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.     

A novel role for the floral homeotic gene APETALA2 during Arabidopsis fruit development

The majority of the Arabidopsis fruit comprises an ovary with three primary tissue types: the valves, the replum and the valve margins. The valves, which are derived from the ovary walls, are separated along their entire length by the replum. The valve margin, which consists of a separation layer and a lignified layer, forms as a narrow stripe of cells at the valve-replum boundaries. The valve margin identity genes are expressed at the valve-replum boundary and are negatively regulated by FUL and RPL in the valves and replum, respectively. In ful rpl double mutants, the valve margin identity genes become ectopically expressed, and, as a result, the entire outer surface of the ovary takes on valve margin identity. We carried out a genetic screen in this sensitized genetic background and identified a suppressor mutation that restored replum development. Surprisingly, we found that the corresponding suppressor gene was AP2, a gene that is well known for its role in floral organ identity, but whose role in Arabidopsis fruit development had not been previously described. We found that AP2 acts to prevent replum overgrowth by negatively regulating BP and RPL, two genes that normally act to promote replum formation. We also determined that AP2 acts to prevent overgrowth of the valve margin by repressing valve margin identity gene expression. We have incorporated AP2 into the current genetic network controlling fruit development in Arabidopsis.     

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.     

Evidence that an evolutionary transition from dehiscent to indehiscent fruits in Lepidium (Brassicaceae) was caused by a change in the control of valve margin identity genes

In the Brassicaceae, indehiscent fruits evolved from dehiscent fruits several times independently. Here we use closely related wild species of the genus Lepidium as a model system to analyse the underlying developmental genetic mechanisms in a candidate gene approach. ALCATRAZ (ALC), INDEHISCENT (IND), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are known fruit developmental genes of Arabidopsis thaliana that are expressed in the fruit valve margin governing dehiscence zone formation. Comparative expression analysis by quantitative RT‐PCR, Northern blot and in situ hybridization show that their orthologues from Lepidium campestre (dehiscent fruits) are similarly expressed at valve margins. In sharp contrast, expression of the respective orthologues is abolished in the corresponding tissue of indehiscent Lepidium appelianum fruits, indicating that changes in the genetic pathway identified in A. thaliana caused the transition from dehiscent to indehiscent fruits in the investigated species. As parallel mutations in different genes are quite unlikely, we conclude that the changes in gene expression patterns are probably caused by changes in upstream regulators of ALC, IND and SHP1/2, possible candidates from A. thaliana being FRUITFULL (FUL), REPLUMLESS (RPL) and APETALA2 (AP2). However, neither expression analyses nor functional tests in transgenic plants provided any evidence that the FUL or RPL orthologues of Lepidium were involved in evolution of fruit indehiscence in Lepidium. In contrast, stronger expression of AP2 in indehiscent compared to dehiscent fruits identifies AP2 as a candidate gene that deserves further investigation.     

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.     

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.     

Silique valves as sails in anemochory of Lunaria (Brassicaceae)

• The generally held opinion that seeds of Lunaria remain at the replum after detachment of the two valves and then wind causes a shaking or rattling of the replum with its diaphragm, thus launching the seeds, is challenged. In a sparse forest in the Swabian Alb, the first author noticed flying valves of Lunaria rediviva to which the narrow‐winged flat seeds are attached.
• Investigations with SEM and histology have shown that the valves secrete a glue only at those sites where the seeds rest on the valves before valve tissues die. Further analysis has shown (using the periodic acid‐Schiff reaction) that the glue consists of polysaccharides. After detachment and dispersal of the valves, the adhesive strength continuously decreases.
• This is the first report for a sticky valve exudate in the Brassicaceae. Because of the adhesion of Lunaria seeds to their valves for some time, the 1st order diaspore is a mericarp, in a broad sense, and can be interpreted as an adaptation to long‐distance dispersal by stronger winds. In this context, the ‘flying carpets’ of Lunaria are more effective and transport more than one seed.
• Molecular studies assigned Lunaria to the tribe Biscutelleae, which now contains the angustiseptate genera Biscutella and Megadenia as well as the latiseptate genera Lunaria and Ricotia. The valves in Ricotia can easily be detached (studied in herbarium material and a living plant), but, in contrast to Lunaria, the ripe seeds remain at the replum and its diaphragm, respectively.

     

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.     

The vernalization gene FRIGIDA in cultivated Brassica species

The repressor FLOWERING LOCUS C (FLC) holds a key position among the genes, which drive Arabidopsis floral transition along the vernalization pathway. The FRIGIDA (FRI) gene activates FLC expression, and the interplay of strong and weak alleles of FLC and FRI in many cases explains the variations in Arabidopsis requirement for cold induction. In annual and biennial life forms of Brassica, the variations in time to flower have been also related to FLC; whereas the place of FRI in the vernalization process has not been sufficiently elucidated. In contrast to Arabidopsis, FRI in Brassica genomes A and C and presumably B is represented by two expressible loci, FRI.a and FRI.b, each of them manifesting genome-specific polymorphisms. FRI.a and FRI.b sequences from diploid species B. rapa (genome A) and B. oleracea (genome C) are conserved (96–99% similarity) in subgenomes A and C of tetraploid species B. carinata (genome BC), B. juncea (genome AB), and B. napus (genome AC). Phylogenetic analysis of FRI sequences in the genus Brassica clearly discerns the lineages A/C and B, while in the family Brassicaceae, two FRI clusters discriminated by such analysis correspond to the lineages I (including the genus Arabidopsis) and II (including the genus Brassica). The origin of two FRI loci is discussed in the context of the Brassicaceae evolution via paleopolyploidy and subsequent genome reorganization.