LEAFY, TERMINAL FLOWER1 and AGAMOUS are functionally conserved but do not regulate terminal flowering and floral determinacy in Impatiens balsamina

In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.     

Mutual regulation of Arabidopsis thaliana ethylene-responsive element binding protein and a plant floral homeotic gene, APETALA2

BACKGROUND AND AIMS: It has previously been shown that Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) contributed to resistance to abiotic stresses. Interestingly, it has also been reported that expression of ethylene-responsive factor (ERF) genes including AtEBP were regulated by the activity of APETALA2 (AP2), a floral homeotic factor. AP2 is known to regulate expression of several floral-specific homeotic genes such as AGAMOUS. The aim of this study was to clarify the relationship between AP2 and AtEBP in gene expression. METHODS: Northern blot analysis was performed on ap2 mutants, ethylene-related Arabidopsis mutants and transgenic Arabidopsis plants over-expressing AtEBP, and a T-DNA insertional mutant of AtEBP. Phenotypic analysis of these plants was performed. KEY RESULTS: Expression levels of ERF genes such as AtEBP and AtERF1 were increased in ap2 mutants. Over-expression of AtEBP caused upregulation of AP2 expression in leaves. AP2 expression was suppressed by the null-function of ethylene-insensitive2 (EIN2), although AP2 expression was not affected by ethylene treatment. Loss of AtEBP function slightly reduced the average number of stamens. CONCLUSIONS: AP2 and AtEBP are mutually regulated in terms of gene expression. AP2 expression was affected by EIN2 but was not regulated by ethylene treatment.     

APETALA 2‐like genes AP2L2 and Q specify lemma identity and axillary floral meristem development in wheat

The spikelet is the basic unit of the grass inflorescence. In tetraploid (Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss‐of‐function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma‐like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2‐like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield.     

The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins,APETALA3 and PISTILLATA

MADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.     

An APETALA3 homolog controls both petal identity and floral meristem patterning in Nigella damascena L. (Ranunculaceae)

Flower architecture mutants provide a unique opportunity to address the genetic origin of flower diversity. Here we study a naturally occurring floral dimorphism in Nigella damascena (Ranunculaceae), involving replacement of the petals by numerous sepal‐like and chimeric sepal/stamen organs. We performed a comparative study of floral morphology and floral development, and characterized the expression of APETALA3 and PISTILLATA homologs in both morphs. Segregation analyses and gene silencing were used to determine the involvement of an APETALA3 paralog (NdAP3–3) in the floral dimorphism. We demonstrate that the complex floral dimorphism is controlled by a single locus, which perfectly co‐segregates with the NdAP3–3 gene. This gene is not expressed in the apetalous morph and exhibits a particular expression dynamic during early floral development in the petalous morph. NdAP3–3 silencing in petalous plants perfectly phenocopies the apetalous morph. Our results show that NdAP3–3 is fully responsible for the complex N. damascena floral dimorphism, suggesting that it plays a role not only in petal identity but also in meristem patterning, possibly through regulation of perianth organ number and the perianth/stamen boundary.     

落叶松APETALA2-Like转录因子基因的分离及功能分析

以落叶松转录组测序数据为基础,利用RT-PCR从落叶松中分离出一个1590 bp的APETALA2-Like转录因子基因全长编码序列,命名为La AP2L1。蛋白质特性分析显示La AP2L1编码529个氨基酸,为亲水性蛋白,相对分子质量为58.327 k D,等电点为6.45。二级结构主要由α-螺旋(alpha helix)、β-折叠(strand)和环肽链(loop)组成。多重序列比对和系统进化分析表明La AP2L1所编码的氨基酸与黑松、云杉等亲缘关系最近。同时,通过构建植物表达载体,利用浸花法转化模式植物拟南芥的功能研究发现,与转空载体对照拟南芥相比,过量表达La AP2L1的转基因拟南芥的叶片、茎、花和株高都显著增大、增高,表明落叶松La AP2L1转录因子可能参与了植物器官发育的调节。     

Minimal regions in the Arabidopsis PISTILLATA promoter responsive to the APETALA3/PISTILLATA feedback control do not contain a CArG box

PISTILLATA (PI) is a floral homeotic B function gene in Arabidopsis and together with the other B function gene, APETALA3 (AP3), is involved in specifying petal and stamen identities. The expression of PI and AP3 is under similar developmental control. The initiation of AP3 and PI expression is at least partly caused by the floral meristem identity gene LEAFY, but the maintenance of AP3 and PI expression involves an autoregulatory loop requiring the activity of both genes. PI and AP3 are MADS domain proteins that form, and appear to function as, a heterodimer. AP3/PI binds in vitro to a sequence motif, CC(A/T)₆GG, a MADS domain protein consensus binding site also known as the CArG box. We identified a 481-bp PI promoter region that confers both the initiation and the maintenance of PI expression patterns. We further dissected the promoter and identified minimal regions responsible for the AP3/PI-dependent expression. No CArG box is present in these minimal regions, suggesting that either AP3/PI does not bind directly to the PI promoter for the maintenance control, or that it requires additional factors to bind to the PI promoter. Our results suggest that the mechanisms of regulation of the two B function genes, AP3 and PI, are different, because CArG boxes are present in the AP3 promoter and are necessary for the AP3 feedback control. Received: 1 March 2000 / Revision accepted: 15 June 2000     

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.     

The peu‐miR160a−PeARF17.1/PeARF17.2 module participates in the adventitious root development of poplar

Deep roots give rise to flourishing leaves, and the two complement each other. However, the genetic mechanisms underlying adventitious rooting for forest trees have remained elusive. In this study, we verified that peu‐miR160a targets six poplar genes AUXIN RESPONSE FACTORS (ARFs), PeARF10.1, PeARF16.1, PeARF16.2, PeARF16.3, PeARF17.1 and PeARF17.2, using 5’RLM‐RACE. Interaction experiments with peu‐miR160a and PeARFs in poplar protoplasts further confirmed that peu‐miR160a targets and negatively regulates the six PeARFs. Peu‐miR160a and its target genes exhibited obvious temporal expression in different stages of adventitious root development, and they could also be induced by IAA and abscisic acid. Peu‐miR160a‐overexpressing lines exhibited a significant shortening of adventitious root length, an increase in the number of lateral roots, severe dwarfing and shortened internodes. In addition, the overexpression of PeARF17.1 or mPeARF17.2 (peu‐miR160a‐resistant version of PeARF17.2) significantly increased the number of adventitious roots. Furthermore, PeARF17.1‐overexpressing lines had multiple branches with no visible trunk, although the adventitious root length of the PeARF17.1‐overexpressing lines was significantly increased. Our findings reveal that the peu‐miR160a ? PeARF17.1/PeARF17.2 module is an important regulator involved in the development of the adventitious roots of poplar.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.     

miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems

In Arabidopsis, two floral homeotic genes APETALA2 (AP2) and AGAMOUS (AG) specify the identities of perianth and reproductive organs, respectively, in flower development. The two genes act antagonistically to restrict each other to their proper domains of action within the floral meristem. In addition to AG, which antagonizes AP2, miR172, a microRNA, serves as a negative regulator of AP2. In this study, we showed that AG and miR172 have distinct functions in flower development and that they largely act independently in the negative regulation of AP2. We uncovered functions of miR172-mediated repression of AP2 in the regulation of floral stem cells and in the delineation of the expression domain of another class of floral homeotic genes. Given the antiquity of miR172 in land plants, our findings have implications for the recruitment of a microRNA in the building of a flower in evolution.     

Early floral development and androecium organization in the sarracenioid clade (Actinidiaceae,Roridulaceae and Sarraceniaceae) of Ericales

The early floral development of Actinidia (A. arguta, A. callosa, A. chinensis and A. kolomikta; Actinidiaceae), Saurauia (S. montana, S. oldhamii, S. pittieri and S. subspinosa; Actinidiaceae), Roridula gorgonias (Roridulaceae) and Heliamphora nutans (Sarraceniaceae) was studied comparatively using scanning electron microscopy. Late stages of androecium development are additionally presented for Clematoclethra scandens (Actinidiaceae), Darlingtonia californica and Sarracenia leucophylla (Sarraceniaceae). Flowers are typically pentamerous and share a number of developmental features: perianth organs emerge in a clockwise or anticlockwise spiral sequence on the floral apex with relatively long plastochrons between successive organs, resulting in conspicuous size differences among perianth organs in early development; the perianth always consists of two differentiated whorls (unlike earlier interpretations of the perianth in Heliamphora); the androecium is polystemonous in most species and is initiated with leading stamens in alternipetalous positions; successive stamen primordia appear in a lateral succession until a ring‐like structure is formed; and the anthers become inverted shortly before anthesis. Later androecial development differs conspicuously between taxa and further proliferation may be centrifugal, centripetal and/or lateral. For Ericales, unusual features of floral development include: petals initiated in a spiral sequence (but later organized in a whorl) with comparatively long plastochrons between individual petals (except Saurauia); common occurrence of perianth organs in double positions in Actinidiaceae; and anthers that become inverted close to anthesis. The floral development in the sarracenioids is additionally compared with that of other families and clades in Ericales, further emphasizing the highly variable patterns of androecium development in the order.