A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos

AGAMOUS-like-15 (AGL15) is a member of the MADS-domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChIP) approach to isolate genes regulated directly by AGL15. ChIP allows purification of in vivo protein-DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding protein. The DNA-binding regulatory protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans-acting regulatory factor. The cis element should be able to confer regulation in response to the trans-acting factor. We describe, in this report, our ChIP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChIP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2). Expression of DTA1, which encodes a protein with high similarity to GA-2 oxidase-like proteins, is induced by AGL15. DTA2 encodes a novel protein and expression of this target is repressed by AGL15.     

The MADS domain protein AGL15 localizes to the nucleus during early stages of seed development.

Little is known about regulatory factors that act during the earliest stages of plant embryogenesis. The MADS domain protein AGL15 (for AGAMOUS-like) is expressed preferentially during embryogenesis and accumulates during early seed development in monocotyledonous and dicotyledonous flowering plants. AGL15-specific antibodies and immunohistochemistry were used to demonstrate that AGL15 accumulates before fertilization in the cytoplasm in the cells of the egg apparatus and moves into the nucleus during early stages of development in the suspensor, embryo, and endosperms. Relatively high levels of AGL15 are present in the nuclei during embryo morphogenesis and until the seeds start to dry in Brassica, maize, and Arabidopsis. AGL15 is associated with the chromosomes during mitosis, and gel mobility shift assays were used to demonstrate that AGL15 binds DNA in a sequence-specific manner. To assess whether AGL15 is likely to play a role in specifying the seed or embryonic phase of development, AGL15 accumulation was examined in Arabidopsis mutants that prematurely exit embryogenesis. lec1-2 mutants show an embryo-specific loss of AGL15 at the transition stage, suggesting that AGL15 interacts with regulators in the leafy cotyledons pathway.     

The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis

The developmental roles of AGL15 and AGL18, members of the AGL15-like clade of MADS domain regulatory factors, have not been defined previously. Analysis of transgenic Arabidopsis plants showed that overexpression of AGL18 produces the same phenotypic changes as overexpression of AGL15, and the two genes have partially overlapping expression patterns. Functional redundancy was confirmed through analysis of loss-of-function mutants. agl15 agl18 double mutants, but not single mutants, flower early under non-inductive conditions, indicating that AGL15 and AGL18 act in a redundant fashion as repressors of the floral transition. Further genetic analyses and expression studies were used to examine the relationship between AGL15 and AGL18 activity and other regulators of the floral transition. AGL15 and AGL18 act upstream of the floral integrator FT, and a combination of agl15 and agl18 mutations partially suppresses defects in the photoperiod pathway. agl15 agl18 mutations show an additive relationship with mutations in genes encoding other MADS domain floral repressors, and further acceleration of flowering is seen in triple and quadruple mutants under both inductive and non-inductive conditions. Thus, flowering time is determined by the additive effect of multiple MADS domain floral repressors, with important contributions from AGL15 and AGL18.     

The embryo MADS domain factor AGL15 acts postembryonically. Inhibition of perianth senescence and abscission via constitutive expression

AGL15 (AGAMOUS-like 15), a member of the MADS domain family of regulatory factors, accumulates preferentially throughout the early stages of the plant life cycle. In this study, we investigated the expression pattern and possible roles of postembryonic accumulation of AGL15. Using a combination of reporter genes, RNA gel blot analysis, and immunochemistry, we found that the AGL15 protein accumulates transiently in the shoot apex in young Arabidopsis and Brassica seedlings and that promoter activity is associated with the shoot apex and the base of leaf petioles throughout the vegetative phase. During the reproductive phase, AGL15 accumulates transiently in floral buds. When AGL15 was expressed in Arabidopsis under the control of a strong constitutive promoter, we noted a striking increase in the longevity of the sepals and petals as well as delays in a selected set of age-dependent developmental processes, including the transition to flowering and fruit maturation. Although ethylene has been implicated in many of these same processes, the effects of AGL15 could be clearly distinguished from the effects of the ethylene resistant1-1 mutation, which confers dominant insensitivity to ethylene. By comparing the petal breakstrength (the force needed to remove petals) for flowers of different ages, we determined that ectopic AGL15 had a novel effect: the breakstrength of petals initially declined, as occurs in the wild type, but was then maintained at an intermediate value over a prolonged period. Abscission-associated gene expression and structural changes were also altered in the presence of ectopic AGL15.     

Effect of regulated overexpression of the MADS domain factor AGL15 on flower senescence and fruit maturation

We have examined the effect of regulated overexpression of AGL15, a member of the MADS domain family of regulatory factors, on reproductive tissues. Using molecular and physiological markers, we show that constitutive overexpression of AGL15 in Arabidopsis leads to delay and down-regulation of senescence programs in perianth organs and developing fruits and alters the process of seed desiccation. Through genetic crosses, we show that the rate of water loss in the maturing seeds is dictated by the genetic composition and physiological state of the maternal tissue, rather than the embryo. To define the developmental time and/or place when senescence programs are most affected by elevated AGL15 levels, we expressed AGL15 under the control of various promoters. Expression during senescence or in abscission zone cells did not produce delays in floral organ senescence or abscission. Using a glucocorticoid-inducible expression system, we show that an increase in AGL15 levels around the time of flower opening is necessary to delay senescence and increase floral organ longevity.     

The Arabidopsis AGL9 MADS box gene is expressed in young flower primordia

 MADS box genes are likely involved in many different steps of plant development, since their RNAs accumulate in a wide variety of tissues, including roots, stems, leaves, flowers and embryos. In flowers, MADS box genes regulate the early step of specifying floral meristem identity as well as the later step of determining the fate of floral organ primordia. Here we describe the isolation and characterization of a new MADS box gene from Arabidopsis, AGL9. Sequence analyses indicate that AGL9 represents the putative ortholog of the FBP2 and TM5 genes from petunia and tomato, respectively. In situ hybridization analyses show that AGL9 RNA begins to accumulate after the onset of expression of the floral meristem identity genes, but before the activation of the organ identity genes. These data indicate that AGL9 functions early in flower development to mediate between the interaction of these two classes of genes. Later in flower development, AGL9 RNA accumulates in petals, stamens, and carpels, suggesting a role for AGL9 in controlling the development of these organs. Received: 4 May 1997 / Accepted: 14 July 1997     

MADS box genes expressed in developing inflorescences of rice and sorghum

With the aim of elucidating the complex genetic system controlling flower morphogenesis in cereals, we have characterized two rice and two sorghum MADS box genes isolated from cDNA libraries made from developing inflorescences. The rice clones OsMADS24 and OsMADS45, which share high homology with the Arabidopsis AGL2 and AGL4 MADS box genes, are expressed in the floral meristem, in all the primordia, and in mature floral organs. High expression levels have also been found in developing kernels. The sorghum clone SbMADS1 is also homologous to AGL2 and AGL4: expression analysis and mapping data suggest that it is the ortholog of OsMADS24. The pattern of expression of SbMADS2, the other sorghum MADS box gene, suggests that it may play a role as a meristem identity gene, as does AP1 in Arabidopsis, to which it shows considerable homology. The four genes have been mapped on a rice RFLP genetic map: the results are discussed in terms of synteny among cereals. Received: 25 April 1996 / Accepted: 29 August 1996     

FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.

Winter-annual ecotypes of Arabidopsis are relatively late flowering, unless the flowering of these ecotypes is promoted by exposure to cold (vernalization). This vernalization-suppressible, late-flowering phenotype results from the presence of dominant, late-flowering alleles at two loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). In this study, we report that flc null mutations result in early flowering, demonstrating that the role of active FLC alleles is to repress flowering. FLC was isolated by positional cloning and found to encode a novel MADS domain protein. The levels of FLC mRNA are regulated positively by FRI and negatively by LUMINIDEPENDENS. FLC is also negatively regulated by vernalization. Overexpression of FLC from a heterologous promoter is sufficient to delay flowering in the absence of an active FRI allele. We propose that the level of FLC activity acts through a rheostat-like mechanism to control flowering time in Arabidopsis and that modulation of FLC expression is a component of the vernalization response.     

DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.     

PAL31, a novel nuclear protein, expressed in the developing brain

We cloned a cDNA encoding a novel protein (PAL31) predominantly expressed in the fetal rat brain by differential display. PAL31 contains leucine-rich repeat domains, a highly acidic region and a putative nuclear localization signal. PAL31 has 50-70% similarity to SSP29, APRIL, LANP, PHAP I, and PP32. Expression of PAL31 mRNA in the brain was high during the fetal period and decreased after birth. Immunohistochemical studies showed that PAL31 is expressed in the entire embryonic brain, whereas in the adult brain its expression is restricted to the subventricular zone where there are neural progenitor cells. It was also revealed that PAL31 is colocalized with PCNA in the nucleus, indicating that the PAL31 expression is developmentally regulated. Considering the primary structure of PAL31 and its spatiotemporal expression pattern, PAL31 is a novel nuclear protein related to the development of the brain through the proliferation of neuronal cells.     

CaMADS1, a MADS box gene expressed in the carpel of hazelnut

Hazelnut (Corylus avellana L.) is a species of economic interest that shows a peculiar floral biology. Unlike most of the angiosperms, which produce ovules during floral development such that they are ready for pollen at anthesis, hazelnut ovary development is delayed and triggered by compatible pollination. In order to elucidate the mechanisms regulating this unusual process and the role of the MADS box genes in ovary development, a cDNA library from pollinated styles of hazelnut was screened with a mixture of MADS box genes from different plant species. CaMADS1 (Corylus avellana MADS box), a floral-specific MADS box gene, was isolated, and characterized as belonging to the sub-family of the AGAMOUS genes. Northern blot, RT-PCR analyses and in situ hybridization experiments show a precise correlation between ovary development and CaMADS1 expression, indicating a role of this MADS box gene in the processes of floral organogenesis.     

rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.