MADS-box protein complexes control carpel and ovule development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.     

The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice

Genes that control ovule identity were first identified in Petunia. Co-suppression of both FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, two D-lineage genes, resulted in the homeotic transformation of ovules into carpelloid structures. Later in Arabidopsis it was shown that three genes, SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK), redundantly control ovule identity, because in the stk shp1 shp2 triple mutant ovules lose identity and are transformed into carpel and leaf-like structures. Of these three Arabidopsis genes STK is the only D-lineage gene, and its expression, like FBP7 and FBP11, is restricted to ovules. OsMADS13 is the rice ortholog of STK, FBP7, and FBP11. Its amino acid sequence is similar to the Arabidopsis and Petunia proteins, and its expression is also restricted to ovules. We show that the osmads13 mutant is female sterile and that ovules are converted into carpelloid structures. Furthermore, making carpels inside carpels, the osmads13 flower is indeterminate, showing that OsMADS13 also has a function in floral meristem determinacy. OsMADS21 is most likely to be a paralog of OsMADS13, although its expression is not restricted to ovules. Interestingly, the osmads21 mutant did not show any obvious phenotype. Furthermore, combining the osmads13 and the osmads21 mutants did not result in any additive ovule defect, indicating that osmads21 does not control ovule identity. These results suggest that during evolution the D-lineage gene OsMADS21 has lost its ability to determine ovule identity.     

Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.     

Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis

In Arabidopsis thaliana and many other plant species, ovules arise from carpel tissue as new meristematic formations. Cell fate in proliferating ovule primordia is specified by particular ovule identity factors, such as the homeodomain factor BELL1 (BEL1) and MADS box family members SEEDSTICK (STK), SHATTERPROOF1 (SHP1), SHP2, and AGAMOUS. Both in the bel1 mutant and the stk shp1 shp2 triple mutant, integuments are transformed into carpelloid structures. Combining these mutants in a bel1 stk shp1 shp2 quadruple mutant, we showed that the bel1 phenotype is significantly enhanced. We also demonstrate that ovule differentiation requires the regulation of the stem cell maintenance gene WUSCHEL, repression of which is predominantly maintained by BEL1 during ovule development. Based on yeast three-hybrid assays and genetic data, we show that BEL1 interacts with the ovule identity MADS box factors when they dimerize with SEPALLATA proteins. We propose a model for ovule development that explains how the balance between carpel identity activity and ovule identity activity is established by a MADS box homeodomain protein complex.     

A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.     

APETALA1 and SEPALLATA3 interact to promote flower development

In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting flower meristem identity. Later in flower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during flower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the five MADS-box genes identified in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early flowering phenotype of plants constitutively expressing AP1 is significantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal flower development.     

Specific expression of the AGL1 MADS-box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development

Several members of the MADS-box gene family have been shown to be important regulators of flower development, controlling such well-studied early events as the formation of the floral meristem and the specification of floral organ identity. Other floral-specific MADS-box genes, of as yet unknown function, have been isolated by homology and are proposed to be part of a regulatory hierarchy controlling flower development. Some of these genes might regulate later aspects of flower development, such as development of individual floral organs, which is less well studied at the molecular level. This paper presents a detailed analysis of the expression pattern of one such gene from Arabidopsis , AGL1 , using RNA in situ hybridization. It is found that AGL1 is specifically expressed in particular regions of the gynoecium and ovule, only during and after floral development stage 7. AGL1 expression at the tip of the growing carpel primordia, along the margins of the ovary valves in developing and mature gynoecia and in specific regions of developing and mature ovules provides important insights into the possible roles of AGL1 . It is proposed that AGL1 may have regulatory functions in the structural definition and/or function of the valve margins, in axis maintenance during ovule development, in nutritional supply to the growing ovule and embryo sac, and in pollen tube guidance. In the floral homeotic mutants ag-1 , ap3-3 and ap2-2 , AGL1 mRNA is expressed in an organ-dependent manner, suggesting that AGL1 is a carpel-specific gene and as such ultimately depends upon the carpel identity gene AG for proper gene expression.     

Morphological analysis of female gametophyte development in the bel1 stk shp1 shp2 mutant

Abstract

In Arabidopsis thaliana, cell fate in developing ovules is determined by the action of the homeodomain factor BELL1 (BEL1) and of the MADS-box factors SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2. The analysis of the bel1 and the stk shp1 shp2 mutants revealed that the functional megaspore is formed, however, it does not proceed into megagametogenesis. In the bel1 stk shp1 shp2, quadruple mutant megasporogenesis does not take place. In this article we describe a detailed morphological analysis of the quadruple mutant, and we discuss the possibility that BELL1, STK, SHP1 and SHP2 not only control integument identity determination and development, but that they might also play a role during megasporogenesis.     

The Arabidopsis SUPERMAN Gene Mediates Asymmetric Growth of the Outer Integument of Ovules

Arabidopsis superman (sup, also referred to as floral mutant10) mutants have previously been shown to have flowers with supernumerary stamens and reduced carpels as a result of ectopic expression of the floral homeotic gene APETALA3 (AP3). Here, we report that sup mutations also cause specific alterations in ovule development. Growth of the outer integument of wild-type ovules occurs almost exclusively on the abaxial side of the ovule, resulting in a bilaterally symmetrical hoodlike structure. In contrast, the outer integument of sup mutant ovules grows equally on all sides of the ovule, resulting in a nearly radially symmetrical tubular shape. Thus, one role of SUP is to suppress growth of the outer integument on the adaxial side of the ovule. Genetic analyses showed that the effects of sup mutations on ovule development are independent of the presence or absence of AP3 activity. Thus, SUP acts through different mechanisms in its early role in ensuring proper determination of carpel identity and in its later role in asymmetric suppression of outer integument growth.     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆（Eustoma grandiflorum）花器官发育的分子调控机制,根据MADS-box基因保守序列设计简并引物,用3＇-RACE方法从草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明,这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性,分别属DEF/GLO、AG-like和SEP-like亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示：EgDEF1在萼片、花瓣、雄蕊及胚珠中高丰度表达,在心皮中微量表达;而EgGLO1在花瓣和雄蕊中高丰度表达,在萼片中微量表达;在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达,但表达的丰度存在差异,在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

The petunia AGL6 gene has a SEPALLATA-like function in floral patterning

SEPALLATA ( SEP ) MADS-box genes are required for the regulation of floral meristem determinacy and the specification of sepals, petals, stamens, carpels and ovules, specifically in angiosperms. The SEP subfamily is closely related to the AGAMOUS LIKE6 ( AGL6 ) and SQUAMOSA ( SQUA ) subfamilies. So far, of these three groups only AGL6 -like genes have been found in extant gymnosperms. AGL6 genes are more similar to SEP than to SQUA genes, both in sequence and in expression pattern. Despite the ancestry and wide distribution of AGL6 -like MADS-box genes, not a single loss-of-function mutant exhibiting a clear phenotype has yet been reported; consequently the function of AGL6 -like genes has remained elusive. Here, we characterize the Petunia hybrida AGL6 ( PhAGL6 , formerly called PETUNIA MADS BOX GENE4 / pMADS4 ) gene, and show that it functions redundantly with the SEP genes FLORAL BINDING PROTEIN2 ( FBP2 ) and FBP5 in petal and anther development. Moreover, expression analysis suggests a function for PhAGL6 in ovary and ovule development. The PhAGL6 and FBP2 proteins interact in in vitro experiments overall with the same partners, indicating that the two proteins are biochemically quite similar. It will be interesting to determine the functions of AGL6 -like genes of other species, especially those of gymnosperms.     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

AGAMOUS‐LIKE13, a putative ancestor for the E functional genes,specifies male and female gametophyte morphogenesis

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant‐negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet‐like complexes (AGL13–AG)₂ and interact with AG–AP3–PI to form a higher‐order heterotetrameric complex (AGL13–AG–AP3–PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.     

Class D and Bsister MADS-box genes are associated with ectopic ovule formation in the pistil-like stamens of alloplasmic wheat (Triticum aestivum L.)

Homeotic transformation of stamens into pistil-like structures (pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum L.) that have the cytoplasm of a related wild species, Aegilops crassa. An ectopic ovule differentiates in the pistil-like stamen in the alloplasmic wheat. The SEEDSTICK (STK)—like class D MADS-box gene, wheat STK (WSTK), was expressed in the primordia of ectopic ovules in the pistil-like stamens as well as in the true pistil, suggesting that ectopic ovule formation results from WSTK expression in the pistil-like stamens of alloplasmic wheat. The ectopic ovule is abnormal as it fails to form complete integuments. Based on the expression pattern of WSTK and B_sister MADS-box gene, WBsis (wheat B _sister ), we conclude that WSTK plays a role in determination of ovule identity in the pistil-like stamen, but complete ovule development fails due to aberrant expression of WBsis.     

KNAT2: evidence for a link between knotted-like genes and carpel development

The KNAT2 (for KNOTTED-like from Arabidopsis thaliana 2) homeobox gene is expressed in the vegetative apical meristem. It is also active during flower development, suggesting a function in the structuring of flowers. To investigate its role, we used a DEXAMETHASONE (DEX)-inducible system to generate transgenic plants that overexpressed a fusion of KNAT2 with the hormone binding domain of the glucocorticoid receptor. DEX-induced plants were similar to plants overexpressing the closely related KNAT1 gene, indicating overlapping functions, although we observed differences as well. In particular, KNAT2-GR activation induced ectopic carpel features. First, KNAT2 induced the homeotic conversion of nucellus into carpel-like structures. Second, KNAT2 induced stigmatic papillae on rosette leaves in the ap2-5 background. Third, ectopic expression of the carpel identity gene AGAMOUS (AG) was observed in carpels and ovules. Interestingly, the homeotic conversion was not dependent on AG activity, because it was maintained in the ag-1 ap2-5 double mutant. Therefore, our data indicate that KNAT2 also must activate other carpel regulators. Together, these results suggest that KNAT2 plays a role in carpel development.     

Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia

The Norway spruce MADS-box genes DAL11, DAL12 and DAL13 are phylogenetically related to the angiosperm B-function MADS-box genes: genes that act together with A-function genes in specifying petal identity and with C-function genes in specifying stamen identity to floral organs. In this report we present evidence to suggest that the B-gene function in the specification of identity of the pollen-bearing organs has been conserved between conifers and angiosperms. Expression of DAL11 or DAL12 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expression of the endogenous B-genes. In similar experiments, flowers of Arabidopsis plants expressing DAL13 showed a different homeotic change in that they formed ectopic anthers in whorls one, two or four. We also demonstrate the capacity of the spruce gene products to form homodimers, and that DAL11 and DAL13 may form heterodimers with each other and with the Arabidopsis B-protein AP3, but not with PI, the second B-gene product in Arabidopsis. In situ hybridization experiments show that the conifer B-like genes are expressed specifically in developing pollen cones, but differ in both temporal and spatial distribution patterns. These results suggest that the B-function in conifers is dual and is separated into a meristem identity and an organ identity function, the latter function possibly being independent of an interaction with the C-function. Thus, even though an ancestral B-function may have acted in combination with C to specify micro- and megasporangia, the B-function has evolved differently in conifers and angiosperms.