APETALA1 Research Articles

An APETALA3 MADS-box linked SCAR marker associated with male specific sex expression in Coccinia grandis (L). Voigt

Coccinia grandis L. (Family – Cucurbitaceae), a perennial dioecious herb with heteromorphic sex chromosomes has the quality of a model plant for analysis of sexual evolution in angiosperms. Yet, little is known about the physical side, genetic orientation and substitution behavior of key gender-defining factors in this important plant species. Screening of genomic DNA with RAPD primers was used for sex diagnosis and gender specificity of C. grandis in this study. Bulk analyses of pooled DNA from male and female genotypes resulted in the isolation of a putative male specific sex marker CgMSM. A sequence characterized amplified region (SCAR) marker CgY1 designed from CgMSM amplified an 829bp fragment in the male C. grandis but not in the female plants. Southern blotting confirmed it as a single copy locus in the male genome. Further, the CgY1 marker accurately identified 11 male and 6 female genotypes from 17 individuals of C. grandis with unknown sex. Sequence homology revealed high similarity between CgMSM (CgY1) and the putative MADS-box domain of the APETALA3 (AP3) MADS-box gene which is responsible for development of petals and stamens in flowering plants. Thus, the SCAR marker CgY1 could be used as an efficient tool for early sex identification in C. grandis and forms the basis for characterization of genes associated with evolutionary pathways in sexual dimorphism.

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression.

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators. Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype. A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants. The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of ‘phyllogens’.

MiRNA-Mediated Posttranscriptional Regulation of Gene Expression in ABR17-Transgenic Arabidopsis thaliana Under Salt Stress

MicroRNAs (miRNAs) are a class of small endogenous RNAs conserved in eukaryotic organisms including plants. They suppress gene expression posttranscriptionally in many different biological processes. Previously, we reported salinity-induced changes in gene expression in transgenic Arabidopsis thaliana plants that constitutively expressed a pea abscisic acid-responsive (ABR17) gene. In the current study, we used microarrays to investigate the role of miRNA-mediated posttranscriptional gene regulation in these same transgenic plants in the presence and absence of salinity stress. We identified nine miRNAs that were significantly modulated due to ABR17 gene expression and seven miRNAs that were modulated in response to salt stress. The target genes regulated by these miRNAs were identified using sRNA target Base (starBase) degradome analysis and through 5′-RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). Our findings revealed miRNA–mRNA interactions comprising regulatory networks of auxin response factor (ARF), argonaute 1 (AGO1), dicer-like proteins 1 (DCL1), SQUAMOSA promoter binding (SPB), NAC, APETALA 2 (AP2), nuclear factor Y (NFY), RNA-binding proteins, A. thaliana vacuolar phyrophosphate 1 (AVP1), and pentatricopeptide repeat (PPR) in ABR17-transgenic A. thaliana, which control physiological, biochemical, and stress signaling cascades due to the imposition of salt stress. Our results are discussed within the context of the effect of the transgene, ABR17, and the roles of miRNA expression may play in mediating plant responses to salinity.

Flower development in Coffea arabica L.: new insights into MADS-box genes

Coffea arabica L. shows peculiar characteristics during reproductive development, such as flowering asynchrony, periods of floral bud dormancy, mucilage secretion and epipetalous stamens. The MADS-box transcription factors are known to control several developmental processes in plants, including flower and fruit development. Significant differences are found among plant species regarding reproductive development and little is known about the role of MADS-box genes in Coffea reproductive development. Thus, we used anatomical and comparative molecular analyses to explore the flowering process in coffee. The main morphological changes during flower development in coffee were observed by optical and scanning electron microscopy. Flowering asynchrony seems to be related to two independent processes: the asynchronous development of distinct buds before the reproductive induction and the asynchronous development of floral meristems within each bud after the reproductive induction. A total of 23 C. arabica MADS-box genes were characterized by sequence comparison with putative Arabidopsis orthologs and their expression profiles were analyzed by RT-PCR in different tissues. The expression of the ABC model orthologs in Coffea during floral development was determined by in situ hybridization. The APETALA1 (AP1) ortholog is expressed only late in the perianth, which is also observed for the APETALA3 and TM6 orthologs. Conversely, the PISTILLATA ortholog is widely expressed in early stages, but restrict to stamens and carpels in later stages of flower development, while the expression of the AGAMOUS ortholog is always restricted to fertile organs. The AP1 and PISTILLATA orthologs are also expressed at specific floral organs, such as bracts and colleters, respectively, suggesting a potential role in the development of such structures. Altogether, the results from our comprehensive expression analyses showed significant differences between the spatiotemporal expression profiles of C. arabica MADS-box genes and their orthologs, which suggests differential functionalization in coffee. Moreover, these differences might also partially explain the particular characteristics of floral development in coffee, such as mucilage secretion and formation of epipetalous stamens.

Arabidopsis thaliana peroxidases involved in lignin biosynthesis: In silico promoter analysis and hormonal regulation

Phytohormones such as auxins, cytokinins, and brassinosteroids, act by means of a signaling cascade of transcription factors of the families NAC, MYB, AP2 (APETALA2), MADS and class III HD (homeodomain) Zip, regulating secondary growth. When the hormonal regulation of Zinnia elegans peroxidase (ZePrx), an enzyme involved in lignin biosynthesis, was studied, it was found that this peroxidase is sensitive to a plethora of hormones which control xylem lignification.In a previous study we sought Arabidopsis thaliana homologues to ZePrx. Peroxidases 4, 52, 49 and 72 are the four peroxidases that fulfill the restrictive conditions that a peroxidase involved in lignification must have. In the present study, we focus our attention on hormonal regulation in order to establish the minimal structural and regulatory elements contained in the promoter region which an AtPrx involved in lignification must have. The results indicate that of the four peroxidases selected in our previous study, the one most likely to be homologous to ZePrx is AtPrx52. The results suggest that hormones such as auxins, cytokinins and BRs directly regulate AtPrx52, and that the AtPrx52 promoter may be the target of the set of transcription factors (NAC, MYB, AP2 and class I and III HD Zip) which are up-regulated by these hormones during secondary growth. In addition, the AtPrx52 promoter contains multiple copies of all the putative cis-elements (the ACGT box, the OCS box, the OPAQ box, the L1BX, the MYCL box and the W box) known to confer regulation by NO and H2O2.

A Cucumber DELLA Homolog CsGAIP May Inhibit Staminate Development through Transcriptional Repression of B Class Floral Homeotic Genes

In hermaphroditic Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis.

The Arabidopsis thaliana At4g13040 gene, a unique member of the AP2/EREBP family, is a positive regulator for salicylic acid accumulation and basal defense against bacterial pathogens

The Arabidopsis genome contains a large number of putative transcription factors, containing a DNA binding domain similar to APETALA2/ethylene response element binding protein (AP2/EREBP), for most of which a function is not known. Phylogenetic analysis divides the Apetala 2 (AP2) super-family into 5 major groups: AP2, RAV, ethylene response factor (ERF), dehydration response element binding protein (DREB) and At4g13040. Similar to ERF and DREB, the At4g13040 protein contains only one AP2 domain; however, its structural uniqueness places it into a distinct group. In this article, we report that At4g13040 (referred herein as Apetala 2 family protein involved in SA mediated disease defense 1 - APD1) is an important regulator for SA mediated plant defense. The APD1 gene is upregulated upon pathogen inoculation, exogenous SA application and in the mutant that constitutively activates SA signaling. The T-DNA insertion lines (inserted in the APD1 promoter), which fail to induce expression upon pathogen inoculation, are compromised for resistance against virulent bacterial pathogens and show reduced induction of pathogenesis related 1 gene. Our results suggest that APD1 functions downstream of PAD4 in Arabidopsis and promotes pathogen-induced SA accumulation. Exogenous SA application completely restores the loss-of-resistance phenotype of the apd1 mutant. Thus, APD1 is a positive regulator of disease defense that functions upstream of SA accumulation.

A Novel AP2-Type Transcription Factor, SMALL ORGAN SIZE1, Controls Organ Size Downstream of an Auxin Signaling Pathway

The organ size of flowering plants is determined by two post-embryonic developmental events: cell proliferation and cell expansion. In this study, we identified a new rice loss-of-function mutant, small organ size1 (smos1), that decreases the final size of various organs due to decreased cell size and abnormal microtubule orientation. SMOS1 encodes an unusual APETALA2 (AP2)-type transcription factor with an imperfect AP2 domain, and its product belongs to the basal AINTEGUMENTA (ANT) lineage, including WRINKLED1 (WRI1) and ADAP. SMOS1 expression was induced by exogenous auxin treatment, and the auxin response element (AuxRE) of the SMOS1 promoter acts as a cis-motif through interaction with auxin response factor (ARF). Furthermore, a functional fluorophore-tagged SMOS1 was localized to the nucleus, supporting the role of SMOS1 as a transcriptional regulator for organ size control. Microarray analysis showed that the smos1 mutation represses expression of several genes involved in microtubule-based movement and DNA replication. Among the down-regulated genes, we demonstrated by gel-shift and chromatin immunoprecipitation (ChIP) experiments that OsPHI-1, which is involved in cell expansion, is a target of SMOS1. SMOS1 homologs in early-diverged land plants partially rescued the smos1 phenotype of rice. We propose that SMOS1 acts as an auxin-dependent regulator for cell expansion during organ size control, and that its function is conserved among land plants.

A Tree Ortholog of APETALA1 Mediates Photoperiodic Control of Seasonal Growth

Photoperiodic control of development plays a key role in adaptation of plants to seasonal changes. A signaling module consisting of CONSTANS (CO) and FLOWERING LOCUS T (FT) mediates in photoperiodic control of a variety of developmental transitions (e.g., flowering, tuberization, and seasonal growth cessation in trees). How this conserved CO/FT module can mediate in the photoperiodic control of diverse unrelated developmental programs is poorly understood. We show that Like-AP1 (LAP1), a tree ortholog of Arabidopsis floral meristem identity gene APETALA1 (AP1), mediates in photoperiodic control of seasonal growth cessation downstream of the CO/FT module in hybrid aspen. Using LAP1 overexpressors and RNAi-suppressed transgenic trees, we demonstrate that short day (SD)-mediated downregulation of LAP1 expression is required for growth cessation. In contrast with AP1 targets in flowering, LAP1 acts on AINTEGUMENTA-like 1 transcription factor, which is implicated in SD-mediated growth cessation. Intriguingly, unlike AP1 in Arabidopsis, ectopic expression of LAP1 fails to induce early flowering in hybrid aspen trees. These results indicate that AP1 ortholog in trees has acquired a novel function in photoperiodic regulation of seasonal growth. Thus, photoperiodic signaling pathway may have diverged downstream of AP1/LAP1 rather than the CO/FT module during evolution. Moreover, control of flowering by the CO/FT module can be uncoupled from its role in photoperiodic control of seasonal growth in trees. Thus, our findings can explain mechanistically how a conserved signaling module can mediate in the control of a highly diverse set of developmental transitions by a similar input signal, namely photoperiod.

Global small RNA analysis in fast-growing Arabidopsis thaliana with elevated concentrations of ATP and sugars

BackgroundIn higher eukaryotes, small RNAs play a role in regulating gene expression. Overexpression (OE) lines of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) were shown to grow faster and exhibit higher ATP and sugar contents. Leaf microarray studies showed that many genes involved in microRNAs (miRNAs) and trans-acting siRNAs (tasiRNAs) biogenesis were significantly changed in the fast-growing lines. In this study, the sRNA profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on sRNA expression were analyzed.Results9-13 million reads from each library were mapped to genome. miRNAs, tasiRNAs and natural antisense transcripts-generated small interfering RNAs (natsiRNAs) were identified and compared between libraries. In the leaf of OE lines, 15 known miRNAs increased in abundance and 9 miRNAs decreased in abundance, whereas in the root of OE lines, 2 known miRNAs increased in abundance and 9 miRNAs decreased in abundance. miRNAs with increased abundance in the leaf and root samples of both OE lines (miR158b and miR172a/b) were predicted to target mRNAs coding for Dof zinc finger protein and Apetala 2 (AP2) proteins, respectively. Furthermore, a significant change in the miR173-tasiRNAs-PPR/TPR network was observed in the leaves of both OE lines.ConclusionIn this study, the impact of high energy content on the sRNA profiles of Arabidopsis is reported. While the abundance of many stress-induced miRNAs is unaltered, the abundance of some miRNAs related to plant growth and development (miR172 and miR319) is elevated in the fast-growing lines. An induction of miR173-tasiRNAs-PPR/TPR network was also observed in the OE lines. In contrast, only few cis- and trans-natsiRNAs are altered in the fast-growing lines.

CsPI from the perianthless early-diverging Chloranthus spicatus show function on petal development in Arabidopsis thaliana.

BackgroundIn the floral ABC model, B-class genes comprised of DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI) had been proposed to involve in second and third whorl floral organ development. However, less is known about the function of B-class genes from early-diverging angiosperms. Chloranthaceae is one of the early-diverging angiosperm families. In this study, we characterized the role of the PI-like gene CsPI cloned from Chloranthus spicatus which have the simplest perianthless bisexual flowers.ResultsThe expression profile analysis reveals high levels of CsPI mRNA in stamens in Chloranthus spicatus, with weak distribution in leaves and other floral organs. Nevertheless, CsPI rescued both stamen and petal development in Arabidopsis thaliana pi-1 mutants and caused partially conversion of sepals into petaloid organs in wild-type Arabidopsis thaliana plants. Yeast two-hybrid analysis showed that CsPI can form not only homodimers but also heterodimers with proteins encoded by Arabidopsis thaliana and Chloranthus spicatus AP3-like genes.ConclusionsThese results suggested that CsPI has an ancestral function on stamen development and that CsPI has capability to specify petal development in Arabidopsis thaliana. The finding indicates that the activity of the encoded PI-like proteins is highly conserved between the early-diverging Chloranthus and Arabidopsis. Moreover, our results appear to suggest that B-function genes may not play a role in perianth development in Chloranthus spicatus.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-21) contains supplementary material, which is available to authorized users.

Overexpression of BpAP1 induces early flowering and produces dwarfism in Betula platyphylla × Betula pendula

The involvement of APETALA1 (AP1) in the flowering transition has been the focus of much research. Here, we produced Betula platyphylla × Betula pendula (birch) lines that overexpressed BpAP1 using Agrobacterium-mediated transformation; we obtained five independent 35S::BpAP1 transgenic lines. Polymerase chain reaction (PCR), Southern, northern and western analyses were used to identify the transformants. As determined by quantitative real-time PCR (qRT-PCR), BpAP1 expression in roots, shoots, leaves and terminal buds of 35S::BpAP1 transgenic lines was significantly higher than that in the wild type (WT, P < 0.01). The average height of 2-year-old 35S::BpAP1 plants was significantly lower (41.17%) than that of non-transgenic plants. In the 35S::BpAP1 lines, inflorescences emerged successively beginning 2 months after transplanting. In addition, the length-diameter ratio of fully developed male and female inflorescences were both significantly less than those of the WT (P < 0.05), i.e. the morphological characteristic was stubby. The male inflorescences emerged early, with empty, draped anthers, and pollen was rarely produced, whereas the female floret structure was not different from WT. The pistils developed normally and could accept pollen, leading to the production of hybrid progeny (F1 ). F1 plants completed flowering within only 1 year after sowing. We demonstrate that BpAP1 can be inherited through sexual reproduction. Overexpression of BpAP1 caused early flowering and dwarfism; these lines had an obviously shortened juvenile phase. These results greatly increase our understanding of the mechanisms underlying the flowering transition and enhance genetic studies of birch traits, and they open up new possibilities for the breeding of birch and other woody plants.

Expression Pattern of Class B Gene PAP3 in Flower Development of Pepper

Class B gene APETALA3 (AP3) plays a key role in the development of petals and stamens. Here, we investigated the expression pattern of PAP3 gene (genbank accession number: HM104635) in the buds of cytoplasmic male sterility line 121A and its near-isogenic restorer line 121C at four developmental stages and analyzed the possible association between Class B genes and cytoplasmic male sterility of pepper. Semi-quantitative PCR and quantitative real-time RT-PCR (qRT-PCR) as well as RNA in situ hybridization showed increased expression of PAP3 at late phase of anther development and its higher expression in restorer line compared with sterility line indicating PAP3’s role at late developmental stage of anther and suppressed expression in sterility line. RNA in situ hybridization showed Class B gene features: high abundance in stamen and petal; lower expression in pistil; no expression in sepal. Results of transient expression in onion epidermal cells also showed PAP3 localized in the nucleus, which is consistent with the expression pattern of transcription factors of MADS-box gene family.

Two ancestral APETALA3 homologs from the basal angiosperm Magnolia wufengensis (Magnoliaceae) can affect flower development of Arabidopsis

APETALA3 (AP3) homologs are involved in specifying petal and stamen identities in core eudicot model organisms. In order to investigate the functional conservation of AP3 homologs between core eudicots and basal angiosperm, we isolated and identified two AP3 homologs from Magnolia wufengensis, a woody basal angiosperm belonging to the family Magnoliaceae. Sequence and phylogenetic analyses revealed that both genes are clade members of the paleoAP3 lineage. Moreover, a highly conserved motif of paleoAP3 is found in the C-terminal regions of MAwuAP3_1/2 proteins, but the PI-derived motif, usually present in AP3/DEF-like lineage members, is missing. Semi-quantitative and real time PCR analyses showed that the expression of MAwuAP3_1/2 was restricted to tepals and stamens. However, the MAwuAP3_1 expression was maintained at a high level during the rapid increased in size of tepals and stamens, while MAwuAP3_2 mRNA was only detected at the early stage of tepal and stamen development. Furthermore, the expression of MAwuAP3_1/2 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expressions of the endogenous AP3 gene. Moreover, the 35S::MAwuAP3_1/2 transgenic Arabidopsis can be used partially to rescue the loss-of-function ap3 mutant (ap3-3) of Arabidopsis. These findings call for a more comprehensive understanding of the B-functional evolution from basal angiosperm to core eudicot clades.

Abiotic and Biotic Stress Responses in Solanum tuberosum Group Phureja DM1‐3 516 R44 as Measured through Whole Transcriptome Sequencing

This study conducted an in‐depth analysis of the potato (Solanum tuberosum L.) transcriptome in response to abiotic (salinity, drought, and heat) and biotic (Phytophthora infestans, DL‐β‐amino‐n‐butyric acid, and acibenzolar‐s‐methyl) stresses and plant hormone treatment (abscisic acid, 6‐benzylaminopurine, gibberellic acid, and indole‐3‐acetic acid) using ribonucleic acid sequencing (RNA‐seq) of the doubled monoploid S. tuberosum Group Phureja DM1‐3 516 R44 clone. Extensive changes in gene expression were observed with 37% of the expressed genes being differentially expressed in at least one comparison of stress to control tissue. Stress‐inducible genes known to function in stress tolerance or to be involved in the regulation of gene expression were among the highest differentially expressed. Members of the MYB, APETALA2 (AP2)/ethylene‐responsive element binding factor (ERF), and NAM, ATAF1/2, and CUC2 (NAC) transcription factor families were the most represented regulatory genes. A weighted gene co‐expression network analysis yielded 37 co‐expression modules containing 9198 genes. Fifty percent of the genes within these co‐expression modules were specific to a stress condition indicating condition‐specific responses. Cross‐species comparison between potato and Arabidopsis thaliana (L.) Heynh. uncovered differentially expressed orthologs and defined evolutionary conserved genes. Collectively, the transcriptional profiling of RNA‐seq data presented here provide a valuable reference for potato stress responses to environmental factors that is supported by statistically significant differences in expression changes, highly interconnected genes in co‐expression networks, and evidence of evolutionary conservation.

The miR172 target TOE3 represses AGAMOUS expression during Arabidopsis floral patterning

microRNA172 (miR172) regulates phase transition and floral patterning in Arabidopsis by repressing targets that encode the APETALA2 (AP2) and AP2-like transcription factors. The miR172-mediated repression of the AP2 gene restricts AGAMOUS (AG) expression. In addition, most miR172 targets, including AP2, redundantly act as floral repressors, and the overexpression of the target genes causes delayed flowering. However, how miR172 targets other than AP2 regulate both of the developmental processes remains unclear. Here, we demonstrate that miR172-mediated repression of the TARGET OF EAT 3 (TOE3) gene is critical for floral patterning in Arabidopsis. Transgenic plants that overexpress a miR172-resistant TOE3 gene (rTOE3-ox) exhibit indeterminate flowers with numerous stamens and carpelloid organs, which is consistent with previous observations in transgenic plants that overexpress a miR172-resistant AP2 gene. TOE3 binds to the second intron of the AG gene. Accordingly, AG expression is significantly reduced in rTOE3-ox plants. TOE3 also interacts with AP2 in the nucleus. Given the major role of AP2 in floral patterning, miR172 likely regulates TOE3 in floral patterning, at least in part via AP2. In addition, a miR156 target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 directly activates TOE3 expression, revealing a novel signaling interaction between miR156 and miR172 in floral patterning.

Isolation and functional analysis of the ClM8-FRUITFULL-like MADS-box gene from Chrysanthemum lavandulifolium

AP1/FUL-like genes encode class-specific MADS box transcription factors in angiosperms and play important roles in the plant floral transition and floral organ identity. Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is one of the original species of Chrysanthemum. Understanding the function of AP1/FUL-like genes in C. lavandulifolium provide molecular-level insight into the flowering mechanism of Chrysanthemum. Here, an AP1/FUL-like gene was isolated from C. lavandulifolium. Sequence and phylogenetic analyses indicated that this gene, ClM8, belongs to the euFUL clade of the AP1/FUL-like genes. We observed different expression levels of ClM8 in C. lavandulifolium roots, stems, fruits and leaves and high expression in the flowers. A subcellular localisation assay showed that ClM8 is localised to the nucleus in vivo. The over-expression of ClM8 in Arabidopsis resulted in altered flowering time and inflorescence structure. These aberrant, transgenic Arabidopsis plants showed early flowering following the production of four to six small rosette leaves, lateral flowers and a composite terminal flower. The terminal flower consisted of two or four pistils surrounded by an abnormal number of sepals, petals and stamens. In addition, the morphology of the style of 35S::ClM8 plants silique differed from wild type, appearing thinner and longer. Further investigation suggested that the expression levels of FLOWERING LOCUS T (FT), LEAFY (LFY) and APETALA1 (AP1) were apparently increased in ClM8-transgenic Arabidopsis. Thus, we speculate that ClM8 promoted flowering in Arabidopsis due to the upregulation of FT. Taken together, our results suggest that ClM8 is involved in flowering induction and floral development that might also play a role in the development of fruits and leaves.

MlWRKY12, a novel Miscanthus transcription factor, participates in pith secondary cell wall formation and promotes flowering

WRKY proteins play crucial roles in various plant processes. An AtWRKY12 homologous gene, named MlWRKY12, was isolated from Miscanthus lutarioriparius. The MlWRKY12 gene encodes a WRKY transcription factor belonging to the group IIc subfamily. MlWRKY12 is a nuclear protein. Gene expression pattern analysis revealed a relatively high MlWRKY12 expression level in rhizomes, stems and leaf sheaths. In situ hybridization analysis further demonstrated that MlWRKY12 was expressed in vascular bundle sheath, sclerenchyma and parenchyma tissues. The heterologous expression of MlWRKY12 in an atwrky12 background mutant successfully rescued the phenotype of pith cell walls caused by the defect of AtWRKY12. Most strikingly, the transgenic Arabidopsis plants overexpressing MlWRKY12 exhibited early flowering. The transcript abundance of flowering related genes was measured by quantitative RT-PCR analysis, suggesting that overexpression of MlWRKY12 in Arabidopsis had a significant impact on the expression level of CONSTANS (CO). Moreover, the expression levels of FLOWERING LOCUS T (FT), LFY (LEAFY), APETALA1 (AP1), CAULIFLOWER (CAL) and FRUITFULL (FUL) were upregulated in transgenic plants. These results demonstrated the conserved function of MlWRKY12 existing in secondary cell wall formation of monocotyledonous species and implied a possible impact of MlWRKY12 on flowering control.

The roles of two transcription factors, ABI4 and CBFA, in ABA and plastid signalling and stress responses

Genetic and physiological studies have revealed evidences for multiple signaling pathways by which the plastid exerts retrograde control over photosynthesis-associated-nuclear-genes. In this study we have examined the mechanisms of control of transcription by plastid signals, focusing on transcription factors. We have also further addressed the physical nature of plastid signals and the physiological role, in stress acclimation of this regulatory pathway. ABI4, a master Apetala 2 (AP2)-type transcription factor (TF), is targeted by multiple signalling pathways in plant cells, such as abscisic acid (ABA) signals, sugar signals and plastid signals derived from reactive oxygen species (ROS) and chlorophyll intermediates. ABI4 binds the promoter of target genes to prevent their transcription by competing with other competitive TFs. However, we found that once ABI4 bound the element (CCACGT), it may not be bound by other TFs, therefore making the signalling long-lasting. Downstream of ABI4, CBFA (CCAAT binding factor A) is a subunit of the HAP2/HAP3/HAP5 (Heme activator protein) trimeric transcription complex. CBFA however is a redundant HAP3 subunit. When emergency occurs (such as herbicide treatments or environmental stresses followed by ABA and ROS accumulation), the master transcription factor ABI4 down-regulates some TFs, like CBFA, and then some other TF subunits enter the transcription complex and transcriptional efficiency of stress-responsive genes (including the transcription co-factor CBP) is improved instantaneously. abi4, cbfA and cbp mutants showed weaker drought-tolerance after a herbicide norflurazon treatment, which indicated the physiological role of these key transcription factors.