The MADS box gene, FOREVER YOUNG FLOWER, acts as a repressor controlling floral organ senescence and abscission in Arabidopsis

The ectopic expression of a MADS box gene FOREVER YOUNG FLOWER (FYF) caused a significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. The defect in floral abscission was found to be due to a deficiency in the timing of cell separation of the abscission zone cells. Down-regulation of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) may contribute to the delay of the floral abscission in 35S:FYF flowers. FYF was found to be highly expressed in young flowers prior to pollination and was significantly decreased after pollination, a pattern that correlated with its function. Ethylene insensitivity in senescence/abscission and the down-regulation of ETHYLENE RESPONSE DNA-BINDING FACTOR 1 (EDF1) and EDF2, downstream genes in the ethylene response, in 35S:FYF Arabidopsis suggested a role for FYF in regulating senescence/abscission by suppressing the ethylene response. This role was further supported by the fact that 35S:FYF enhanced the delay of flower senescence/abscission in ethylene response 1 (etr1), ethylene-insensitive 2 (ein2) and constitutive triple response 1 (ctr1) mutants, which have defects in upstream genes of the ethylene signaling pathway. The presence of a repressor domain in the C-terminus of FYF and the enhancement of the delay of senescence/abscission in FYF+SRDX (containing a suppression motif) transgenic plants suggested that FYF acts as a repressor. Indeed, in FYF-DR+VP16 transgenic dominant-negative mutant plants, in which FYF was converted to a potent activator by fusion to a VP16-AD motif, the senescence/abscission of the flower organs was significantly promoted, and the expression of BOP2, IDA and EDF1/2 was up-regulated. Our data suggest a role for FYF in controlling floral senescence/abscission by repressing ethylene responses and regulating the expression of BOP2 and IDA in Arabidopsis.     

Arabidopsis class I KNOTTED-like homeobox proteins act downstream in the IDA-HAE/HSL2 floral abscission signaling pathway

Floral organ abscission in Arabidopsis thaliana is regulated by the putative ligand-receptor system comprising the signaling peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the two receptor-like kinases HAESA and HAESA-LIKE2. The IDA signaling pathway presumably activates a MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade to induce separation between abscission zone (AZ) cells. Misexpression of IDA effectuates precocious floral abscission and ectopic cell separation in latent AZ cell regions, which suggests that negative regulators are in place to prevent unrestricted and untimely AZ cell separation. Through a screen for mutations that restore floral organ abscission in ida mutants, we identified three new mutant alleles of the KNOTTED-LIKE HOMEOBOX gene BREVIPEDICELLUS (BP)/KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1). Here, we show that bp mutants, in addition to shedding their floral organs prematurely, have phenotypic commonalities with plants misexpressing IDA, such as enlarged AZ cells. We propose that BP/KNAT1 inhibits floral organ cell separation by restricting AZ cell size and number and put forward a model whereby IDA signaling suppresses BP/KNAT1, which in turn allows KNAT2 and KNAT6 to induce floral organ abscission.     

Ethylene-dependent and -independent pathways controlling floral abscission are revealed to converge using promoter::reporter gene constructs in the ida abscission mutant

The process of floral organ abscission in Arabidopsis thaliana can be modulated by ethylene and involves numerous genes contributing to cell separation. One gene that is absolutely required for abscission is INFLORESCENCE DEFICIENT IN ABSCISSION, IDA, as the ida mutant is completely blocked in abscission. To elucidate the genetic pathways regulating floral abscission, molecular markers expressed in the floral abscission zone have been studied in an ida mutant background. Using plants with promoter-reporter gene constructs including promoters of a novel FLORAL ABSCISSION ASSOCIATED gene (FAA) encoding a putative single-stranded binding protein (BASIL), chitinase (CHIT::GUS) and cellulase (BAC::GUS), it is shown that IDA acts in the last steps of the abscission process. These markers, as well as HAESA, encoding a receptor-like kinase, were unaffected in their temporal expression patterns in ida compared with wild-type plants; thus showing that different regulatory pathways are active in the abscission process. In contrast to BASIL, CHIT::GUS and BAC::GUS showed, however, much weaker induction of expression in an ida background, consistent with a reduction in pathogen-associated responses and a lack of total dissolution of cell walls in the mutant. IDA, encoding a putative secreted peptide ligand, and HAESA appeared to have identical patterns of expression in floral abscission zones. Lastly, to address the role of ethylene, IDA::GUS expression in the wild type and the ethylene-insensitive mutant etr1-1 was compared. Similar temporal patterns, yet restricted spatial expression patterns were observed in etr1-1, suggesting that the pathways regulated by IDA and by ethylene act in parallel, but are, to some degree, interdependent.     

Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants

Abscission is an active process that enables plants to shed unwanted organs. Because the purpose of the flower is to facilitate pollination, it often is abscised after fertilization. We have identified an Arabidopsis ethylene-sensitive mutant, inflorescence deficient in abscission (ida), in which floral organs remain attached to the plant body after the shedding of mature seeds, even though a floral abscission zone develops. The IDA gene, positioned in the genomic DNA flanking the single T-DNA present in the ida line, was identified by complementation. The gene encodes a small protein with an N-terminal signal peptide, suggesting that the IDA protein is the ligand of an unknown receptor involved in the developmental control of floral abscission. We have identified Arabidopsis genes, and cDNAs from a variety of plant species, that encode similar proteins, which are distinct from known ligands. IDA and the IDA-like proteins may represent a new class of ligands in plants.     

KNAT1, KNAT2 and KNAT6 act downstream in the IDA-HAE/HSL2 signaling pathway to regulate floral organ abscission

Cell separation processes, such as abscission, are critical for plant development and play key roles from sculpting the form of the plant to scattering seeds. It is however essential that such processes are under tight temporal and spatial regulation. Floral organ abscission in Arabidopsis thaliana is regulated by a ligand-receptor module consisting of the signaling peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the two receptor-like kinases HAESA (HAE) and HAESA-LIKE 2 (HSL2), and it is the restricted expression pattern of IDA that hinders cell separation from occurring in the abscission zones (AZs) of other organs where HAE and HSL2 are present. In the July issue of The Plant Cell we report on the identification of additional components acting downstream in the IDA signaling pathway. Through a screen for mutations that restore floral organ abscission in ida mutants, we identified two new alleles of the KNOTTED-LIKE HOMEOBOX gene BREVIPEDICELLUS (BP)/KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1) and show that BP/KNAT1 is important in regulating the timing of floral abscission by controlling AZ cell size and by regulating KNAT2 and KNAT6.     

Hawaiian skirt: an F-box gene that regulates organ fusion and growth in Arabidopsis

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, Pro(PGAZAT):beta-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using Pro(HWS):beta-glucuronidase or Pro(HWS):green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro(35S):HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.     

The EPIP peptide of INFLORESCENCE DEFICIENT IN ABSCISSION is sufficient to induce abscission in arabidopsis through the receptor-like kinases HAESA and HAESA-LIKE2

In Arabidopsis thaliana, the final step of floral organ abscission is regulated by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA): ida mutants fail to abscise floral organs, and plants overexpressing IDA display earlier abscission. We show that five IDA-LIKE (IDL) genes are expressed in different tissues, but plants overexpressing these genes have phenotypes similar to IDA-overexpressing plants, suggesting functional redundancy. IDA/IDL proteins have N-terminal signal peptides and a C-terminal conserved motif (extended PIP [EPIP]) at the C terminus (EPIP-C). IDA can, similar to CLAVATA3, be processed by an activity from cauliflower meristems. The EPIP-C of IDA and IDL1 replaced IDA function in vivo, when the signal peptide was present. In addition, synthetic IDA and IDL1 EPIP peptides rescued ida and induced early floral abscission in wild-type flowers. The EPIP-C of the other IDL proteins could partially substitute for IDA function. Similarly to ida, a double mutant between the receptor-like kinases (RLKs) HAESA (HAE) and HAESA-LIKE2 (HSL2) displays nonabscising flowers. Neither overexpression of IDA nor synthetic EPIP or EPIP-C peptides could rescue the hae hsl2 abscission deficiency. We propose that IDA and the IDL proteins constitute a family of putative ligands that act through RLKs to regulate different events during plant development.     

The SERK1 receptor‐like kinase regulates organ separation in Arabidopsis flowers

Through a sensitized screen for novel components of pathways regulating organ separation in Arabidopsis flowers, we have found that the leucine‐rich repeat receptor‐like kinase SOMATIC EMBRYOGENESIS RECEPTOR‐LIKE KINASE1 (SERK1) acts as a negative regulator of abscission. Mutations in SERK1 dominantly rescue abscission in flowers without functional NEVERSHED (NEV), an ADP‐ribosylation factor GTPase‐activating protein required for floral organ shedding. We previously reported that the organization of the Golgi apparatus and location of the trans‐Golgi network (TGN) are altered in nev mutant flowers. Disruption of SERK1 restores Golgi structure and the close association of the TGN in nev flowers, suggesting that defects in these organelles may be responsible for the block in abscission. We have also found that the abscission zones of nev serk1 flowers are enlarged compared to wild‐type. A similar phenotype was previously observed in plants constitutively expressing a putative ligand required for organ separation, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), suggesting that signalling through IDA and its proposed receptors, HAESA and HAESA‐LIKE2, may be deregulated in nev serk1 abscission zone cells. Our studies indicate that in addition to its previously characterized roles in stamen development and brassinosteroid perception, SERK1 plays a unique role in modulating the loss of cell adhesion that occurs during organ abscission.     

Identification of a putative receptor-ligand pair controlling cell separation in plants

Cell separation events are important throughout the lifespan of a plant. To assure that the plant''s integrity is not compromised, such events, which depend on cell wall degradation, have to be tightly controlled both in time and space. The final step of floral organ abscission in Arabidopsis is controlled by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), in that mutation of IDA causes a block in abscission. Overexpression results in early abscission of floral organs. In a recent article we show that this is also the case when overexpressing the related IDA-LIKE (IDL) proteins, indicating a degree of functional redundancy. Based on gene swap and deletion constructs introduced in the ida mutant and synthetic peptide assays we demonstrated that the conserved C-terminal motif (EPIP) of IDA and IDL1 was sufficient to replace IDA function. This function is dependent on the presence of the receptor-like kinases (RLK) HAESA (HAE) and HAESA-LIKE2 (HSL2), suggesting that an IDA peptide acts as a ligand interacting with these receptors. Our study further revealed that the five IDL genes are expressed at various sites where cell separation takes place. We suggest that the IDL proteins constitute a family of ligands that act through RLKs similar to HAESA and control cell separation at different sites and development stages during the life of the plant.Key words: Arabidopsis, signaling, receptor, ligand, gene-family, cell separation, HAE, IDA     

Two alpha-L-arabinofuranosidase genes in Arabidopsis thaliana are differentially expressed during vegetative growth and flower development

Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. The cloning and expression analysis of two genes, AtASD1 and AtASD2, encoding putative alpha-L-arabinofuranosidases in Arabidopsis thaliana are reported here. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Using RT-PCR, RNA gel blot analysis and reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. AtASD1 expression is particularly intense in zones of cell proliferation, the vascular system, developing and regressing floral tissues, and floral abscission zones. By comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones.     

Characterization of a non-abscission mutant in Lupinus angustifolius. I. Genetic and structural aspects

A spontaneous mutant, Abs, that does not abscise any organs despite an apparently normal pattern of growth and senescence was isolated from among plants of Lupinus angustifolius cv. 'Danja'. Abs was found to be a recessive single gene mutation, and it was proposed that the gene for the original mutant phenotype, referred to as Abs, be designated abs1. An artificially induced mutant allelic to abs1 was also obtained and a non-allelic mutant phenotype, Delabs (delayed abscission), which was designated abs2. Morphological and cytological features of the abscission process under conditions of natural and ethylene-induced senescence were compared in the wild-type parent and Abs mutant. In the parent genotype abscission under natural conditions is similar to many other species, consisting of a stage of cell division forming an abscission zone, activation of the cytoplasm of zone cells, dissolution of the middle lamella, disorganization of fibrillar wall structure, and cell separation. A slightly different pattern of abscission zone development was observed for ethylene-treated explants of the parent, mainly with respect to features of cell division and cell enlargement. In Abs no abscission occurred for any abscission sites under conditions of natural senescence or with ethylene treatment of small shoot explants. However, relatively normal abscission zones were differentiated at all sites in the mutant except that extensive cell wall disorganization did not occur. Ethylene production by leaves or other organs of the mutant was no different from that of Danja. Application of copper salts or hydrogen peroxide, droughting, waterlogging, or application of abscisic acid (ABA) increased ethylene production equally in both genotypes but did not result in abscission in the mutant. Release of root cap border cells, the only other cell separation process examined, was similar in each genotype. The study concludes that the mutation is quite specific to the abscission process and may be due to a lack of or delay in the expression of hydrolytic enzyme(s) associated specifically with abscission zone differentiation and separation.     

Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs.

AINTEGUMENTA (ANT) was previously shown to be involved in floral organ initiation and growth in Arabidopsis. ant flowers have fewer and smaller floral organs and possess ovules that lack integuments and a functional embryo sac. The present work shows that young floral meristems of ant plants are smaller than those in wild type. Failure to initiate the full number of organ primordia in ant flowers may result from insufficient numbers of meristematic cells. The decreased size of ant floral organs appears to be a consequence of decreased cell division within organ primordia. Ectopic expression of ANT under the control of the constitutive 35S promoter results in the development of larger floral organs. The number and shape of these organs is not altered and the size of vegetative organs is normal. Microscopic and molecular analyses indicate that the increased size of 35S::ANT sepals is the result of increased cell division, whereas the increased sizes of 35S::ANT petals, stamens, and carpels are primarily attributable to increased cell expansion. In addition, 35S::ANT ovules often exhibit increased growth of the nucellus and the funiculus. These results suggest that ANT stimulates cell growth in floral organs.     

Molecular characterization of two highly homologous receptor-like kinase genes, RLK902 and RKL1, in Arabidopsis thaliana

Receptor-like kinases (RLKs) constitute a large family of signal perception molecules. We characterized two highly homologous RLK genes, RLK902 and RKL1, in Arabidopsis. RLK902 and RKL1 showed a 75% amino acid sequence identity over their entire regions. In the RLK902 pro::GUS transgenic lines, GUS activity was strong in the root tips, lateral root primordia, stipules, and floral organ abscission zones, while the RKL1 promoter activity was dominant in the stomata cells, hydathodes and trichomes of young rosette leaves, and floral organ abscission zones. Neither the rlk902 mutant line, rkl1 mutant line nor rlk902/rkl1 double-knockout mutant line showed any significant phenotypes under normal growth conditions. These results suggest that RLK902 and RKL1 might mediate the signal transduction pathway in which at least one other complementary signaling pathway to these two RLKs might exist.