Keywords: Arabidopsis, ovule primordia, ovule number, development, transcription factors, hormones

Genetic factors controlling carpel margin meristem formation

As already mentioned, the establishment and maintenance of the meristematic tissues of the CMM is inherently correlated to the generation of ovule primordia. CMM development is known to be controlled at the transcriptional level and by hormones as reviewed by Reyes-Olalde et al. (2013). Several single and higher order mutant combinations with strongly reduced carpel marginal tissue development have been described in literature. One of the them is theaintegumenta (ant) mutant. ANT is a transcription factor that contains two AP2 domains that controls organ initiation and promotes cellular divisions during organ development (Klucher et al.,1996). Interestingly, theant-9 mutant has medial ridges that are frequently unfused to each other with a consequent reduction in functional CMM tissue. It has also been reported that theant mutant displays enhanced morphological defects when combined with a mutation inREVOLUTA (REV), a member of the class III Homeodomain-Leucine Zipper (HD-ZIP III) family. In theant rev double mutant a partial disruption of CMM and placenta development causes the reduced development of ovule primordia (Nole-Wilson et al.,2010).

An unfused carpels phenotype due to the compromised fusion between the two medial ridges was also observed in the mutants forLEUNIG (LUG), a floral organ identity gene that encodes a glutamine-rich protein with seven WD repeats, typical of transcriptional co-repressors (Liu et al.,2000). Despite this failure in ridge fusion, ovules are formed from the placenta although in a markedly decreased number in bothlug-1 (intermediate-strength allele) andlug-3 (strong allele) mutants (Table1). The simultaneous loss ofLUG andANT functions enhanced the defects in flower development in respect to the singlelug andant mutants. While the double mutantlug-3 ant-9 did not form any ovules, septum or stigma, nearly 50% of thelug-1 ant-9 pistils could develop normal medial ridges, that gave rise to partially formed septal tissues, although ovules, stigma and style were never present (Liu et al.,2000) (Table1).

ANT also interacts synergistically with SEUSS (SEU), a transcriptional coregulator functionally similar to LEU, in the control of organ size of the flower. While theseu-3 single mutant shows on average ovule numbers not significantly different from wild-type Col-0, the double mutantseu-3 ant-1 results in a complete loss of ovule initiation, caused by severe defects in early gynoecia development. In the weaker allelic combinationseu-3 ant-3, employing theant-3 hypomorphic allele, placenta formation is not compromised but defects such as ovule initiation and gametogenesis are present at later stages (Table1) (Azhakanandam et al.,2008).

Other two players in CMM development are CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, two transcription factors that belong to the NAC transcription factor family. Thecuc1 andcuc2 single mutants display almost no phenotype, while thecuc1 cuc2 double mutant completely lacks the shoot apical meristem (SAM) and the cotyledons are fused along their margin forming a cup-shaped structure. These seedlings die a few days after germination (Aida et al.,1997). Studying gynoecium development in thecuc1 cuc2 double mutant was only possible using plants obtained byin vitro regeneration. They presented defects in the formation of the septum and in ovule development (Ishida et al.,2000). A gene that has been described to play a role withCUC1 andCUC2 in promoting the formation of carpel marginal structures and thus facilitating septum and ovule development isSPATULA (SPT), which encodes a basic helix–loop–helix (bHLH) transcription factor. Mutations inSPT cause a split carpel phenotype in the apical part of the gynoecium. Moreover,spt plants have slightly fewer ovules than the wild type, from which only a small fraction develop into seeds (Nahar et al.,2012). When combined withcuc1 andcuc2 single mutants, the average number of ovules decreases. Thus, while thespt single mutant shows an average of 48 ovules per carpel,spt cuc1 andspt cuc2 present 36 and 32 respectively (Table1), indicating that CUC1, CUC2, and SPT are together required for ovule development. Another mutant that displays an unfused gynoecium at the apex iscrabs claw (crc) (Alvarez and Smyth,1999).CRC encodes a transcription factor of the YABBY family and the characterization of different mutant alleles showed that, besides the failure of the fusion of the stylar region,crc mutants present a gradation of phenotypes with wider and shorter gynoecia that contain fewer ovules compared to the wild type (Alvarez and Smyth,1999; Bowman and Smyth,1999).

Thereby, the phenotype of ovule reduction that we frequently observe in the mutants defective in medial ridge fusion and thus in CMM formation could be due, at least in part, to their role in regulating cell proliferation in the medial ridges, from which septum and ovules originate.

CMM formation and the auxin gradient

Auxin is a key hormone for plant development, and it is also fundamental for gynoecium and thereby CMM and ovule development. In the last two decades several studies have demonstrated that local auxin biosynthesis and polar transport are responsible for the correct apical–basal patterning of the gynoecium. The auxin gradient hypothesis supports that high levels of auxin in the gynoecium apical regions control stigma and style formation; medium levels direct ovary formation whereas low levels of the hormone are responsible of gynophore development at the gynoecium base (Nemhauser et al.,2000). Indeed, all mutants in which the auxin synthetic pathway or transport are compromised have a similar severe gynoecium phenotype forming a pistil-like structure with reduction/absence of the valves, expansion of the gynophore and stylar regions and serious vasculature defects (reviewed in Balanzá et al.,2006; Larsson et al.,2013). This phenotype was characterized for the first time in the flowers ofpin-formed1-1 (pin1-1), a strong mutant allele of the auxin efflux carrier PIN1 (Okada et al.,1991) and in thepinoid mutant, a knock-out line for a serine/threonine kinase that regulates PINs polarity (Bennett et al.,1995). Other examples of mutants with similar pistil-like structure phenotypes are theyucca1 yucca4 (yuc1 yuc4) andweak ethylene insensitive8 tryptophan aminotransferase related2 (wei8 tar2) double mutants, in which local auxin production is impaired (Cheng et al.,2006; Stepanova et al.,2008). Predictably, in most of these auxin-related mutants the severe defects in gynoecium formation lead to a pistil with a reduction or complete absence of ovules and the consequent complete sterility.

Nemhauser et al. (2000) confirmed the importance of polar auxin transport (PAT) in gynoecium development through an experiment in which they used 1-napthylphthalamic acid (NPA), an inhibitor of the auxin transport. They showed that NPA application caused significant loss of ovules. The authors also highlighted that ovules seemed more sensitive to disruption in PAT, with respect to the other tissues of the gynoecium. Indeed, treated carpels were largely devoid of ovules but were still able to produce valves. In 2010 Nole-Wilson and collaborators proposed the connection between ANT and the hormone auxin on the base of the observation that theant mutant is more sensitive than the wild type to alteration in PAT. Moreover, the expression of a subset of auxin-related genes was altered in theant single andant rev double mutant gynoecia, indicating that the morphological defects of theant rev double mutants, at least in part, are due to an alteration in auxin homeostasis in these plants.

Auxin signaling is primarily regulated by theAUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) proteins. The phenotype ofARF5/MONOPTEROS (MP) strong mutant alleles results in an embryo lethal phenotype, whilemp partial loss of function mutants have normal embryo development whereas that their reproductive development is compromised (Hardtke and Berleth,1998). In the pistil of thempS319 weak allele the CMM does not develop, and placenta and ovules are completely missing (Cole et al.,2009; Galbiati et al.,2013). Interestingly, MP has been demonstrated to directly activate theANT, CUC1 andCUC2 transcription factors encoding genes (Galbiati et al.,2013) Their role as major players in ovule primordia initiation and ovule number determination will be discussed in the following sections.

The establishment of the boundaries

When new organ primordia are originated in the plant, two different regions, the boundaries and the zone of primordia outgrowth, need to be defined. The organ boundary is defined as the region between the meristem and the developing organ, or, as in the case of ovules, as the region between two adjacent ovule primordia. As Aida and Tasaka nicely reviewed in 2006, the “boundary cells” need to have peculiar characteristics respect to the surrounding cells, usually displaying reduced cell division and expansion. Another important aspect is the arrangement of the plasmodesmata that regulates the movement of transcription factors between cells. For example, the boundaries in the inflorescence meristem seem to restrict the passage of proteins into flower primordia (Wu,2003).

The boundary-specific regulatory genes play a critical role in orchestrating several morphogenetic and patterning events and their spatial coordination. When this coordination is missing, fusion between organs is the most frequent observed phenotype (Aida et al.,1997). TheCUC gene family was the first discovered to have a fundamental role in organ boundary establishment. In fact, in thecuc1cuc2 double mutant embryo the cotyledons do not separate (Aida et al.,1997).

The transcripts ofCUC1 andCUC2 were detected byin situ hybridization in the anlagen placenta and in ovules at stage 1-II and later on, starting from stage 2-I, restricted to the boundary between two ovules (Ishida et al.,2000; Galbiati et al.,2013). As we already mentioned, the study of the gynoecium phenotype of thecuc1 cuc2 double mutant was only possible on plants regeneratedin vitro. They showed defects in the formation of the septum and in ovule development; most of the gynoecia having less than 10 ovules (Table1). However, thecuc1 cuc2 double mutant plants never gave seeds (Ishida et al.,2000). A further demonstration that CUC1 and CUC2 are directly linked to the determination of ovule number in a direct way came from the work of Galbiati et al. (2013). In order to study the ovule phenotype in absence of bothCUC1 andCUC2, CUC1 was silenced in acuc2-1 mutant background using aCUC1 specific RNAi construct under the control of the ovule-specificSEEDSTICK promoter (pSTK:CUC1_RNAi) which is already active in the placenta before ovule primordia arise. The analysis ofcuc2-1 pSTK:CUC1_RNAi plants revealed a reduction in ovule number of 20% (Table1). Furthermore,ant-4 cuc2-1 pSTK::CUC1_RNAi plants were generated in order to analyze the possible additive role of ANT to CUC function in the regulation of ovule primordia formation. Theant-4 cuc2-1 pSTK::CUC1_RNAi plants displayed a further dramatic reduction in the number of developing ovules (a mean of seven ovule primordia per pistil), while the single mutantant-4 and the plantscuc2-1 pSTK::CUC1_RNAi showed 20 and 30 ovules per pistil, respectively (Table1). Despite the reduction in ovule number in the different mutant backgrounds, the size of the pistils was not reduced. Therefore, the ovules were more distantly spaced compared to those in wild-type pistils (Galbiati et al.,2013). These studies of the characterization of theant single andcuc1 cuc2 double mutants, as well asant-4 cuc2-1 pSTK::CUC1_RNAi plants prove that ANT, CUC1 and CUC2 are key players in the control of the number of ovule primordia that develop from the placenta and that they act additively (Elliott et al.,1996; Ishida et al.,2000; Galbiati et al.,2013). All the information about these factors taken together indicates that they work in different ways: while ANT promotes ovule primordia growth, the CUCs play a role in the establishment of the ovule primordia boundaries (Figure2).

CUC3, another putative NAC-domain transcription factor member of theCUC family, is expressed in an extensive range of boundaries in adult plants. Besides, the function of CUC3 is partially redundant with that of its homologous CUC1 and CUC2 in the establishment of the cotyledon boundary (Vroemen et al.,2003). Several studies revealed thatCUC expression is controlled and restricted to the boundaries in several ways. For instance, in the SAMCUC1 andCUC2 but notCUC3 are regulated bymiR164, which restricts the expression ofCUC1 andCUC2 mRNAs to the boundary domain (Laufs et al.,2004). In the carpel, in a similar way to the pattern already described forCUC1 andCUC2, CUC3 expression marks the boundaries between ovule primordia. Therefore, it would be interesting to study also the contribution ofCUC3 in the regulation of defining ovule boundaries.

In 2008, Xu and Shen showed that three different transcription factors, ASSYMETRIC LEAVES 1 (AS1), AS2, and JAGGED (JAG), support normal sepal and petal growth by restricting the expression domain of the boundary-specifying genesCUC1 andCUC2. AS1 and AS2 were already suggested to have roles in boundary control, given that they positive regulate, within the shoot apex, the members of theLATERAL ORGAN BOUNDARIES (LOB) gene family, a plant-specific family of transcription factors that are expressed in the boundaries (Byrne et al.,2002).LOB, the gene that names the family, is expressed at the base of all lateral organs. Interestingly, plants overexpressingLOB produced abnormal flowers with reduced floral organs and they were sterile even when fertilized with wild-type pollen (Shuai et al.,2002). Lee et al. in 2009 identified two new MYB transcription factors involved in lateral organ separation:LATERAL ORGAN FUSION 1 (LOF1) andLOF2. The single mutantlof1 exhibits a novel fusion between the axillary stem and the cauline leaf. Additional fusions resulted whenlof1 was combined withlof2, cuc2 orcuc3, indicating the existence of overlapping roles forLOF1, CUC2, andCUC3 to control organ separation during reproductive development.

Despite the identification of a number of boundary-specific transcription factors, boundary formation and maintenance is still a poorly understood process, and only CUC1 and CUC2 have been demonstrated to have a role in ovule boundary establishment. The factors that have been described to regulate or interact with the CUCs in a different developmental context could also have a role during ovule initiation, and some of them, likeAS1 andAS2 are already known to be expressed in the gynoecium and ovules (Xu and Shen,2008).

Aintegumenta, a master regulator of primordia formation

In Arabidopsis many genes have been described to play roles in the different phases of ovule development, although most of them do not determine directly the number of ovules (Schneitz et al.,1997; reviewed in Shi and Yang,2011). However, the ANT transcription factor has been described to have a clear role in ovule primordia formation.In situ hybridization experiments showed that within the carpel it is expressed in the placenta and in the integuments of the developing ovules. Inant plants ovules do not develop integuments and megasporogenesis is blocked at the tetrad stage leading to complete female-sterility (Elliott et al.,1996). ANT is not only required for ovule development but it is also involved in ovule primordia formation. Indeed, in theant-9 mutant the number of ovules per carpel is reduced by more than half in respect to the wild-type (Table1). Given that theant gynoecia have the same length as those of wild type, the ovules that do arise inant are more distantly spaced than in wild-type plants (Liu et al.,2000).

In addition toANT, another essential gene for the regulation of ovule primordia outgrowth and for the control of integument formation isHUELLENLOS (HLL), a gene that encodes a mitochondrial ribosomal protein. Thus, plants presenting mutations inHLL display a phenotype similar toant at the level of ovule integuments (Schneitz et al.,1997,1998). Moreover,hll-1 andhll-3 mutant alleles display a reduction of about 10% in the number of ovules, although the authors also describe thathll plants display smaller gynoecia, which could contribute to the development of fewer ovules (Table1). The phenotype of the double mutanthll ant was more severe at the level of primordia outgrowth however, nothing was described regarding ovule number (Schneitz et al.,1998; Skinner et al.,2001). A similar phenotype tohll was observed in theshort integuments 2 (sin2) mutants. Apart of an arrest in cell division in both ovule integuments,sin2 plants presented shorter pistils bearing less ovules than the wild type (Table1). Moreover, the authors describe an abnormal distribution of the ovules along the placenta, being the distance between ovules bigger than in wild-type plants (Broadhvest et al.,2000). Thus, in this particular case the shorter carpel might not be the only cause of reduced ovule numbers. The double mutantsin2 ant-5 was not different fromant-5 single mutant, indicating thatANT is epistatic toSIN2 with respect to ovule development. On the contrary,sin2 hll-1 double mutant had a stronger effect on ovule development thansin2 orhll-1 single mutants (Broadhvest et al.,2000). All these experiments taken together indicate that although ANT plays a master role, SIN2 and HLL also contribute to ovule primordia formation.

Auxin is required for ovule primordia formation

As we previously underlined, the boundary region and the primordia formation zone are highly interconnected. It has been demonstrated that a fundamental role of the “boundary transcription factors” is to organize PAT, mediated by PIN proteins, in order to create a zone of auxin maximum where organ founder cells will be selected. Auxin maxima are fundamental for the formation of primordia, and auxin action has been well described for lateral roots (LR) and flower primordia (reviewed in Benková et al.,2003,2009; Yamaguchi et al.,2013). The directionality of auxin flux depends principally on the polar localization of the PIN proteins. In Arabidopsis there are eight PIN proteins (PIN1-8), from which onlyPIN1 andPIN3 are expressed in the pistil and ovules (Benková et al.,2003; Ceccato et al.,2013). PIN1 protein is localized at the membrane of placenta cells and later on, in the developing ovules, it is restricted to the lateral-apical membranes of nucellus cells. PIN3 is also present in few cells at the tip of the developing nucellus shortly after ovule primordia emergence but, contrary to PIN1, it is not expressed in the placenta cells (Ceccato et al.,2013). PIN-dependent efflux mediates primordium development by supplying auxin to the tip creating an auxin maxima; indeed in plants expressing theGFP reporter gene downstream the auxin-responsiveDR5 promoter (pDR5::GFP), the GFP signal is detected at the tip of all ovule primordia (Benková et al.,2003). The weakpin1-5 mutant allele is able to develop some flowers in which the pistils have slightly reduced valves but normal styles and stigmas (Sohlberg et al.,2006). The pistils of thepin1-5 weak allele have an average of 9 ovules per carpel (Table1) (Bencivenga et al.,2012). In addition, Galbiati et al. (2013) demonstrated that the reduced number of ovules incuc2-1 pSTK:CUC1_RNAi was caused by a down-regulation ofPIN1 and an incorrect PIN1 protein localization. CUC1 and CUC2 promotePIN1 expression and localization to correctly form the auxin maximum where primordium will form (Figure2). In the same way, a CUC2-dependent regulatory pathway controlling PIN1-mediated auxin efflux has been described to explain leaf serrations (Bilsborough et al.,2011). Moreover, in the newly formed primordia of the SAM the auxin maxima, in a negative feed-back loop, repressCUC2 expression and restricts it to the boundaries (Vernoux et al.,2000; Heisler et al.,2005; and reviewed in Aida and Tasaka,2006; Rast and Simon,2008). A similar inhibitory loop could controlCUC expression at the ovule boundaries (Figure2). The phenotype ofcuc2-1 pSTK:CUC1_RNAi was completely recovered by cytokinin (CK) application, since CK has been demonstrated to increasePIN1 expression in the ovules (Bencivenga et al.,2012). These experiments evidence a convergence of two different plant hormones in the regulation of ovule primordia formation. In the next paragraph we will delve deeper into the role of CK in the formation and determination of ovule number.

Cytokinin positively regulates ovule number

CK is an essential hormone for plant growth and development as it has a central role in the regulation of cell division and differentiation. In the last 10 years, several studies have clearly proven that CK has also a significant role during ovule development. As it will be explained in this paragraph, it has been demonstrated that in plants that are defective in the production or perception of this hormone, correct ovule formation is compromised and/or the number of ovule is drastically reduced. CK signaling, which has been recently summarized in a detailed review article (Hwang et al.,2012), is mediated by a two-component signaling pathway: histidine protein kinases (AHKs) work as CK receptors, while histidine phosphotransfer proteins (AHPs) transmit the signal from AHKs to nuclear response regulators (ARRs), which are able to regulate transcription. InArabidopsis the CK signal is perceived by three histidine kinases:ARABIDOPSIS HISTIDINE KINASE4 (AHK4, also known asCYTOKININ RESPONSE1, CRE1/WOODEN LEG, WOL),AHK2 andAHK3. These three genes are all expressed in inflorescences, carpels and developing ovules (Higuchi et al.,2004; Nishimura et al.,2004). More precisely,AHK2 andAHK3 are expressed during all stages of ovule development, starting from early primordia stages to ovule maturity, whereasCRE1 expression remains restricted to the chalazal region and later to the integuments of ovules during all the developmental stages (Bencivenga et al.,2012). The single and double mutants ofAHKs do not present any phenotype at the level of the ovules (Higuchi et al.,2004). However, mutants lacking all three receptors exhibit no perception of CK and present a strong slowdown of shoot and root growth. The resulting miniature plants also show delayed flower induction and impaired fertility (Higuchi et al.,2004; Nishimura et al.,2004; Riefler et al.,2006). Thus, the triple mutantcre1-12 ahk2-2 ahk3-3 do not produce seeds (Higuchi et al.,2004) because the gametophyte arrests at stage FG1-FG2 (Bencivenga et al.,2012). Moreover, a severe reduction in the ovule number, an average of 5 ovule per pistil, was noticed in these triple mutant plants (Table1) (Bencivenga et al.,2012). A similar sterile phenotype was also observed for another allelic combination: theahk4-1 ahk2-1 ahk3-1 triple mutant (Nishimura et al.,2004). Differently, Riefler et al. (2006) obtained a weaker triple mutantcre1-2 ahk2-5 ahk3-7 that self-fertilized and formed few seeds, suggesting that infertility of the histidine kinase triple mutants is a phenotype associated with specific mutant alleles.

Attention has also been given to the importance of CK catabolism. InArabidopsis the irreversible degradation of CK is catalyzed by the oxidase/dehydrogenase (CKX). TheCKX gene family of Arabidopsis consists of seven members (CKX1 toCKX7), and bypromoter:GUS fusion constructs it was shown thatCKX1, CKX5, andCKX6 (At3g63440, previously calledAtCKX7) are expressed in flower tissues, beingCKX6 the only one reported to be expressed in the carpel and ovules, in particular in the funiculus (Werner et al.,2003). Werner and colleagues engineered transgenicArabidopsis plants that individually overexpressed six differentCKXs in order to enhance CK degradation. As expected, these plants manifested phenotypes linked to CK deficiency, like delayed vegetative growth and leaf expansion, diminished activity and size of the SAM but increased overall root system. The reproductive development of CK-deficient plants was also altered. In35S::CKX1 and35S::CKX3 plants, flowering was strongly delayed and furthermore the fertility of flowers was heavily reduced, partially due to the lack of pollen.35S::CKX1 and35S::CKX3 siliques were not filled completely and they formed approximately 8–20 viable seeds, whereas the wild-type siliques harbored up to 60 seeds. Although the number of ovules formed in these plants was not reported in this work, the expression patterns together with the phenotypes in the flowers and fruits indicate once more that CK play a role during reproduction. Moreover, the authors suggest a role for ANT in the observed reduced cell division in the leaves ofckx plants. Considering the documented role of ANT in ovule primordia initiation already introduced in this article, it will be very interesting to analyze also its role in the reproductive tissues of these plants.

With an opposite experimental approach, the simultaneous mutations of twoCKX genes, it was demonstrated that plants with an increased level of CK had an enhanced activity of the reproductive meristem (Bartrina et al.,2011). Indeed, theckx3-1 ckx5-1 double mutant produced more flowers due to a larger inflorescence meristem with more cells than the wild type. Moreover, flowers were bigger and so were the gynoecia. Besides, double mutant gynoecia contained twice as many ovules as wild-type ones, indicating an increased activity of their placental tissue. Theckx3-1 andckx5-1 single mutants already developed more ovules than the wild-type, and the flower size and the number of ovules was reflected into the length of the fruits (siliques ofckx3 ckx5 were 20 mm long compared with the 17 mm of the wild type) and the seed number (110 seeds in theckx3 ckx5 mutant siliques compared with an average of 65 seeds in wild-type siliques, Table1) The authors suggested that CKX3 and CKX5 may regulate the activity of meristematic cells in the placenta thus affecting organogenic capacity and ovule primordia formation.

A conclusive evidence about the relationship between the levels of CK and the initiation of ovule formation was obtained from experiments in which inflorescences were treated with synthetic CK (6-Benzylaminopurine, BAP). The treatment resulted in the formation of new primordia, 20 ± 3 primordia in average in each pistil, positioned between the ovules already formed before the CK application (Bencivenga et al.,2012). An equivalent CK treatment was also able to increase the ovule number inpSTK::CUC1_RNAi cuc2 plants already described in this review, by acting on the expression and localization of the auxin efflux carrier PIN1 (Galbiati et al.,2013). These results point out the importance of the cross-talk between CK and auxin during ovule primordia formation. However, the hormonal cross-talk is not limited to auxin and CK since very recently it has been demonstrated that also brassinosteroids (BR) play a crucial role in ovule and seed formation by regulating the expression of genes that control ovule development (Huang et al.,2012), as will be explained in the next paragraph.

The role of brassinosteroids

BRs are hormones known to control general plant development. More specifically, they have been described as involved in the control of the initiation and formation of reproductive organs (Szekeres et al.,1996; Kim et al.,2005). Huang et al. (2012) found that the BR-deficient and -insensitive mutants have smaller and less seeds, while BR-enhanced mutants have more seeds. The analysis of the number of ovules and seeds and the morphological analysis of the siliques ofdet-2 (a BR-deficient mutant involved in BR biosynthesis),bri1-5 (the mutant for the BR receptor), heterozygous plants forbin2-1 (a gain of function mutant deficient in BR signaling) andbzr1-1D (a BR signal-enhanced mutant) leaded to the conclusion that BR signaling positively regulates ovule number (Table1) (Huang et al.,2012). Specifically, it was found that the transcription factor BRZ1 plays an important role in ovule and seed number determination, depending on its state of phosphorylation/dephosphorylation (more dephosphorylation implying more activity and more ovules and seeds).

By treating plants with BR it was shown that BR influences ovule development through regulating the transcription of genes such asHLL andANT, which are redundant in the control of ovule primordia growth as already introduced in this review (Schneitz et al.,1998), andAP2, that affects floral organ (including ovule) pattern formation (Modrusan et al.,1994).HLL andANT are clearly induced by BR, whileAP2 is slightly repressed by BR. These genes appeared to be targets of BRZ1, and its state of phosphorylation/dephosphorylation influences the expression of these genes. Further analysis indicated thatAP2 andANT are direct targets of BRZ1, whileHLL is regulated by an indirect way. The analysis of ovule number ofbzr1-1D andap2-5 single mutants andbzr1-1D ap2-5 double mutant (Table1), together with other molecular proofs, indicate that BZR1 and AP2 play antagonistic effects in ovule number determination, being BZR1 (and HLL and ANT) promoters and AP2 inhibitor of ovule primordia formation (Huang et al.,2012).

A model for ovule primordia formation that integrates the molecular and hormonal networks has been proposed by Galbiati et al. (2013): MP is required forANT, CUC1 andCUC2 expression during the early stages of placenta development and ovule primordia formation, being ANT expressed in the ovule primordia, whereasCUC1 andCUC2 in the ovule boundaries. CUC1 and CUC2 may be involved in the increase of CKs required for properPIN1 expression needed for primordia formation. Once the primordia have formed, auxin accumulates at the edge of the developing ovule. This model can be easily extended with the recently discovered role of the plant hormones BR, which positively regulate the number of ovule primordia, in part by the direct regulation ofANT by BZR1 (Figure2).

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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