Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers

The AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3) gene of Arabidopsis thaliana is a key regulator of growth and patterning in both shoots and roots. AIL6 encodes an AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor that is expressed in the root stem cell niche, the peripheral region of the shoot apical meristem and young lateral organ primordia. In flowers, AIL6 acts redundantly with AINTEGUMENTA (ANT) to regulate floral organ positioning, growth, identity and patterning. Experiments were undertaken to define the genomic regions required for AIL6 function and expression in flowers. Transgenic plants expressing a copy of the coding region of AIL6 in the context of 7.7 kb of 5′ sequence and 919 bp of 3′ sequence (AIL6:cAIL6-3′) fail to fully complement AIL6 function when assayed in the ant-4 ail6-2 double mutant background. In contrast, a genomic copy of AIL6 with the same amount of 5′ and 3′ sequence (AIL6:gAIL6-3′) can fully complement ant-4 ail6-2. In addition, a genomic copy of AIL6 with 590 bp of 5′ sequence and 919 bp of 3′ sequence (AIL6m:gAIL6-3′) complements ant-4 ail6-2 and contains all regulatory elements needed to confer normal AIL6 expression in inflorescences. Efforts to map cis-regulatory elements reveal that the third intron of AIL6 contains enhancer elements that confer expression in young flowers but in a broader pattern than that of AIL6 mRNA in wild-type flowers. Some AIL6:gAIL6-3′ and AIL6m:gAIL6-3′ lines confer an over-rescue phenotype in the ant-4 ail6-2 background that is correlated with higher levels of AIL6 mRNA accumulation. The results presented here indicate that AIL6 intronic sequences serve as transcriptional enhancer elements. In addition, the results show that increased expression of AIL6 can partially compensate for loss of ANT function in flowers.


Background
AIL6 encodes a member of the small subfamily of AIL/ PLT transcription factors that are part of the large AP2/ ERF family in Arabidopsis thaliana [1]. AIL proteins are key regulators of developmental processes throughout the plant life cycle (reviewed in [2]). AIL6 regulates multiple processes during Arabidopsis root and shoot development, largely in a redundant fashion with other AIL genes. Loss of AIL6 function on its own has no obvious phenotype in the shoot and only results in a slightly shorter root and root apical meristem [3,4]. Within shoots, AIL6 acts with ANT and AINTEGUMENTA-LIKE7/PLETHORA7 (AIL7/PLT7) to maintain the shoot apical meristem during vegetative development and works in a redundant fashion with AINTEGUMENTA-LIKE5/PLETHORA5 (AIL5/PLT5) and AIL7 to control shoot phyllotaxy [5,6]. In flowers, AIL6 acts redundantly with ANT to regulate floral organ initiation, growth, identity specification and patterning [4]. AIL6 function is required for root formation in combination with PLETHORA1 (PLT1) and PLETHORA2 (PLT2) and controls the positioning of lateral roots in a redundant fashion with AIL5 and AIL7 [3,7].
As expected from its functions throughout the plant, AIL6 is expressed at the mRNA level in multiple tissues. In roots, AIL6 mRNA is detected in the stem cell niche, pericycle cells prior to lateral root initiation, and lateral root primordia [3,7]. In the shoot, AIL6 mRNA is detected in young lateral organ primordia and throughout the shoot apical meristem with expression higher in the periphery of the meristem and incipient lateral organ as compared with the center of the meristem [1,5]. In flowers, AIL6 expression is associated with young flower primordium and early stages of floral organ development. AIL6 is expressed throughout stage one and two flower primordia, becoming primarily restricted to the floral meristem dome in stage three flowers with only low levels present in sepal primordia [1]. In stage six flowers, some AIL6 mRNA is present within petal, stamen and carpel primordia; only low amounts of AIL6 mRNA are detected after this stage of development [1].
In several of these tissues, AIL6 expression is linked with the activity of AUXIN RESPONSE FACTORs (ARFs), transcription factors that regulate gene expression in response to auxin. In lateral roots, AIL6 appears to act downstream of ARF7 and ARF19 [7]. AIL6 is not expressed in any pericycle cells of arf7 arf19 double mutants, which lack most lateral roots, although it is not known whether AIL6 is a direct target of these transcription factors [7]. Chromatin immunoprecipitation (ChIP) experiments show that MONOPTEROS/AUXIN RESPONSE FACTOR 5 (MP/ARF5) directly activates AIL6 in the periphery of the shoot apical meristem to promote flower primordium initiation [8]. Two other potential regulators of AIL6 are the floral meristem identity protein LEAFY (LFY) and APETALA1 (AP1) which were shown to bind to AIL6 in genome-wide ChIP experiments [9,10].
Despite the importance of AIL6 in Arabidopsis vegetative and reproductive development, sequences required for proper AIL6 expression have not been identified. Here I define the genomic regions necessary for AIL6 function in flowers by complementation of ant-4 ail6-2 double mutants with transgenes containing different amounts of AIL6 sequence. These experiments show that introns are required for AIL6 function and expression in flowers. In particular, intron three was found to contain enhancer elements that drive AIL6 expression in early stages of flower development. The importance of intron three to AIL6 regulation is also demonstrated by work from other labs showing binding of LFY and AP1 to the third intron of AIL6 [9,10]. Furthermore, this work demonstrates that increased expression of AIL6 can partially compensate for loss of ANT function.
To determine whether the lack of complementation by the AIL6:cAIL6-3′ transgene was a consequence of the absence of introns or insufficient 5′ and 3′ sequence, ant-4/+ ail6-2 plants were transformed with a transgene corresponding to a genomic copy of AIL6 in the same context of 5′ and 3′ sequence (AIL6:gAIL6-3′) (Fig. 1a). Of the 5 lines obtained, one line (line 12) exhibited partial rescue of ant-4 ail6-2, two lines (lines 11 and 17) exhibited complete rescue such that the flowers resembled ant-4, and two lines (lines 4 and 21) exhibited an overrescue phenotype such that the plants had a less severe phenotype than ant-4 ( Fig. 2e, f ). The phenotypic variation in the degree of complementation in these lines is presumably a consequence of variation in transgene insertion sites. AIL6:gAIL6-3′ ant-4 ail6-2 line 17 flowers closely resemble ant-4 flowers with regard to the identity, numbers, and size of the floral organs ( Fig. 2e; Table 1). In flowers of the over-rescue AIL6:gAIL6-3′ ant-4 ail6-2 line 4, the petals are larger than those of ant-4 and the stamen anthers consist of four locules (Fig. 2f ). However, AIL6:gAIL6-4′ ant-4 ail6-2 line 4 petals are not as big as those of wild-type flowers, and the flowers are female sterile and consist of fewer petals and stamens compared to wild-type ( Fig. 2f; Table 1). Similar effects were observed with regard to complementation of leaf growth defects in AIL6:gAIL6-3′ ant-4 ail6-2 lines 17 and 4; line 17 leaves resemble those of ant-4 while line 4 leaves are larger (Fig. 2k, l, m).

AIL6 mRNA accumulation depends on intronic sequences
To determine whether the distinct phenotypes conferred by the AIL6:cAIL6-3′ and AIL6:gAIL6-3′ transgenes correlated with the levels of AIL6 mRNA accumulation in these plants, AIL6 mRNA levels were examined by reverse transcriptase quantitative PCR (RT-qPCR). AIL6:cAIL6-3′ ant-4 ail6-2 line 2 inflorescences accumulate less AIL6 mRNA compared with Ler (Fig. 2n). AIL6:gAIL6-3′ ant-4 ail6-2 lines 17 and 4 accumulate more AIL6 mRNA than Ler, approximately 2.3 and 6.5 fold higher levels than Ler, respectively (Fig. 2n). Since the ant-4 ail6-2 double mutant is in a mixed Ler/ Col background (see "Methods"), I also examined AIL6 mRNA levels in Col and found them to be similar to those in Ler (Fig. 2n). AIL6 mRNA levels were slightly increased in the ant-4 background, suggesting possible cross-regulation of AIL6 expression. The increased levels of AIL6 mRNA in lines 17 and 4 compared to Ler, Col and ant-4 is likely a consequence of the chromosomal position of the transgene insertion site. The RT-qPCR results indicate that intronic sequences increase steady-state AIL6 mRNA levels. They also suggest that the inability of AIL6:cAIL6-3′ to fully complement AIL6 function in the ant-4 ail6-2 double mutant results from insufficient AIL6 mRNA. Furthermore, the higher AIL6 mRNA levels in AIL6:gAIL6-3′ ant-4 ail6-2 line 4 as compared with line 17 suggest that increased AIL6 activity can partially compensate for loss of ANT function.

Complementation of AIL6 function in ant ail6 flowers by a smaller genomic fragment
To refine the amount of 5′ sequence required for AIL6 function, ant-4/+ ail6-2 plants were transformed with a smaller AIL6 genomic fragment containing 590 bp of 5′ sequence and 919 bp of 3′ sequence (AIL6m:gAIL6-3′) (Fig. 1a). Of the 14 transgenic lines obtained, six lines conferred partial rescue of ant-4 ail6-2, five lines conferred a full rescue of ant-4 ail6-2 to the ant-4 phenotype, and four lines had an over-rescue phenotype with larger petals and some stamen anthers with four locules. This range of phenotypes closely parallels that of AIL6:gAIL6-3′ lines and once again is likely a consequence of variation in transgene insertion sites.    To investigate whether this amount of AIL6 5′ sequence was sufficient for normal AIL6 expression in flowers, the gene encoding the reporter β-glucuronidase (GUS) was fused in frame to AIL6 in the AIL6m:gAIL6-3′ context (i.e. AIL6m:gAIL6-GUS-3′; Fig. 1b) was made. Of 16 transgenic lines, GUS activity was detected in young flowers of seven lines while no staining was observed in nine lines. A representative line (AIL6m:gAIL6-GUS-3′ line 15) was chosen for further characterization (Fig. 4e). Examination of GUS stained and sectioned AIL6m:gAIL6-GUS-3′ line 15 inflorescences under bright and dark-field illumination shows GUS activity in a pattern that matches AIL6 in situ hybridization data (Fig. 4h-k). GUS activity is detected throughout stage 1 and 2 flower primordia, becoming restricted to stamen and carpel primordia in stage 4 and 5 flowers (Fig. 4h-k). These results show that this AIL6 genomic fragment is sufficient to confer a normal pattern of AIL6 expression. In contrast, no GUS activity was detected in inflorescences of any of eight lines containing an AIL6:GUS reporter in which GUS is under the control of 7.7 kb of AIL6 5′ sequence (Figs. 1b,   4f ). In addition, no GUS activity was detected in young flowers of nine AIL6m:GUS-3′ lines in which GUS is present in the context of 590 bp of AIL6 5′ sequence and 919 bp of AIL6 3′ sequence (Figs. 1b, 4g). These results indicate that introns contain cis-regulatory elements responsible for AIL6 expression in inflorescences.

The third intron of AIL6 contains enhancer elements that drive expression in young flowers
To begin to map intronic sequences responsible for AIL6 expression in inflorescences, additional GUS reporters were made in which either intron three or intron one was placed upstream of 590 bp of AIL6 5′ sequence (Fig. 1b). These constructs also contain 919 bp of AIL6 3′ sequence. Introns three and one correspond to the largest and second largest introns, respectively, within AIL6. GUS staining of inflorescences from the intron three constructs (i.e. I3F-AIL6m:GUS-3′ and I3R-AIL6m:GUS-3′; Fig. 1b) showed staining throughout the inflorescence meristem and young flowers in a broader pattern than that observed in AIL6m:gAIL6-GUS-3′ (compare Fig. 5a, b, e-h to Fig. 4e, h-k). In contrast, no GUS signal was observed in the young flowers of the intron one constructs (i.e. I1F-AIL6m:GUS-3′ and I1R-AIL6m:GUS-3′; Figs. 1b, 5c, d). These results suggest that intron three contains enhancer elements that promote AIL6 mRNA expression in young flowers but that additional regulatory elements are present in other regions that restrict AIL6 expression within the inflorescence meristem and young flowers.
ChIP experiments have identified several transcription factors that appear to regulate AIL6 expression within inflorescences. The auxin response factor MP/ ARF5 promotes AIL6 expression in groups of cells on the periphery of the inflorescence meristem to promote flower primordium initiation [8]. MP binds to several regions of AIL6 including 5′ sequence, exon one, intron one, and intron three [8]. Two other putative regulators of AIL6 are the floral meristem identity proteins AP1 and LFY [9,10]. ChIP-Seq identified a binding peak for AP1 within intron three (Fig. 6) [9]. ChIP-chip identified a wide LFY binding region with two peaks that overlap exon two, intron two, exon three, and intron three (Fig. 6) [10]. These results are consistent with the identification of intron three as important in AIL6 regulation during early stages of flower development. Further experiments will be necessary to map additional regulatory elements that in combination with intron three confer a normal AIL6 expression pattern.

Conclusions
This study shows the importance of intronic sequences in regulating AIL6 transcription in flowers. Intron three of AIL6 is sufficient to drive expression of a reporter gene in early stages of flower development. The identification of introns as important for AIL6 regulation is consistent with ChIP data showing that several AIL6 regulators can bind to these intronic regions. In addition, increased expression of AIL6 is shown to partially compensate for loss of ANT, a gene with which AIL6 shares some functions.

RNA extraction and RT-qPCR
RNA extraction from inflorescences, cDNA synthesis and qPCR reactions were performed as described previously except that primers that do not amplify an AIL6 transcript in the ail6-2 background: RTAIL6-8 and RTAIL6-9 (Table 2) were used and in some cases RNA was extracted with Trizol with cleanup and DNase treatment performed on an RNeasy column (Qiagen) [12]. Data was normalized using AT5G15710 with the primers (RTFbox-1, RTFbox-2) shown in Table 2 [13]. The data shown are the average of two biological replicates.

In situ hybridization
Inflorescences were fixed, embedded, sectioned, hybridized and washed as described previously except that a hybridization temperature of 53 °C was used [14]. The AIL6 probe was made from a template corresponding to nucleotides 497-1691 of AIL6 that was PCR amplified with AIL6-FW2 and AIL6-RV2 (Table 2) using Phusion DNA polymerase and cloned into the BamHI/EcoRI sites of pGEM3Z to create longAIL6/pGEM3Z. LongAIL6/ pGEM3Z was linearized with HindIII and transcribed with T7 RNA polymerase.

Organ counts
The first 30 flowers on five plants of each genotype were counted.

GUS staining
The GUS assays were performed as described in [15]. The tissue was incubated in 2 mM 5-bromo-4-chloro-3indolyl-β-glucuronic acid for 22 h. After taking pictures of whole inflorescences, the tissue was embedded in paraplast, sectioned, mounted on slides and observed under bright-field and dark-field illumination.