AINTEGUMENTA-LIKE6 regulates cellular differentiation in flowers
- First Online:
- Cite this article as:
- Krizek, B.A. & Eaddy, M. Plant Mol Biol (2012) 78: 199. doi:10.1007/s11103-011-9844-3
- 422 Views
During flower development, pluripotent stem cells within the floral meristem give rise to proliferative precursor cells whose progeny eventually acquire specialized functions within each floral organ. The regulatory mechanisms by which plant cells transition from a proliferating state to a differentiated state are not well characterized. Several members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family, including AINTEGUMENTA (ANT) and AIL6/PLT3, are important regulators of cell proliferation in flowers. To further investigate the role of AIL6 during flower development, we have characterized transgenic plants in which the coding region of AIL6 was expressed under the control of the constitutive 35S promoter (35S:cAIL6). These plants display changes in floral organ size and morphology that are associated with alterations in the pattern and duration of cell divisions within developing organs. In addition, we find that very high levels of AIL6 expression inhibit cellular differentiation. In contrast, ant ail6 double mutants display premature differentiation of floral meristem cells. These results indicate that these two transcription factors regulate both proliferation and differentiation in flowers.
During reproductive development in Arabidopsis thaliana, pluripotent stem cells within the floral meristem give rise to highly proliferative progeny cells which transition through various stages of differentiation before becoming fully differentiated and ceasing cell division. The exact mechanisms controlling the transition of cells within developing floral organ primordia from a proliferating state to a differentiated state and how they are integrated within the programs of floral organ identity specification are not well understood. Floral organ primordia adopt distinct fates in response to the activities of unique combinations of floral organ identity proteins [reviewed in (Irish 2010)]. These proteins appear to regulate the entire process of floral organogenesis, from the progressive elaboration of organ morphology to cell and tissue-type patterning [reviewed in (Sablowski 2010; Ito 2011)].
According to the ABCE model, four different classes of floral organ identity proteins act in different combinations to specify sepal, petal, stamen and carpel identity in whorls one to four of the flower, respectively [reviewed in (Krizek and Fletcher 2005)]. The combination of A and E gene activities specifies sepal identity, the combination of A, B and E activities specifies petal identity, the combination of B, C and E activities specifies stamen identity and the combination of C and E activities specifies carpel identity. With the exception of the class A protein APETALA2 (AP2), the remaining floral organ identity proteins [the class A protein APETALA1 (AP1), the class B proteins APETALA3 (AP3) and PISTILLATA (PI), the class C protein AGAMOUS (AG), and the class E SEPALATA (SEP) proteins] are MADS domain transcription factors. According to the quartet model, these MADS domain proteins form higher order protein complexes, likely tetramers, that bind to CC(A/T)6GG (CArG boxes) sequences in the promoters of target genes (Theissen 2001; Melzer and Theissen 2009; Melzer et al. 2009). The floral organ identity proteins regulate distinct genes at different times in flower development with identified targets including genes involved in hormonal pathways, growth control and patterning (Kaufmann et al. 2009, 2010).
A number of genes regulating floral organ growth in Arabidopsis have been identified with some genes primarily controlling cell proliferation and others cell elongation [reviewed in (Breuninger and Lenhard 2010)]. Promoters of organ growth that regulate cell division include AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3), two members of the AIL/PLT gene family that encode AP2/ERF type transcription factors. ant mutants produce smaller leaves and flowers than wild type and these organ growth defects are enhanced in ant ail6 double mutants (Elliott et al. 1996; Klucher et al. 1996; Krizek 2009). Both genes are expressed throughout the floral meristem and newly initiated floral organ primordia, but ANT expression persists longer than that of AIL6 in developing floral organs with little or no expression in mature organs (Elliott et al. 1996; Nole-Wilson et al. 2005). ANT has been proposed to regulate organ size by controlling the length of time in which cells within developing organs are competent to undergo proliferation (Mizukami and Fischer 2000). Constitutive expression of ANT under the 35S promoter resulted in persistent expression of the G1 cyclin CycD3 in maturing leaves, a prolonged period of cell division and larger organs (Mizukami and Fischer 2000).
Besides its role in regulating organ growth in the shoot, AIL6/PLT3 acts redundantly with PLT1 and PLT2 in root development (Galinha et al. 2007). plt1plt2 ail6 seedlings have a rootless phenotype with fully differentiated cells present in the root pole of these seedlings at 3 days post germination (Galinha et al. 2007). Four AIL/PLT proteins (PLT1, PLT2, AIL6 and BBM) are detected in gradients within the root with the highest levels present in the stem cell niche and lower levels in the proliferating cells above the meristem (Galinha et al. 2007). Low levels of PLT2 and PLT3 are also detected in cells of the elongation zone. This gradient of PLT activity has been proposed to be instructive for distinct cellular behaviors with high PLT activity specifying stem cell identity, intermediate levels specifying mitotic activity and low levels required for cellular differentiation (Galinha et al. 2007).
To further investigate the role of AIL6 during flower development, we expressed the coding region of AIL6 under the control of the constitutive 35S promoter (35S:cAIL6). These plants exhibit alterations in floral organ morphology that are associated with persistent cell divisions within some developing floral organs. The severity of these phenotypic alterations is correlated with AIL6 mRNA levels. In the highest expressing 35S:cAIL6 line characterized, we also observed negative effects on cellular differentiation. The characteristic surface morphologies of sepal, petal, stamen and carpel epidermal cells were absent in this 35S:cAIL6 line suggesting that cells did not undergo complete differentiation. Correspondingly, we observe that floral meristem cells undergo premature differentiation in ant ail6 flowers. These findings suggest that ANT and AIL6 regulate the transition of cells between proliferation and differentiation.
Materials and methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Landsberg erecta (Ler) was used as the wild type. Plants were grown on a soil mixture of Metro-Mix 360:perlite:vermiculite (5:1:1) under continuous light or in 16 h days (100–150 μmol/m2/s) at a temperature of 22°C.
Plasmid construction and Agrobacterium transformation
The coding region of AIL6 (cAIL6), corresponding to the most upstream in-frame ATG (Feng et al. 2005), was PCR amplified from cDNA and cloned into the KpnI/BamHI sites of pART7. The resulting 35S:cAIL6 construct was digested with NotI and cloned into pMLBart. The 35S:cAIL6 plasmid was transformed into Agrobacterium tumefaciens strain ASE by electroporation. Arabidopsis Ler plants were transformed with this Agrobacterium strain by vacuum infiltration (Bechtold et al. 1993). Transformants containing the 35S:cAIL6 transgene were selected using basta.
RNA extraction and real time RT-PCR
RNA was extracted from inflorescences (Verwoerd et al. 1989) and treated with Turbo DNase (Applied Biosystems) following the manufacturer’s instructions. First strand cDNA synthesis was performed using Quanta qScript cDNA Super Mix (Quanta BioSciences) following the manufacturer’s instructions. The real time PCR reactions were performed on an iCycler (Biorad) using B-R SYBR Green SuperMix for iQ (Quanta BioSciences) and the following primers for AIL6: RTAIL6-4: 5′-cgagttgctgggaacaaag-3′ and RTAIL6-5: 5′-tcatacgcttcagctgcttc-3′, which amplified a 71 bp fragment. Melt curve analyses were performed at the end of each experiment to check the specificity of the reactions. Experiments were performed twice using two or three biological tissue replicates and reactions in triplicate. The raw fluorescence data was analyzed using Miner, a software program that calculates Ct (threshold cycle) and amplification efficiencies (Zhao and Fernald 2005). Normalization was performed using two reference genes (At5g15710, At5g12240) that are expressed stably and at lower levels than traditional reference genes, since AIL6 expression is low (Czechowski et al. 2005). The fold difference in AIL6 expression was calculated using the Pfaffl method (Pfaffl 2001). Data reported in the figures correspond to those normalized to At5g12240 but similar values were obtained when normalizing to At5g15710.
Tissue for SEM was fixed, dried, dissected and coated as described previously (Krizek 1999). SEM analyses were performed on a FEI Quanta 200 ESEM.
Paraffin and epoxy sectioning
Tissue for paraffin sectioning was prepared and sectioned as described below for in situ hybridization. After sectioning, slides were placed in 0.05% Toluidine Blue O for 10 min, rinsed with water and allowed to air dry (Sakai 1973). The paraffin was removed with xylenes and slides mounted with permount. For embedding in epoxy, tissue was fixed in 0.2 M sodium cacodylate buffer for 2 h at room temperature, rinsed with 0.1 M cacodylate buffer, postfixed in 0.1 M cacodylate buffered 1% osmium tetroxide for 1 h at 4°C, rinsed with 0.1 M cacodylate buffer, dehydrated in ethanol and acetone, and incubated overnight in a 1:1 mixture of EMBed-812 (Electron Microscopy Sciences) and acetone. The next day the tissue was incubated for 5 h in a 3:1 mixture of EMBed-812 and acetone and then incubated overnight in 100% EMBed-812. The samples were embedded the next day in 100% EMBed-812 and cured for approximately 24 h. Samples were sectioned on an ultramicrotome and stained with Toluidine Blue O (Epoxy tissue stain; Electron Microscopy Sciences).
In situ hybridization
35S:cAIL6 plants exhibit alterations in floral organ development
Some later-arising flowers in both 35S:cAIL6 line 22 and 31 have a more normal appearance and are fertile, allowing us to obtain some seeds from these lines. The seeds obtained are sometimes larger than normal with light brown or greenish seed coats that never take on the brown color of wild-type mature seeds (Fig. 1d–f). The seed phenotype, like the flower phenotype, is more severe in line 22 compared to line 31.
To determine whether the phenotypic differences between lines 22 and 31 might be a consequence of different steady-state levels of AIL6 mRNA, we characterized AIL6 expression in three 35S:cAIL6 lines (1, 22, 31) and wild type inflorescences by real-time RT-PCR. 35S:cAIL6 line 1 produces flowers with a wild-type appearance and accumulates similar amounts of AIL6 mRNA as wild-type inflorescences (Fig. 1g). In contrast, AIL6 mRNA levels were much higher in lines 22 and 31 as compared with wild type. AIL6 mRNA levels were approximately 55 fold higher in line 22 and approximately 30 fold higher in line 31 (Fig. 1g). These results suggest that the stronger floral and seed phenotypes of line 22 are a consequence of higher levels of AIL6 mRNA.
To better characterize the altered morphology of 35S:cAIL6 line 22 carpels, we examined these organs throughout flower development by scanning electron microscopy. In stage 11 and younger flowers, 35S:cAIL6 carpels appear normal in morphology (Fig. 2i, j). During stage 12 of wild-type flower development, stigmatic papillae appear at the apex of the carpel and the stylar region become clearly distinct from the stigma above and the ovary valves below (Fig. 2k). In 35S:cAIL6 flowers, the stylar region is tapered but not as well-defined and distinct from the ovary valve tissue (Fig. 2l). This continues to be true through opening of the floral bud in stage 13 flowers. Bulging from the stylar region begins to occur in 35S:cAIL6 line 22 carpels during stage 13 and continues with the bulges developing into larger outgrowths (Fig. 2m–p).
Dosage dependent effects of AIL6 misexpression on cellular differentiation in floral organs
AIL6 overexpression does not affect leaf development
Persistent cell division is associated with the altered development of 35S:cAIL6 floral organs
35S:cAIL6 lines exhibit uneven AIL6 expression
AIL6 expression in developing floral organs affects organ size and morphology
Misexpression of AIL6 in flowers can result in increase growth of floral organs similar to what has been observed in plants in which ANT or AIL5 are constitutively expressed (Krizek 1999; Mizukami and Fischer 2000; Nole-Wilson et al. 2005). However in the case of 35S:cAIL6 plants, organ morphology is also affected, something not observed in 35S:ANT or 35S:AIL5 flowers. These changes in organ morphology include bumps or protrusions on sepals and carpels and fringe on the distal end of sepals and petals. The uneven growth and bulging observed in some 35S:cAIL6 floral organs may be a consequence of the uneven accumulation of AIL6 mRNA within developing floral organs. We observed overlap between cells exhibiting persistent histone H4 expression and AIL6 misexpression, suggesting that AIL6 activity can promote cell proliferation. Patchiness in the distribution of AIL6 mRNA could lead to uncoordinated cell division within an organ and consequently affect organ morphology.
The different consequences of ANT and AIL6 overexpression suggest that these genes have somewhat distinct functions during flower development. This is consistent with the different expression patterns of these genes; ANT expression is maintained in developing floral organs much longer than AIL6 (Elliott et al. 1996; Nole-Wilson et al. 2005). In addition, previous work has revealed other differences between ANT and AIL6 suggesting that AIL6 does not just act similarly to ANT in a subset of ANT functions within the shoot. For example, ail6 mutants are more sensitive to the effects of the auxin transport inhibitor NPA on floral meristem initiation than ant mutants (Krizek 2011).
High AIL6 mRNA levels are sufficient to inhibit cell differentiation
We observed somewhat distinct phenotypes in two 35S:cAIL6 lines that accumulated high levels of AIL6 mRNA. Floral organs of 35S:cAIL6 line 31, which had 30 fold higher AIL6 mRNA levels as compared with wild type, were increased in size but had fairly normal morphology of sepals, petals and stamens. Cell differentiation occurred normally in this line with epidermal cells of floral organs displaying characteristics of fully differentiated cells. In contrast, floral organs of 35S:cAIL6 line 22, which had 55 fold higher AIL6 mRNA levels as compared with wild type, exhibited changes in organ morphology and incomplete cell differentiation. Our results suggest that high levels of AIL6 activity in developing floral organs promotes cell proliferation while extremely high levels of AIL6 activity can inhibit cell differentiation. These results suggest that AIL6 regulates both cell proliferation and differentiation and that it can affect cell behaviors in flowers in a dose-dependent manner. The observed premature differentiation of floral meristem cells in ant ail6 flowers further supports roles for AIL6 and ANT in inhibition of cellular differentiation during early stages of flower development. These functions are consistent with the expression pattern of these genes. While ANT expression persists longer than AIL6 in developing floral organs, neither is expressed in mature organs (Elliott et al. 1996; Nole-Wilson et al. 2005). We hypothesize that changes in the combined AIL6 and ANT activity level regulate the transitioning of cells from a proliferating state toward complete differentiation.
Although AIL6 mRNA levels in 35S:cAIL6 lines 22 and 31 were higher in leaves as compared with inflorescences, no effect on organ size, morphology or cellular differentiation was observed in the leaves of these plants. This indicates that high AIL6 activity is not sufficient to promote cell proliferation or inhibit cell differentiation in leaves.
Previous work has suggested that four AIL/PLT proteins (PLT1, PLT2, AIL6/PLT3 and BBM) act in a dosage dependent manner in the root with different levels of overall PLT activity specifying distinct cell behaviors along the longitudinal axis of the root (Galinha et al. 2007). Manipulation of this gradient by misexpression of PLT2 under the control of the 35S promoter resulted in a larger meristem due to continued division of cells that normally would have stopped proliferating and started elongating (Galinha et al. 2007). Our work is consistent with AIL/PLT proteins acting in a similar dose-dependent fashion within flowers with different AIL/PLT activity levels associated with distinct cellular behaviors.
AIL6 expression under the 35S promoter led to uneven accumulation of AIL6 mRNA
Surprisingly, we found that AIL6 does not accumulate to high levels in all tissues of 35S:cAIL6 inflorescences even though it is expressed under a constitutive promoter. It is possible that the 35S promoter used in this experiment does not confer constitutive or nearly constitutive expression. Although the pART7 plasmid containing the 35S promoter has been commonly used to misexpress genes, we were unable to find published data showing tissue-specific expression resulting from the use of this promoter. Thus, it is possible that uneven AIL6 accumulation results from uneven expression of the transgene. Alternatively, AIL6 may be subject to post-transcriptional regulation. Further work examining the expression of other genes expressed under the control of this 35S promoter is needed to distinguish between these possibilities.
We thank Soumitra Ghoshroy and the Electron Microscopy Center staff for advice on the use of the SEM and the ABRC for the F12B17 BAC clone. This work was supported by National Science Foundation (NSF) grant IOS 0922367.