Differences in both expression and protein activity contribute to the distinct functions of AINTEGUMENTA compared with AINTEGUMENTA-LIKE 5 and AINTEGUMENTA-LIKE 7.

Three members of the Arabidopsis AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family, AIL5/PLT5, AIL6/PLT3, and AIL7/PLT7, exhibit partially overlapping roles with AINTEGUMENTA (ANT) during flower development. Loss of ANT function alone results in smaller floral organs and female sterility indicating that some ANT functions cannot be provided by these related transcription factors. Previously, we showed that expression of AIL6 at the same levels and spatial pattern as ANT could largely rescue the defects of ant mutants. This suggested that the functional differences between ANT and AIL6 were primarily a consequence of expression differences. Here, we investigated the functional differences between ANT and both AIL5 and AIL7 by expressing these two AILs under the control of the ANT promoter. We found that only ANT:gAIL5 lines with much higher amounts of AIL5 mRNA as compared with ANT could compensate for loss of ANT function. ANT:gAIL7 lines with AIL7 mRNA levels similar to those of ANT were able to rescue some but not all aspects of the ant mutant phenotype. Thus, expression differences alone cannot explain the functional differences between ANT and these two related proteins. Studies in yeast show that AIL5 and AIL7 have lower transcriptional activation activities as compared with ANT and AIL6 when bound to the consensus ANT DNA binding site. Our results suggest that differences in both expression and protein activity contribute to the functional specificity of ANT compared with AIL5 and AIL7.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

My favorite flowering image: 'giant' Arabidopsis flowers.

A fascinating aspect of floral diversity is the dramatic difference in flower size observed in nature. The largest flowers in the world, Rafflesia arnoldii, span several feet while flowers of the genus Wolffia are microscopic. My own particular interest in flower size started when I overexpressed the Arabidopsis gene AINTEGUMENTA (ANT) and observed a larger flower phenotype.

The Arabidopsis transcription factor AINTEGUMENTA orchestrates patterning genes and auxin signaling in the establishment of floral growth and form.

Understanding how flowers form is an important problem in plant biology, as human food supply depends on flower and seed production. Flower development also provides an excellent model for understanding how cell division, expansion and differentiation are coordinated during organogenesis. In the model plant Arabidopsis thaliana, floral organogenesis requires AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE 6 (AIL6)/PLETHORA 3 (PLT3), two members of the Arabidopsis AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family. Together, ANT and AIL6/PLT3 regulate aspects of floral organogenesis, including floral organ initiation, growth, identity specification and patterning. Previously, we used RNA-Seq to identify thousands of genes with disrupted expression in ant ail6 mutant flowers, indicating that ANT and AIL6/PLT3 influence a vast transcriptional network. The immediate downstream targets of ANT and AIL6/PLT3 in flowers are unknown, however. To identify direct targets of ANT regulation, we performed an RNA-Seq time-course experiment in which we induced ANT activity in transgenic plants bearing an ANT-glucocorticoid receptor fusion construct. In addition, we performed a ChIP-Seq experiment that identified ANT binding sites in developing flowers. These experiments identified 200 potential ANT target genes based on their proximity to ANT binding sites and differential expression in response to ANT. These 200 candidate target genes were involved in functions such as polarity specification, floral organ development, meristem development and auxin signaling. In addition, we identified several genes associated with lateral organ growth that may mediate the role of ANT in organ size control. These results reveal new features of the ANT transcriptional network by linking ANT to previously unknown regulatory targets.

AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic, growth, and vascular development genes in young Arabidopsis flowers.

Arabidopsis flower primordia give rise to organ primordia in stereotypical positions within four concentric whorls. Floral organ primordia in each whorl undergo distinct developmental programs to become one of four organ types (sepals, petals, stamens, and carpels). The Arabidopsis transcription factors AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are required for correct positioning of floral organ initiation, contribute to the specification of floral organ identity, and regulate the growth and morphogenesis of developing floral organs. To gain insight into the molecular mechanisms by which ANT and AIL6 contribute to floral organogenesis, we identified the genome-wide binding sites of both ANT and AIL6 in stage 3 flower primordia, the developmental stage at which sepal primordia become visible and class B and C floral homeotic genes are first expressed. AIL6 binds to a subset of ANT sites, suggesting that AIL6 regulates some but not all of the same target genes as ANT. ANT- and AIL6-binding sites are associated with genes involved in many biological processes related to meristem and flower organ development. Comparison of genes associated with both ANT and AIL6 ChIP-Seq peaks and those differentially expressed after perturbation of ANT and/or AIL6 activity identified likely direct targets of ANT and AIL6 regulation. These include class B and C floral homeotic genes, growth regulatory genes, and genes involved in vascular development.

AINTEGUMENTA and AINTEGUMENTA-LIKE6/PLETHORA3 Induce LEAFY Expression in Response to Auxin to Promote the Onset of Flower Formation in Arabidopsis.

Proper timing of the onset to flower formation is critical for reproductive success. Monocarpic plants like Arabidopsis (Arabidopsis thaliana) switch from production of branches in the axils of leaves to that of flowers once in their lifecycle, during the meristem identity transition. The plant-specific transcription factor LEAFY (LFY) is necessary and sufficient for this transition. Previously, we reported that the plant hormone auxin induces LFY expression through AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP). It is not known whether MP is solely responsible for auxin-directed transcriptional activation of LFY. Here, we show that two transcription factors belonging to the AINTEGUMENTA-LIKE/PLETHORA family, AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3), act in parallel with MP to upregulate LFY in response to auxin. ant ail6 mutants display a delay in the meristem identity transition and in LFY induction. ANT and AIL6/PLT3 are expressed prior to LFY and bind to the LFY promoter to control LFY mRNA accumulation. Genetic and promoter/reporter studies suggest that ANT/AIL6 act in parallel with MP to promote LFY induction in response to auxin sensing. Our study highlights the importance of two separate auxin-controlled pathways in the meristem identity transition.

RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways.

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

AINTEGUMENTA-LIKE6 can functionally replace AINTEGUMENTA but alters Arabidopsis flower development when misexpressed at high levels.

KEY MESSAGE: Expression differences underlie the functional differences between two related transcription factors: AINTEGUMENTA and AINTEGUMENTA-LIKE6. Ectopic expression of AINTEGUMENTA-LIKE6 at high levels alters floral organ initiation, growth and identity specification. AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) encode related transcription factors with partially overlapping roles in floral organ development in Arabidopsis thaliana. To investigate whether the functional differences between ANT and AIL6 are a consequence of differences in gene expression and/or protein activity, we made transgenic plants in which a genomic copy of AIL6 was expressed under the control of the ANT promoter. ANT:gAIL6 can rescue the floral organ size defects of ant mutants when AIL6 is expressed at similar levels as ANT in wild type. Thus, the functional differences between ANT and AIL6 result primarily from gene expression differences. However, lines that express AIL6 at higher levels display additional phenotypes that include reduced numbers of floral organs and the production of mosaic floral organs. These phenotypes were also observed in two different inducible AIL6 transgenic lines but not in 35S:ANT, suggesting that AIL6 protein may have activities distinct from ANT, although the in vivo relevance of such differences is not clear. Similar to 35S:ANT plants, overexpression of AIL6 in the inducible lines also results in the production of larger flowers. The distinct phenotypes resulting from AIL6 misexpression in the transgenic lines described here and those previously characterized appear to result from different levels and patterns of AIL6 expression.

AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development.

AINTEGUMENTA (ANT) is an important regulator of Arabidopsis flower development that has overlapping functions with the related AINTEGUMENTA-LIKE6 (AIL6) gene in floral organ initiation, identity specification, growth, and patterning. Two other AINTEGUMENTA-LIKE (AIL) genes, AIL5 and AIL7, are expressed in developing flowers in spatial domains that partly overlap with those of ANT. Here, it is shown that AIL5 and AIL7 also act in a partially redundant manner with ANT. The results demonstrate that AIL genes exhibit unequal genetic redundancy with roles for AIL5, AIL6, and AIL7 only revealed in the absence of ANT function. ant ail5 and ant ail7 double mutant flowers show alterations in floral organ positioning and growth, sepal fusion, and reductions in petal number. In ant ail5, petals are often replaced by filaments or dramatically reduced in size. ant ail7 double mutants produce increased numbers of carpels, which have defects in valve fusion and a loss of apical tissues. The distinct phenotypes of ant ail5, ant ail7 and the previously characterized ant ail6 indicate that AIL5, AIL6, and AIL7 make unique contributions to flower development. These distinct roles are also supported by genetic analyses of ant ail triple mutants. While ant ail5 ail6 triple mutants closely resemble ant ail6 double mutants, ant ail5 ail7 triple mutants exhibit more severe deviations from the wild type than either ant ail5 or ant ail7 double mutants. Furthermore, it is shown that AIL5, AIL6, and AIL7 act in a dose dependent manners in ant and other mutant backgrounds.

Three Arabidopsis AIL/PLT genes act in combination to regulate shoot apical meristem function.

The shoot apical meristem, a small dome-shaped structure at the shoot apex, is responsible for the initiation of all post-embryonic shoot organs. Pluripotent stem cells within the meristem replenish themselves and provide daughter cells that become incorporated into lateral organ primordia around the meristem periphery. We have identified three novel regulators of shoot apical meristem activity in Arabidopsis thaliana that encode related AIL/PLT transcription factors: AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE6 (AIL6)/PLETHORA3 (PLT3) and AINTEGUMENTA-LIKE7 (AIL7)/PLETHORA7 (PLT7). Loss of these genes results in plants that initiate only a few leaves prior to termination of shoot apical meristem activity. In 7-day-old ant ail6 ail7 seedlings, we observed reduced cell division in the meristem region, differentiation of meristematic cells and altered expression of the meristem regulators WUSCHEL (WUS), CLAVATA3 (CLV3) and SHOOT MERISTEMLESS (STM). Genetic experiments suggest that these three AIL genes do not act specifically in either the WUS/CLV or STM pathway regulating meristem function. Furthermore, these studies indicate that ANT, AIL6 and AIL7 have distinct functions within the meristem rather than acting in a strictly redundant manner. Our study thus identifies three new genes whose distinct functions are together required for continuous shoot apical meristem function.

Control of flower size.

Flowers exhibit amazing morphological diversity in many traits, including their size. In addition to interspecific flower size differences, many species maintain significant variation in flower size within and among populations. Flower size variation can contribute to reproductive isolation of species and thus has clear evolutionary consequences. In this review we integrate information on flower size variation from both evolutionary and developmental biology perspectives. We examine the role of flower size in the context of mating system evolution. In addition, we describe what is currently known about the genetic basis of flower size based on quantitative trait locus (QTL) mapping in several different plant species and molecular genetic studies in model plants, primarily Arabidopsis thaliana. Work in Arabidopsis suggests that many independent pathways regulate floral organ growth via effects on cell proliferation and/or cell expansion.

The GUS Reporter System in Flower Development Studies.

The ß-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development in a broad range of species. Transcriptional and translational GUS fusions are used to characterize gene and protein expression patterns, respectively, during reproductive development. Additionally, GUS reporters can be used to map cis-regulatory elements within promoter sequences and to investigate whether genes are regulated post-transcriptionally. Gene trap/enhancer trap GUS constructs can be used to identify novel genes involved in flower development and marker lines useful in mutant characterization. Flower development studies primarily have used the histochemical assay in which inflorescence tissue from transgenic plants containing GUS reporter genes are stained for GUS activity and examined as whole-mounts or subsequently embedded into wax and examined as tissue sections. In addition, quantitative GUS activity assays can be performed on either floral extracts or intact flowers using a fluorogenic GUS substrate. Another use of GUS reporters is as a screenable marker for plant transformation. A simplified histochemical GUS assay can be used to quickly identify transgenic tissues.

AINTEGUMENTA-LIKE6 regulates cellular differentiation in flowers.

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.

Auxin regulation of Arabidopsis flower development involves members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) family.

Auxin is an important regulator of many aspects of plant growth and development. During reproductive development, auxin specifies the site of flower initiation and subsequently regulates organ growth and patterning as well as later events that determine reproductive success. Underlying auxin action in plant tissues is its uneven distribution, resulting in groups of cells with high auxin levels (auxin maxima) or graded distributions of the hormone (auxin gradients). Dynamic auxin distribution within the periphery of the inflorescence meristems specifies the site of floral meristem initiation, while auxin maxima present at the tips of developing floral organ primordia probably mediate organ growth and patterning. The molecular means by which auxin accumulation patterns are converted into developmental outputs in flowers is not well understood. Members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family are important developmental regulators in both roots and shoots. In roots, the expression of two AIL/PLT genes is regulated by auxin and these genes feed back to regulate auxin distribution. Here, several aspects of flower development involving both auxin and AIL/PLT activity are described, and evidence linking AIL/PLT function with auxin distribution in reproductive tissues is presented.

Coordination of growth in root and shoot apices by AIL/PLT transcription factors.

Growth at the root tip and organ generation at the shoot tip depend on the proper functioning of apical meristems and the transitioning of meristematic cell descendants from a proliferating state to cell elongation and differentiation. Members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family, a clade of two-AP2 domain proteins, specify both stem cell fate and control cellular progression of stem cell daughter cells toward differentiation. Here we highlight the importance of an AIL/PLT protein gradient in controlling distinct cellular behaviors in the root through the regulation of distinct targets in different parts of the root tip. Within the shoot, AIL/PLT proteins also promote organ growth and inhibit differentiation pointing to conserved roles in meristem function. However, they exhibit unequal genetic redundancy in these functions and do not always act in a purely additive manner. Differences in AIL/PLT regulation and perhaps transcriptional targets in roots and shoots suggest that these growth regulators have adapted to mediate growth control in distinct ways in these organ systems.

AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain.

The Arabidopsis protein AINTEGUMENTA (ANT) is an important regulator of organ growth during flower development. ANT is a member of the AP2 subclass of the AP2/ERF family of plant-specific transcription factors. These proteins contain either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that ANT can act as a transcriptional activator in yeast through binding to a consensus ANT-binding site. This activity was used as the basis for a genetic screen to identify amino acids that are critical for the DNA binding ability of ANT. Mutants that showed reduced or no activation of a reporter gene under the control of ANT-binding sites were identified. The mutations identified in the screen as well as additional site-directed mutations suggest that the mode of DNA recognition by members of the AP2 subfamily is distinct from that of ERF proteins. Surprisingly, it appears that each AP2 domain of ANT uses different amino acids to contact DNA. Identification of several linker mutations argues that this sequence acts in the positioning of each AP2 domain on the DNA or makes direct DNA contacts.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Developing transgenic Arabidopsis plants to be metal-specific bioindicators.

Deoxyribonucleic acid (DNA) microarrays provide a means to assess genome-wide expression patterns after exposure of an organism to different xenobiotics. Potential uses for this technology include identification of unknown toxicants, assessment of toxicity of new compounds, and characterization of the cellular mechanisms of toxicant action. Here we describe another use of DNA microarrays in toxicant-specific gene discovery. Combining results from two DNA microarray experiments, we have identified genes from the model plant Arabidopsis thaliana that are induced in response to one but not other heavy metals. The promoters of these genes should be useful in developing metal-specific transgenic biomonitors. To test this idea, we have fused the promoter of one of the newly identified Ni-inducible genes (AHB1) to the beta-glucuronidase (GUS) reporter gene. Arabidopsis plants containing the AHBI::GUS transgene show reporter gene activity when they are grown on media containing Ni but not when grown on media containing Cd, Cu, Zn, or without added metals. Thus, this approach has resulted in the creation of a transgenic strain of Arabidopsis that can report on the presence and concentration of Ni in plant growth media. Such transgenic models can serve as cheap and efficient biomonitors of bioavailable heavy metal contamination in soils and sediments.

The GUS reporter system in flower development studies.

The ß-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development. Transcriptional and translational GUS fusions are used to characterize gene and protein expression patterns, respectively, during reproductive development. Additionally, GUS reporters can be used to map cis-regulatory elements within promoter sequences and to investigate whether genes are regulated posttranscriptionally. Gene trap/enhancer trap GUS constructs can be used to identify novel genes involved in flower development and marker lines useful in mutant characterization. Flower development studies primarily have used the histochemical assay in which inflorescence tissue from transgenic plants containing GUS reporter genes are stained for GUS activity and examined as whole-mounts or subsequently embedded into wax and examined as tissue sections. In addition, quantitative GUS activity assays can be performed on either floral extracts or intact flowers using a fluorogenic GUS substrate.

A molecular framework for auxin-mediated initiation of flower primordia.

A classical role of the hormone auxin is in the formation of flowers at the periphery of the reproductive shoot apex. Mutants in regulators of polar auxin transport or in the auxin-responsive transcription factor MONOPTEROS (MP) form naked inflorescence "pins" lacking flowers. How auxin maxima and MP direct initiation of flower primordia is poorly understood. Here, we identify three genes whose expression is directly induced by auxin-activated MP that furthermore jointly regulate flower primordium initiation. These three genes encode known regulators of flower development: LEAFY (LFY), which specifies floral fate, and two AINTEGUMENTA-LIKE/PLETHORA transcription factors, key regulators of floral growth. Our study thus reveals a mechanistic link between flower primordium initiation and subsequent steps in flower morphogenesis. Finally, we uncover direct positive feedback from LFY to the auxin pathway. The auxin LFY module we describe may have been recruited during evolution to pattern other plant organ systems.