The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.Appropriate timing of the shift from vegetative to reproductive growth is an important determinant of plant fitness. The time at which a plant flowers is determined through integration of signals reflecting extrinsic and intrinsic conditions, such as photoperiod, the duration of cold, plant health, and age (for review, see Amasino, 2010). One of the most important pathways regulating the timing of the floral transition is the photoperiod pathway (for review, see Imaizumi and Kay, 2006). Under long-day (LD) inductive conditions in Arabidopsis (Arabidopsis thaliana), photoperiod pathway components act to promote flowering by inducing CONSTANS (CO) and downstream genes. The floral integrator FLOWERING LOCUS T (FT) is a major target of multiple flowering pathways and the photoperiod pathway in particular. It is directly activated by CO (Samach et al., 2000). Under LD conditions, the peak of CO expression is coincident with the presence of light, and CO activates FT expression in the leaf vascular system (Yanovsky and Kay, 2003). FT travels through the phloem to the shoot apex (Corbesier et al., 2007), where, together with FLOWERING LOCUS D (Abe et al., 2005; Wigge et al., 2005), it activates APETALA1 (AP1) and other floral meristem identity genes, starting the flowering process. Other flowering time pathways converge on FT and/or directly impact gene expression in the meristem. The changes in gene expression that accompany the floral transition must be rapid, robust, largely irreversible, and strictly controlled spatially. This is achieved through positive feed-forward and negative feedback loops involving multiple regulatory factors (for recent review, see Kaufmann et al., 2010).Members of the MADS-box family of regulatory factors are central players in the regulatory loops controlling the floral transition (for a recent review, see Smaczniak et al., 2012a). MADS-domain factors typically act in large multimeric complexes and are well suited for regulation that involves combinatorial action. During the floral transition, MADS-domain proteins can act either as repressors or activators. In Arabidopsis, important floral repressors include SHORT VEGETATIVE PHASE (SVP) and members of the FLOWERING LOCUS C (FLC)-like group, including FLC, FLOWERING LOCUS M (FLM)/MADS AFFECTING FLOWERING1 (MAF1), and MAF2 to MAF5. Promoters of flowering include such MADS-domain factors as SUPPRESSOR OF CONSTANS1 (SOC1) and AGAMOUS-LIKE24 (AGL24). Together with non-MADS-box proteins FT and TWIN SISTER OF FT, SOC1 and AGL24 function as floral integrators. These operate downstream of the flowering time pathways but upstream of the meristem identity regulators such as LEAFY (LFY) and the MADS-domain factor AP1.The MADS-domain factors AGL15 and AGL18 also contribute to regulation of the floral transition in Arabidopsis. While single mutants have no phenotype, agl15 agl18 double mutants flower earlier than the wild type (Adamczyk et al., 2007). Therefore, AGL15 and AGL18 appear to act in a redundant fashion in seedlings, and like SVP, FLC, and MAF1 to MAF5, they act as floral repressors. The contributions of AGL15 and AGL18 are most apparent in the absence of strong photoperiodic induction: the agl15 agl18 double mutant combination partially suppresses the delay in flowering observed in co mutants, as well as the flowering delay associated with growth under short-day (SD) noninductive conditions. The earlier flowering in agl15 agl18 mutants under these conditions is associated with up-regulation of FT, and both AGL15 and AGL18 are expressed in the vascular system and shoot apex of young seedlings (Adamczyk et al., 2007), raising the possibility that AGL15 and AGL18 act directly on FT in leaves, as well as other targets in the meristem.AGL15, and to a lesser extent AGL18, have been further implicated in the networks that control flowering through molecular studies. Zheng et al. (2009) performed a chromatin immunoprecipitation (ChIP) analysis using AGL15-specific antibodies, tissue derived from embryo cultures, and a tiling array. Floral repressors (SVP and FLC), floral integrators (FT and SOC1), and a microRNA targeting AP2-like factors (miR172a) were identified as possible AGL15 targets (Zheng et al., 2009), suggesting that AGL15 may contribute to regulation through multiple avenues during the floral transition. AGL15 itself is directly bound and activated by AP2, which is both an A-class floral identity gene and a floral repressor (Yant et al., 2010). AGL15 is down-regulated in ap2 mutants, which are early flowering, while AGL18 is the nearest locus to multiple AP2-bound sites (Yant et al., 2010). Both AGL15 and AGL18 were identified as SOC1 targets through ChIP analyses (Immink et al., 2009; Tao et al., 2012). In yeast (Saccharomyces cerevisiae) two-hybrid assays, AGL15 interacts with a number of other MADS-domain proteins (de Folter et al., 2005), and in a one-hybrid study based on the SOC1 promoter, AGL15-SVP, AGL15-AGL24, and AGL15-SOC1 heterodimers were shown to bind to regions containing CArG boxes (Immink et al., 2012). AGL18 may act redundantly to AGL15 in these contexts. However, AGL18 either does not interact or only interacts weakly with other proteins in yeast two-hybrid assays (de Folter et al., 2005; Hill et al., 2008; Causier et al., 2012). It remains to be determined whether this truly reflects weaker or nonredundant in planta interactions or a technical problem in the artificial yeast system.Guided by the knowledge gained about AGL15 targets and interactions from molecular studies, we asked the following question: what is the functional significance of these molecular relationships in the context of the floral transition? We performed a series of genetic experiments combining agl15 agl18 mutations and mutations in interacting factors such as SVP, AGL24, and SOC1, as well as targets such as FT and SOC1. We also performed further molecular experiments focused on AGL15, for which a variety of tools are available. Among other things, we show that AGL15 and AGL18, along with AGL24 and SVP, play a role in blocking expression of the floral MADS-domain factor SEPALLATA3 (SEP3) during the vegetative phase. In the absence of these four factors, reproductive programs are initiated early, and floral genes are expressed in the youngest rosette leaf and cauline leaves.     

A MADS domain gene involved in the transition to flowering in Arabidopsis

Flowering time in many plants is triggered by environmental factors that lead to uniform flowering in plant populations, ensuring higher reproductive success. So far, several genes have been identified that are involved in flowering time control. AGL20 (AGAMOUS LIKE 20) is a MADS domain gene from Arabidopsis that is activated in shoot apical meristems during the transition to flowering. By transposon tagging we have identified late flowering agl20 mutants, showing that AGL20 is involved in flowering time control. In previously described late flowering mutants of the long-day and constitutive pathways of floral induction the expression of AGL20 is down-regulated, demonstrating that AGL20 acts downstream to the mutated genes. Moreover, we can show that AGL20 is also regulated by the gibberellin (GA) pathway, indicating that AGL20 integrates signals of different pathways of floral induction and might be a central component for the induction of flowering. In addition, the constitutive expression of AGL20 in Arabidopsis is sufficient for photoperiod independent flowering and the over-expression of the orthologous gene from mustard, MADSA, in the classical short-day tobacco Maryland Mammoth bypasses the strict photoperiodic control of flowering.     

AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis

Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30 degrees C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS.     

HUA2 is required for the expression of floral repressors in Arabidopsis thaliana

The HUA2 gene acts as a repressor of floral transition. Lesions in hua2 were identified through a study of natural variation and through two mutant screens. An allele of HUA2 from Landsberg erecta (Ler) contains a premature stop codon and acts as an enhancer of early flowering 4 (elf4) mutants. hua2 single mutants, in the absence of the elf4 lesion, flower earlier than wild type under short days. hua2 mutations partially suppress late flowering in FRIGIDA (FRI )-containing lines, autonomous pathway mutants, and a photoperiod pathway mutant. hua2 mutations suppress late flowering by reducing the expression of several MADS genes that act as floral repressors including FLOWERING LOCUS C (FLC ) and FLOWERING LOCUS M (FLM ).     

SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy

SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 ( SOC1 ) is one of the flowering pathway integrators and regulates the expression of LEAFY ( LFY ), which links floral induction and floral development. However, the mechanism by which SOC1, a MADS box protein, regulates LFY has proved elusive. Here, we show that SOC1 directly binds to the distal and proximal region of the LFY promoter where critical cis -elements are located. Intragenic suppressor mutant analysis shows that a missense mutation in the MADS box of SOC1 causes loss of binding to the LFY promoter as well as suppression of the flowering promotion function. The full-length SOC1 protein locates in the cytoplasm if expressed alone in protoplast transient expression assay, but relocates to the nucleus if expressed with AGAMOUS-LIKE 24 (AGL24), another flowering pathway integrator and a MADS box protein. The domain analysis shows that co-localization of SOC1 and AGL24 is mediated by the MADS box and the intervening region of SOC1. Finally, we show that LFY is expressed only in those tissues where SOC1 and AGL24 expressions overlap. Thus, we propose that heterodimerization of SOC1 and AGL24 is a key mechanism in activating LFY expression.     

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.     

Overexpression of AGAMOUS-LIKE 28 (AGL28) promotes flowering by upregulating expression of floral promoters within the autonomous pathway

MADS box genes are known to perform important functions in the development of various plant organs. Although the functions of many MADS box genes have previously been elucidated, the biological function of the type I MADS box genes remains poorly understood. In order to understand the function and regulation of the type I MADS box genes, we conducted molecular genetic analyses of AGL28, a member of the Malpha class of type I genes. AGL28 was expressed in vegetative tissues in a photoperiod-independent manner, but not within the reproductive apex. This indicates that AGL28 plays a role in the vegetative phase. Overexpression of AGL28 caused precocious flowering via the upregulation of the expression of FCA and LUMINIDEPENDENS (LD), both floral promoters within the autonomous pathway. However, the loss of AGL28 function did not result in any obvious flowering time phenotype, which suggests that AGL28 may perform a redundant function. Collectively, our data suggest that AGL28 is a positive regulator of known floral promoters within the autonomous pathway in Arabidopsis.     

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 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.     

MOSAIC FLORAL ORGANS1, an AGL6-Like MADS Box Gene,Regulates Floral Organ Identity and Meristem Fate in Rice

Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1.