR156 regulated SPL genes may act similarly as the plastochron is even further shortened in the MIR156b overexpressor plants. A few mutants are known to cause a shortened plastochron and most of them simultaneously affect phyllotaxy. However, we found that shortening of the plastochron due to loss of SPL gene function is neither the cause nor the result of a changed spatial distribution of leaf primordia at the shoot apex. A shorter plastochron without an altered phyllotaxy has also been reported for two rice mutants, plastochron1 and -2 (pla1, -2; Itoh et al. 1998; Miyoshi et al. 2004; Kawakatsu et al. 2006). PLA1 encodes a cytochrome P450 protein, whereas PLA2 encodes a MEI2-like RNA binding protein. In both mutants the reduction in plastochron is accompanied by an increase in the size of the SAM and a higher rate of cell division. However, although the SAM of pla2 is actually smaller than that of pla1, it has a shorter plastochron. Furthermore, higher cell division activity associated with constitutive overexpression of CyclinD shortened the plastochron in tobacco without altering SAM size (Cockcroft et al. 2000). These observations suggest, as already noticed by Kawakatsu et al. (2006), that not SAM size but rather cell division rate is decisive in plastochron duration. Also our results may lend support to this hypothesis as both the spl9spl15 double mutant and the MIR156b overexpressor exhibit a clearly shorter plastochron than wild-type plants but their SAM sizes differ only marginally. Therefore, it will be of interest to determine if SPL9, SPL15 and other miR156 regulated SPL genes control cell division rate in the SAM and, if so, in particular if their role is mediated through the phytohormone cytokinin. Not only is cytokinin a major positive regulator of cell proliferation and division in plants (Werner et al. 2001) it is also, in mutual dependence of auxin, a major determinant in the outgrowth of lateral shoots (Sachs and Thimann 1967; Chatfield et al. 2000). This latter aspect may explain the reduced apical dominance observed for the spl9spl15 double mutant and the MIR156b overexpressor. Finally, mutants disrupting cytokinin signalling are known to result in reduced leaf initiation rates in addition to a smaller SAM and other effects (Nishimura et al. 2004; Higuchi et al. 2004).
Do SPL9 and SPL15 negatively regulate leaf maturation rate?
Based on their observations of the pla mutants in correlation to the expression of the respective genes in leaf primordia but not in the SAM, Kawakatsu et al. (2006) proposed that the rate of leaf maturation plays a significant role in regulating the rate of leaf initiation. In addition, these authors postulated a model in which the inhibitory effect of pre-existing leaf primordia on the initiation of the next leaf is lost as they mature. Similarly, a shortened plastochron in the spl9, spl15 mutants and the MIR156b overexpressor may also be due to precocious maturation of their leaves as suggested by our comparison of cross sections through successive leaf primordia of wild type and the MIR156b overexpressor. Even if the shape, i.e. circularity, of the primordia may not significantly differ after correction for an altered plastochron by comparing primordia based on age and not on serial sequence number, their cross sectional area seems to increase more rapidly in the MIR156b overexpressor. In particular, the cells surrounding the midvein in MIR156b overexpressor leaves appear to enlarge more rapidly.
SPL9 and SPL15 do not modulate the role of GA in the vegetative phase change
Exogenous application of GA3 has been found to accelerate abaxial trichome production in Arabidopsis suggesting that gibberellins function to regulate vegetative phase change (Telfer et al. 1997). These findings are also supported by mutant analysis. For example in spindly (spy) mutants, which undergo constitutive GA response, abaxial trichomes occur on leaves initiated significantly earlier than in wild type (Jakobsen and Olszewski 1993; Telfer et al. 1997). On the other hand, Telfer et al. (1997) found that mutants blocked in GA biosynthesis as well as GA insensitive mutants are significantly delayed in the appearance of abaxial trichomes. Loss-of-function mutants for the here examined SPL9 and SPL15 genes clearly delay the appearance of abaxial trichomes.
Our treatment of spl9spl15 double mutant and 35S::MIR156b transgenic plants with high doses of GA3 showed that, like in wild type, their number of juvenile leaves can be reduced but not to numbers equal to those found for similarly treated wild type. In fact the ratios of juvenile to adult leaves of wild type and mutant phenotypes remain highly comparable to untreated plants. From this we conclude that the role SPL9, SPL15 and other miR156 controlled SPL genes play in the vegetative phase change, is unlikely to be GA mediated although a minor contribution to GA sensitivity can not be excluded.
MiR156 targeted members of the SBP-box family of transcription factors in both mono- and dicots appear to play an important role as positive regulators of shoot maturation and of the vegetative to reproductive phase transition in particular. Both genetic factors, i.e. miR156 and SBP-box genes, have also been suggested to be major determinants in the transition from undifferentiated to differentiated embryogenic calli of rice (Luo et al. 2006). As the interaction between SBP-box genes and miR156 is of ancient origin in land plants (Arazi et al. 2005; Riese et al. 2007) it will be interesting to learn to what extent their molecular interplay is of importance to developmental phase transitions in plants in general.
We are grateful to Dr Detlef Weigel and Dr Rebecca Schwab (MPI, Tübingen, Germany) for the kind gift of the 35S::MIR156b line and Dr Ulrike Unte for providing the spl9-1 allele. The Salk Institute Genomic Analysis Laboratory is acknowledged for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. We thank Susanne Höhmann for excellent technical support.
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