Common regulatory networks in leaf and fruit patterning revealed by mutations in the Arabidopsis ASYMMETRIC LEAVES1 gene

Carpels and leaves are evolutionarily related organs, as the former are thought to be modified leaves. Therefore, developmental pathways that play crucial roles in patterning both organs are presumably conserved. In leaf primordia of Arabidopsis thaliana, the ASYMMETRIC LEAVES1 (AS1) gene interacts with AS2 to repress the class I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP), KNAT2 and KNAT6, restricting the expression of these genes to the meristem. In this report, we describe how AS1, presumably in collaboration with AS2, patterns the Arabidopsis gynoecium by repressing BP, which is expressed in the replum and valve margin, interacts in the replum with REPLUMLESS (RPL), an essential gene for replum development, and positively regulates the expression of this gene. Misexpression of BP in the gynoecium causes an increase in replum size, while the valve width is slightly reduced, and enhances the effect of mutations in FRUITFULL (FUL), a gene with an important function in valve development. Altogether, these findings strongly suggest that BP plays a crucial role in replum development. We propose a model for pattern formation along the mediolateral axis of the ovary, whereby three domains (replum, valve margin and valve) are specified by the opposing gradients of two antagonistic factors, valve factors and replum factors, the class I KNOX genes working as the latter.     

The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit

The Arabidopsis fruit forms a seedpod that develops from the fertilized gynoecium. It is mainly comprised of an ovary in which three distinct tissues can be differentiated: the valves, the valve margins and the replum. Separation of cells at the valve margin allows for the valves to detach from the replum and thus dispersal of the seeds. Valves and valve margins are located in lateral positions whereas the replum is positioned medially and retains meristematic properties resembling the shoot apical meristem (SAM). Members of the WUSCHEL‐related homeobox family have been involved in stem cell maintenance in the SAM, and within this family, we found that WOX13 is expressed mainly in meristematic tissues including the replum. We also show that wox13 loss‐of‐function mutations reduce replum size and enhance the phenotypes of mutants affected in the replum identity gene RPL. Conversely, misexpression of WOX13 produces, independently from BP and RPL, an oversized replum and valve defects that closely resemble those of mutants in JAG/FIL activity genes. Our results suggest that WOX13 promotes replum development by likely preventing the activity of the JAG/FIL genes in medial tissues. This regulation seems to play a role in establishing the gradient of JAG/FIL activity along the medio‐lateral axis of the fruit critical for proper patterning. Our data have allowed us to incorporate the role of WOX13 into the regulatory network that orchestrates fruit patterning.     

Brassicaceae INDEHISCENT genes specify valve margin cell fate and repress replum formation

Members of the Brassicaceae family, including Arabidopsis thaliana and oilseed rape (Brassica napus), produce dry fruits that open upon maturity along a specialised tissue called the valve margin. Proper development of the valve margin in Arabidopsis is dependent on the INDEHISCENT (IND) gene, the role of which in genetic and hormonal regulation has been thoroughly characterised. Here we perform phylogenetic comparison of IND genes in Arabidopsis and Brassica to identify conserved regulatory sequences that are responsible for specific expression at the valve margin. In addition we have taken a comparative development approach to demonstrate that the BraA.IND.a and BolC.IND.a genes from B. rapa and B. oleracea share identical function with Arabidopsis IND since ethyl methanesulphonate (EMS) mutant alleles and silenced transgenic lines have valve margin defects. Furthermore we show that the degree of these defects can be fine‐tuned for crop improvement. Wild‐type Arabidopsis produces an outer replum composed of about six cell files at the medial region of the fruits, whereas Brassica fruits lack this tissue. A strong loss‐of‐function braA.ind.a mutant gained outer replum tissue in addition to its defect in valve margin development. An enlargement of replum size was also observed in the Arabidopsis ind mutant suggesting a general role of Brassicaceae IND genes in preventing valve margin cells from adopting replum identity.     

A genetic framework for fruit patterning in Arabidopsis thaliana

In the model plant Arabidopsis thaliana, the establishment of organ polarity leads to the expression of FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) on one side of an organ. One important question that has remained unanswered is how does this positional information lead to the correct spatial activation of genes controlling tissue identity? We provide the first functional link between polarity establishment and the regulation of tissue identity by showing that FIL and YAB3 control the non-overlapping expression patterns of FRUITFULL (FUL) and SHATTERPROOF (SHP), genes necessary to form stripes of valve margin tissue that allow the fruit to shatter along two defined borders and disperse the seeds. FIL and YAB3 activate FUL and SHP redundantly with JAGGED (JAG), a gene that also promotes growth in organs, indicating that several pathways converge to regulate these genes. These activities are negatively regulated by REPLUMLESS (RPL), which divides FIL/JAG activity, creating two distinct stripes of valve margin.     

The same regulatory point mutation changed seed-dispersal structures in evolution and domestication

It is unclear whether gene regulatory changes that drive evolution at the population and species levels [1-3] can be extrapolated to higher taxonomic levels. Here, we investigated the role of cis-regulatory changes in fruit evolution within the Brassicaceae family. REPLUMLESS (RPL, At5g02030) controls development of the replum, a structure with an important role in fruit opening and seed dispersal [6]. We show that reduced repla resembling the Arabidopsis rpl mutant correlated across the Brassicaceae with a point mutation in a conserved cis-element of RPL. When introduced in Arabidopsis, this nucleotide change specifically reduced RPL expression and function in the fruit. Conversely, Brassica RPL containing the Arabidopsis version of the cis-element was sufficient to convert the Brassica replum to an Arabidopsis-like morphology. A mutation in the same nucleotide position of the same cis-element in a RPL ortholog has been independently selected to reduce seed dispersal during domestication of rice, in spite of its very different fruit anatomy. Thus, single-nucleotide regulatory mutations at the same position explain developmental variation in seed-dispersal structures at the population and family levels and suggest that the same genetic toolkit is relevant to domestication and natural evolution in widely diverged species.     

Antagonistic Gene Activities Determine the Formation of Pattern Elements along the Mediolateral Axis of the Arabidopsis Fruit

The Arabidopsis fruit mainly consists of a mature ovary that shows three well defined territories that are pattern elements along the mediolateral axis: the replum, located at the medial plane of the flower, and the valve and the valve margin, both of lateral nature. JAG/FIL activity, which includes the combined functions of JAGGED (JAG), FILAMENTOUS FLOWER (FIL), and YABBY3 (YAB3), contributes to the formation of the two lateral pattern elements, whereas the cooperating genes BREVIPEDICELLUS (BP) and REPLUMLESS (RPL) promote replum development. A recent model to explain pattern formation along the mediolateral axis hypothesizes that JAG/FIL activity and BP/RPL function as antagonistic lateral and medial factors, respectively, which tend to repress each other. In this work, we demonstrate the existence of mutual exclusion mechanisms between both kinds of factors, and how this determines the formation and size of the three territories. Medial factors autonomously constrain lateral factors so that they only express outside the replum, and lateral factors negatively regulate the medially expressed BP gene in a non-autonomous fashion to ensure correct replum development. We also have found that ASYMMETRIC LEAVES1 (AS1), previously shown to repress BP both in leaves and ovaries, collaborates with JAG/FIL activity, preventing its repression by BP and showing synergistic interactions with JAG/FIL activity genes. Therefore AS gene function (the function of the interacting genes AS1 and AS2) has been incorporated in the model as a new lateral factor. Our model of antagonistic factors provides explanation for mutant fruit phenotypes in Arabidopsis and also may help to understand natural variation of fruit shape in Brassicaceae and other species, since subtle changes in gene expression may cause conspicuous changes in the size of the different tissue types.     

Genetic Interactions That Regulate Inflorescence Development in Arabidopsis

In Arabidopsis, floral meristems arise in continuous succession directly on the flanks of the inflorescence meristem. Thus, the pathways that regulate inflorescence and floral meristem identity must operate both simultaneously and in close spatial proximity. The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis is required for normal inflorescence meristem function, and the LEAFY (LFY), APETALA 1 (AP1), and APETALA 2 (AP2) genes are required for normal floral meristem function. We present evidence that inflorescence meristem identity is promoted by TFL1 and that floral meristem identity is promoted by parallel developmental pathways, one defined by LFY and the other defined by AP1/AP2. Our analysis suggests that the acquisition of meristem identity during inflorescence development is mediated by antagonistic interactions between TFL1 and LFY and between TFL1 and AP1/AP2. Based on this study, we propose a simple model for the genetic regulation of inflorescence development in Arabidopsis. This model is discussed in relation to the proposed interactions between the inflorescence and the floral meristem identity genes and in regard to other genes that are likely to be part of the genetic hierarchy regulating the establishment and maintenance of inflorescence and floral meristems.     

Separation of genetic functions controlling organ identity in flowers

Comparative studies on the ABC model of floral development have revealed extensive conservation of B and C class genes, but have failed to identify similar conservation for A class genes. Using a reverse genetic approach, we show that the previous inability to obtain Antirrhinum mutants corresponding to the A class gene AP2 of Arabidopsis reflects greater genetic redundancy in Antirrhinum . Antirrhinum has two genes corresponding to AP2, termed LIP1 and LIP2, both of which need to be inactivated to give a mutant phenotype. Analysis of interactions between LIP and class B/C genes shows that unlike AP2 in Arabidopsis, LIP genes are not required for repression of C in outer whorls of the flower. However, like AP2, LIP genes play a role in sepal, petal and ovule development, although some of their detailed effects are different, reflecting the diverse morphologies of Antirrhinum and Arabidopsis flowers. The dual functions for which AP2 is required in Arabidopsis are therefore separate in Antirrhinum, showing that the genetic basis of some aspects of organ identity have undergone major evolutionary change.     

Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development

Several MADS box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core eudicot euAP1 and euFUL genes, but notably, the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae specific. Understanding how functional divergence of the core eudicot duplicates occurred requires a careful examination of the function of preduplication (FUL-like) genes. Using virus-induced gene silencing, we show that FUL-like genes in opium poppy (Papaver somniferum) and California poppy (Eschscholzia californica) function in axillary meristem growth and in floral meristem and sepal identity and that they also play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.     

Physiological and morphological changes during early and later stages of fruit growth in Capsicum annuum

Fruit‐set involves a series of physiological and morphological changes that are well described for tomato and Arabidopsis, but largely unknown for sweet pepper (Capsicum annuum). The aim of this paper is to investigate whether mechanisms of fruit‐set observed in Arabidopsis and tomato are also applicable to C. annuum. To do this, we accurately timed the physiological and morphological changes in a post‐pollinated and un‐pollinated ovary. A vascular connection between ovule and replum was observed in fertilized ovaries that undergo fruit development, and this connection was absent in unfertilized ovaries that abort. This indicates that vascular connection between ovule and replum is an early indicator for successful fruit development after pollination and fertilization. Evaluation of histological changes in the carpel of a fertilized and unfertilized ovary indicated that increase in cell number and cell diameter both contribute to early fruit growth. Cell division contributes more during early fruit growth while cell expansion contributes more at later stages of fruit growth in C. annuum. The simultaneous occurrence of a peak in auxin concentration and a strong increase in cell diameter in the carpel of seeded fruits suggest that indole‐3‐acetic acid stimulates a major increase in cell diameter at later stages of fruit growth. The series of physiological and morphological events observed during fruit‐set in C. annuum are similar to what has been reported for tomato and Arabidopsis. This indicates that tomato and Arabidopsis are suitable model plants to understand details of fruit‐set mechanisms in C. annuum.     

The Arabidopsis floral homeotic gene APETALA3 differentially regulates intercellular signaling required for petal and stamen development

Cell-cell signaling is crucial for the coordination of cell division and differentiation during plant organogenesis. We have developed a novel mosaic analysis method for Arabidopsis, based on the maize Ac/Ds transposable element system, to assess the requirements of individual genes in intercellular signaling. Using this strategy, we have shown that the floral homeotic APETALA3 (AP3) gene has distinct roles in regulating intercellular signaling in different tissues. In petals, AP3 acts primarily in a cell-autonomous fashion to regulate cell type differentiation, but its function is also required in a non-cell-autonomous fashion to regulate organ shape. In contrast, AP3-regulated intercellular interactions are required for conferring both cell type identity and organ shape and size in the stamens. Using antibodies raised against AP3, we have shown that the AP3 protein does not traffic between cells. These observations imply that AP3 acts by differentially regulating the production of intercellular signals in a whorl-specific manner.     

Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families - S15a, L7 and P2 - contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.