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.     

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 for the SHATTERPROOF genes during Arabidopsis gynoecium development

Gynoecium development is a complex process which is regulated by key factors that control the spatial formation of the apical, medial and basal parts. SHATTERPROOF1 (SHP1) and SHP2, two closely related MADS-box genes, redundantly control the differentiation of the dehiscence zone and promote the lignification of adjacent cells. Furthermore, SHP1 and SHP2 have shown to play an important role in ovule identity determination. The present work identifies a new function for these two genes in promoting stigma, style and medial tissue development. This new role was discovered by combining the shp1 shp2 double mutant with the aintegumenta (ant) and crabs claw (crc) mutants. In quadruple mutant flowers, the inner whorl is composed of unfused carpels which lack almost completely apical and medial tissues, a phenotype similar to the previously reported fil ant and lug ant double mutants.     

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.     

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 MADS box genes SEEDSTICK and ARABIDOPSIS Bsister play a maternal role in fertilization and seed development

The haploid generation of flowering plants develops within the sporophytic tissues of the ovule. After fertilization, the maternal seed coat develops in a coordinated manner with formation of the embryo and endosperm. In the arabidopsis bsister (abs) mutant, the endothelium, which is the most inner cell layer of the integuments that surround the haploid embryo sac, does not accumulate proanthocyanidins and the cells have an abnormal morphology. However, fertility is not affected in abs single mutants. SEEDSTICK regulates ovule identity redundantly with SHATTERPROOF 1 (SHP1) and SHP2 while a role in the control of fertility was not reported previously. Here we describe the characterization of the abs stk double mutant. This double mutant develops very few seeds due to both a reduced number of fertilized ovules and seed abortions later during development. Morphological analysis revealed a total absence of endothelium in this double mutant. Additionally, massive starch accumulation was observed in the embryo sac. The phenotype of the abs stk double mutant highlights the importance of the maternal-derived tissues, particularly the endothelium, for the development of the next generation.     

Fleshy Fruit Expansion and Ripening Are Regulated by the Tomato SHATTERPROOF Gene TAGL1

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.     

Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanol-inducible alc gene-expression system

In Arabidopsis, different combinations of ABC organ identity proteins interact in the presence of SEPALLATA (SEP) proteins to regulate floral organ differentiation. Ectopic expression of SEP3 in combination with class A and B or B and C genes is sufficient to homeotically convert vegetative leaves into petal-like organs and bracts into stamen-like structures, respectively. Recently, it has been shown that the three MADS-box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 act redundantly to control ovule identity. Protein interaction assays performed in yeast in combination with genetic studies demonstrated that these MADS-box factors only interact in the presence of SEP proteins to form complexes that determine ovule differentiation. Here, we address the question whether the ectopic co-expression of ovule identity proteins is sufficient to induce the homeotic conversion of vegetative leaves into carpel-like structures bearing ovules. We present the phenotypic characterization of Arabidopsis plants that ectopically express ovule identity factors under the regulation of the ethanol inducible gene expression system. These experiments indicate that the ectopic co-expression of SEP3 and SHP1 and/or STK is probably not sufficient to homeotically transform vegetative tissues into carpels with ovules. However, comparing the phenotypes obtained by ectopic expression of STK and/or SHP1 with or without SEP3 shows that co-expression of factors that are able to form complexes in yeast cause more extreme homeotic transformations, confirming the functional role of these complexes in vivo.     

The Tyrosine 343 Residue of Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Is Important for Its Interaction with SHP1, a Cytoplasmic Tyrosine Phosphatase with Tumor Suppressor Functions

The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK^K210R mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr³³⁸, Tyr³⁴², and Tyr³⁴³) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr³³⁸ or Tyr³⁴² did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr³⁴³ abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK^Y343F mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK^Y343F. In conclusion, we identified Tyr³⁴³ of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.     

A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF,is not only expressed in flowers but also throughout berry development

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.     

Beta-chemokine receptor CCR5 signals through SHP1, SHP2, and Syk

The beta-chemokine receptor CCR5 has been shown to modulate cell migration, proliferation, and immune functions and to serve as a co-receptor for the human immunodeficiency virus. We and others have shown that CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-beta. In this study, we further characterize the signaling molecules activated by CCR5 upon binding to its cognate ligand, macrophage inflammatory protein-1beta (MIP1beta). We observed enhanced tyrosine phosphorylation of the phosphatases SHP1 and SHP2 upon MIP1beta stimulation of CCR5 L1.2 transfectants and T-cells derived from peripheral blood mononuclear cells. Furthermore, we observed that SHP1 associated with RAFTK. However, using a dominant-negative phosphatase-binding mutant of RAFTK (RAFTK(m906)), we found that RAFTK does not mediate SHP1 or SHP2 phosphorylation. SHP1 and SHP2 also associated with the adaptor protein Grb2 and the Src-related kinase Syk. Pretreatment of CCR5 L1.2 transfectants or T-cells with the phosphatase inhibitor orthovanadate markedly abolished MIP1beta-induced chemotaxis. Syk was also activated upon MIP1beta stimulation of CCR5 L1.2 transfectants or T-cells and associated with RAFTK. Overexpression of a dominant-negative Src-binding mutant of RAFTK (RAFTK(m402)) significantly attenuated Syk activation, whereas overexpression of wild-type RAFTK enhanced Syk activity, indicating that RAFTK acts upstream of CCR5-mediated Syk activation. Taken together, these results suggest that MIP1beta stimulation mediated by CCR5 induces the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2.     

Increased susceptibility to ischemia-induced brain damage in transgenic mice overexpressing a dominant negative form of SHP2.

Cell culture studies have established SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) as an important factor in growth factor and cytokine-activated signaling pathways. However, the significance of SHP2 in the mammalian central nervous system (CNS) is not known since early embryonic lethality occurs in shp2 null mice. To bypass this embryonic lethality, transgenic animals containing a catalytically inactive mutant of SHP2 (SHP2-CS) under the control of a nestin intron II/thymidine kinase minimal promoter were generated. In the developing CNS of these animals, although high-level transgene expression was detected in the neuroepithelium, there was no obvious abnormality in progenitor cell proliferation or migration. In the adult brain, high-level transgene expression was detected in the subventricular zone, rostral migratory stream, dentate gyrus of hippocampus, and cerebellum. Because SHP2 function is likely important in cell survival pathways, we used a focal cerebral ischemia model to examined whether SHP2 is important during CNS injury. Ischemia-induced damage and neuronal death was found to be significantly greater in nestin-SHP2-CS mice than in wild-type littermates. These findings indicate that SHP2 is a required factor in signaling pathway(s) important for neuronal survival.     

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.