The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

PaWOX3 and PaWOX3B Regulate Flower Number and The Lip Symmetry of Phalaenopsis.

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL-related homeobox (WOX) genes, PaWOX3 and PaWOX3B, were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decrease in mature flower and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium orthologue, OnPRS, exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3/PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2, which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3/PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' can not be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to control lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3/PaWOX3B in maintaining the symmetric axis of lip.

OAF is a DAF-like gene that controls ovule development in plants.

We previously found that the RING-type E3 ligase DEFECTIVE IN ANTHER DEHISCENCE1- (DAD1-) Activating Factor (DAF) controls anther dehiscence by activating the jasmonate biosynthetic pathway in Arabidopsis. Here, we show that in Arabidopsis, the DAF ancestor was duplicated into three genes (DAF, Ovule Activating Factor (OAF), DAFL2), which evolved divergent partial functions from their ancestor through subfunctionalization. In this case, DAF-DAD1-JA signaling regulates anther dehiscence, whereas OAF controls ovule development by negatively regulating cinnamyl alcohol dehydrogenase 9 (CAD9) activity and being negatively regulated by miR847 itself in Arabidopsis. Downregulation of OAF or upregulation of CAD9 and miR847 caused similar abortion of ovule formation due to precocious ovule lignification in transgenic Arabidopsis. Interestingly, only one DAF-like gene, PaOAF, exists in the monocot orchids, which has likely evolved through nonfunctionalization and maintains a conserved function as Arabidopsis OAF in regulating ovule development since defective ovules were observed in the virus-induced gene silencing (VIGS) PaOAF Phalaenopsis orchids. The absence of the DAF ortholog and its function in orchids is likely due to the evolution of stamens to a unique pollinium structure that lacks the feature of anther dehiscence. These findings expand the current knowledge underlying the multifunctional evolution and diverse functionalization of duplicate gene pairs within/among plants.

The AtERF19 gene regulates meristem activity and flower organ size in plants.

Ethylene-responsive factors (ERFs) have diverse functions in the regulation of various plant developmental processes. Here, we demonstrate the dual role of an Arabidopsis ERF gene, AtERF19, in regulating reproductive meristem activity and flower organ size through the regulation of genes involved in CLAVATA-WUSCHEL (CLV-WUS) and auxin signaling, respectively. We found that AtERF19 stimulated the formation of flower primordia and controlled the number of flowers produced by activating WUS and was negatively regulated by CLV3. 35S::AtERF19 expression resulted in significantly more flowers, whereas 35S::AtERF19 + SRDX dominant-negative mutants produced fewer flowers. In addition, AtERF19 also functioned to control flower organ size by promoting the division/expansion of the cells through activating Small Auxin Up RNA Gene 32 (SAUR32), which positively regulated MYB21/24 in the auxin signaling pathway. 35S::AtERF19 and 35S::SAUR32 resulted in similarly larger flowers, whereas 35S::AtERF19 + SRDX and 35S::SAUR32-RNAi mutants produced smaller flowers than the wild type. The functions of AtERF19 were confirmed by the production of similarly more and larger flowers in 35S::AtERF19 transgenic tobacco (Nicotiana benthamiana) and in transgenic Arabidopsis which ectopically expressed the orchid gene (Nicotiana benthamiana) PaERF19 than in wild-type plants. The finding that AtERF19 regulates genes involved in both CLV-WUS and auxin signaling during flower development significantly expands the current knowledge of the multifunctional evolution of ERF genes in plants. The results presented in this work indicate a dual role for the transcription factor AtERF19 in controlling the number of flowers produced and flower organ size through the regulation of genes involved in CLV-WUS and auxin signaling, respectively. Our findings expand the knowledge of the roles of ERF genes in the regulation of reproductive development.

Regulatory network for FOREVER YOUNG FLOWER-like genes in regulating Arabidopsis flower senescence and abscission.

FOREVER YOUNG FLOWER (FYF) has been reported to play an important role in regulating flower senescence/abscission. Here, we functionally analyzed five Arabidopsis FYF-like genes, two in the FYF subgroup (FYL1/AGL71 and FYL2/AGL72) and three in the SOC1 subgroup (SOC1/AGL20, AGL19, and AGL14/XAL2), and showed their involvement in the regulation of flower senescence and/or abscission. We demonstrated that in FYF subgroup, FYF has both functions in suppressing flower senescence and abscission, FYL1 only suppresses flower abscission and FYL2 has been converted as an activator to promote flower senescence. In SOC1 subgroup, AGL19/AGL14/SOC1 have only one function in suppressing flower senescence. We also found that FYF-like proteins can form heterotetrameric complexes with different combinations of A/E functional proteins (such as AGL6 and SEP1) and AGL15/18-like proteins to perform their functions. These findings greatly expand the current knowledge behind the multifunctional evolution of FYF-like genes and uncover their regulatory network in plants.

The Oncidium Ethylene Synthesis Gene Oncidium 1-Aminocyclopropane-1 Carboxylic Acid Synthase 12 and Ethylene Receptor Gene Oncidium ETR1 Affect GA-DELLA and Jasmonic Acid Signaling in Regulating Flowering Time, Anther Dehiscence, and Flower Senescence in Arabidopsis.

In plants, the key enzyme in ethylene biosynthesis is 1-aminocyclopropane-1 carboxylic acid (ACC) synthase (ACS), which catalyzes S-adenosyl-L-methionine (SAM) to ACC, the precursor of ethylene. Ethylene binds to its receptors, such as ethylene response 1 (ETR1), to switch on ethylene signal transduction. To understand the function of ACS and ETR1 in orchids, Oncidium ACC synthase 12 (OnACS12) and Oncidium ETR1 (OnETR1) from Oncidium Gower Ramsey were functionally analyzed in Arabidopsis. 35S::OnACS12 caused late flowering and anther indehiscence phenotypes due to its effect on GA-DELLA signaling pathways. 35S::OnACS12 repressed GA biosynthesis genes (CPS, KS, and GA3ox1), which caused the upregulation of DELLA [GA-INSENSITIVE (GAI), RGA-LIKE1 (RGL1), and RGL2] expression. The increase in DELLAs not only suppressed LEAFY (LFY) expression and caused late flowering but also repressed the jasmonic acid (JA) biosynthesis gene DAD1 and caused anther indehiscence by downregulating the endothecium-thickening-related genes MYB26, NST1, and NST2. The ectopic expression of an OnETR1 dominant-negative mutation (OnETR1-C65Y) caused both ethylene and JA insensitivity in Arabidopsis. 35S::OnETR1-C65Y delayed flower/leaf senescence by suppressing downstream genes in ethylene signaling, including EDF1-4 and ERF1, and in JA signaling, including MYC2 and WRKY33. JA signaling repression also resulted in indehiscent anthers via the downregulation of MYB26, NST1, NST2, and MYB85. These results not only provide new insight into the functions of ACS and ETR1 orthologs but also uncover their functional interactions with other hormone signaling pathways, such as GA-DELLA and JA, in plants.

Multifunctional evolution of B and AGL6 MADS box genes in orchids.

We previously found that B and AGL6 proteins form L (OAP3-2/OAGL6-2/OPI) and SP (OAP3-1/OAGL6-1/OPI) complexes to determine lip/sepal/petal identities in orchids. Here, we show that the functional L' (OAP3-1/OAGL6-2/OPI) and SP' (OAP3-2/OAGL6-1/OPI) complexes likely exist and AP3/PI/AGL6 genes have acquired additional functions during evolution. We demonstrate that the presumed L' complex changes the structure of the lower lateral sepals and helps the lips fit properly in the center of the flower. In addition, we find that OAP3-1/OAGL6-1/OPI in SP along with presumed SP' complexes regulate anthocyanin accumulation and pigmentation, whereas presumed L' along with OAP3-2/OAGL6-2/OPI in L complexes promotes red spot formation in the perianth. Furthermore, the B functional proteins OAP3-1/OPI and OAGL6-1 in the SP complex could function separately to suppress sepal/petal senescence and promote pedicel abscission, respectively. These findings expand the current knowledge behind the multifunctional evolution of the B and AGL6 genes in plants.

Distance-based measurement determines the coexistence of B protein hetero- and homodimers in lily tepal and stamen tetrameric complexes.

The floral quartet model proposes that plant MADS box proteins function as higher order tetrameric complexes. However, in planta evidence for MADS box tetramers remains scarce. Here, we applied a strategy using in vivo fluorescence resonance energy transfer (FRET) based on the distance change and distance symmetry of stable tetrameric complexes in tobacco (Nicotiana benthamiana) leaf cells to improve the accuracy of the estimation of heterotetrameric complex formation. This measuring system precisely verified the stable state of Arabidopsis petal (AP3/PI/SEP3/AP1) and stamen (AP3/PI/SEP3/AG) complexes and showed that the lily (Lilium longiflorum) PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, L8/L9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. Importantly, this system was applied successfully to find complicated tepal and stamen heterotetrameric complexes in lily. We found that heterodimers of B function AP3/PI orthologs (L1/L8) likely coexist with the homodimers of PI orthologs (L8/L8, L9/L9) to form five (two most stable and three stable) tepal- and four (one most stable and three stable) stamen-related heterotetrameric complexes with A/E and C/E function proteins in lily. Among these combinations, L1 preferentially interacted with L8 to form the most stable heterotetrameric complexes, and the importance of the L8/L8 and L9/L9 homodimers in tepal/stamen formation in lily likely decreased to a minor part during evolution. The system provides substantial improvements for successfully estimating the existence of unknown tetrameric complexes in plants.

Silencing of FOREVER YOUNG FLOWER-Like Genes from Phalaenopsis Orchids Promotes Flower Senescence and Abscission.

Ectopic expression of FOREVER YOUNG FLOWER (FYF) delays floral senescence and abscission in transgenic Arabidopsis. To analyze the FYF function in Phalaenopsis orchids, two FYF-like genes (PaFYF1/2) were identified. PaFYF1/2 were highly expressed in young Phalaenopsis flowers, and their expression decreased significantly afterward until flower senescence. This pattern was strongly correlated with the process of flower senescence and revealed that PaFYF1/2 function to suppress senescence/abscission during early flower development. Interestingly, in flowers, PaFYF1 was consistently expressed less in petals than in lips/sepals, whereas PaFYF2 was expressed relatively evenly in all flower organs. This difference suggests a regulatory modification of the functions of PaFYF1 and PaFYF2 during Phalaenopsis flower evolution. Delayed flower senescence and abscission, which were unaffected by ethylene treatment, were observed in 35S::PaFYF1/2 and 35S::PaFYF1/2 + SRDX transgenic Arabidopsis plants due to the downregulation of the ethylene signaling and abscission-associated genes EDF1-4, IDA and BOP1/2. These results suggest a possible repressor role for Phalaenopsis PaFYF1/2 in controlling floral senescence/abscission by suppressing ethylene signaling and abscission-associated genes. To further validate the function of PaFYF1/2, PaFYF1/2-VIGS (virus-induced gene silencing) Phalaenopsis were generated and analyzed. Promotion of senescence and abscission was observed in PaFYF1/2-VIGS Phalaenopsis flowers by the upregulation of PeEDF1/2, PeSAG39 and PeBOP1/2 expression, the early occurrence of greening according to their increased chlorophyll content and the reduction in water content in flower organs. Our results support that PaFYF1/2 function as transcriptional repressors to prohibit flower senescence and abscission in Phalaenopsis.

NAC-Like Gene GIBBERELLIN SUPPRESSING FACTOR Regulates the Gibberellin Metabolic Pathway in Response to Cold and Drought Stresses in Arabidopsis.

To investigate the functions of NAC-like genes, we reported the characterization and functional analysis of one Arabidopsis NAC-like gene which showed a novel function in the regulation of gibberellin biosynthesis and named as GIBBERELLIN SUPPRESSING FACTOR (GSF). GSF acts as a transcriptional activator and has transactivation capacity based on yeast transcription activity assays. YFP + GSF-TM (lacking a transmembrane domain) fusion proteins accumulated in the nuclei, while the YFP + GSF fusion proteins only accumulated in the ER membrane and were absent from the nuclei. These results revealed that GSF requires processing and release from the ER and transportation into the nucleus to perform its function. The ectopic expression of GSF-TM caused a dwarfism phenotype, which was correlated with the upregulation of the gibberellin (GA) deactivation genes GA2-oxidases 2/6 (GA2ox2/6) and the downregulation of the GA biosynthetic genes GA20-oxidases 1-4 (GA20ox1-4). The external application of GA rescued the dwarfism in the 35 S::GSF-TM plants, indicating that GSF affects GA biosynthesis, rather than the GA signaling pathway. Further analysis indicated that the upregulation of GA2ox2/6 is a key factor for the GSF function to regulate the GA level, since 35 S::GA20ox1 could not rescue the dwarfism in the 35 S::GSF-TM plants. Cold treatment induced the processing of the YFP + GSF fusion proteins from the ER membrane and their entry into the nuclei, which is correlated with the cold-induced upregulation of GA2oxs. In addition, the expression of GA2oxs was induced by drought, and the 35 S::GSF-TM plants showed drought tolerance compared to the wild-type plants. Our data suggest a role for GSF in response to abiotic stresses, such as cold and drought, by suppressing the biosynthesis of GA in Arabidopsis.

A tetraspanin gene regulating auxin response and affecting orchid perianth size and various plant developmental processes.

The competition between L (lip) and SP (sepal/petal) complexes in P-code model determines the identity of complex perianth patterns in orchids. Orchid tetraspanin gene Auxin Activation Factor (AAF) orthologs, whose expression strongly correlated with the expansion and size of the perianth after P code established, were identified. Virus-induced gene silencing (VIGS) of OAGL6-2 in L complex resulted in smaller lips and the down-regulation of Oncidium OnAAF. VIGS of PeMADS9 in L complex resulted in the enlarged lips and up-regulation of Phalaenopsis PaAAF. Furthermore, the larger size of Phalaenopsis variety flowers was associated with higher PaAAF expression, larger and more cells in the perianth. Thus, a rule is established that whenever bigger perianth organs are made in orchids, higher OnAAF/PaAAF expression is observed after their identities are determined by P-code complexes. Ectopic expression Arabidopsis AtAAF significantly increased the size of flower organs by promoting cell expansion in transgenic Arabidopsis due to the enhancement of the efficiency of the auxin response and the subsequent suppression of the jasmonic acid (JA) biosynthesis genes (DAD1/OPR3) and BIGPETAL gene during late flower development. In addition, auxin-controlled phenotypes, such as indehiscent anthers, enhanced drought tolerance, and increased lateral root formation, were also observed in 35S::AtAAF plants. Furthermore, 35S::AtAAF root tips maintained gravitropism during auxin treatment. In contrast, the opposite phenotype was observed in palmitoylation-deficient AtAAF mutants. Our data demonstrate an interaction between the tetraspanin AAF and auxin/JA that regulates the size of flower organs and impacts various developmental processes.

The Gene ANTHER DEHISCENCE REPRESSOR (ADR) Controls Male Fertility by Suppressing the ROS Accumulation and Anther Cell Wall Thickening in Arabidopsis.

Male sterility in plants is caused by various stimuli such as hormone changes, stress, cytoplasmic alterations and nuclear gene mutations. The gene ANTHER DEHISCENCE REPRESSOR (ADR), which is involved in regulating male sterility in Arabidopsis, was functionally analyzed in this study. In ADR::GUS flowers, strong GUS activity was detected in the anthers of young flower buds but was low in mature flowers. ADR + GFP fusion proteins, which can be modified by N-myristoylation, were targeted to peroxisomes. Ectopic expression of ADR in transgenic Arabidopsis plants resulted in male sterility due to anther indehiscence. The defect in anther dehiscence in 35S::ADR flowers is due to the reduction of ROS accumulation, alteration of the secondary thickening in the anther endothecium and suppression of the expression of NST1 and NST2, which are required for anther dehiscence through regulation of secondary wall thickening in anther endothecial cells. This defect could be rescued by external application of hydrogen peroxide (H2O2). These results demonstrated that ADR must be N-myristoylated and targeted to the peroxisome during the early stages of flower development to negatively regulate anther dehiscence by suppressing ROS accumulation and NST1/NST2 expression.

Comparative analysis of the pteridophyte Adiantum MFT ortholog reveals the specificity of combined FT/MFT C and N terminal interaction with FD for the regulation of the downstream gene AP1.

To study the evolution of phosphatidylethanolamine-binding protein (PEBP) gene families in non-flowering plants, we performed a functional analysis of the PEBP gene AcMFT of the MFT clade in the pteridophyte Adiantum capillus-veneris. The expression of AcMFT was regulated by photoperiod similar to that for FT under both long day and short day conditions. Ectopic expression of AcMFT in Arabidopsis promotes the floral transition and partially complements the late flowering defect in transgenic Arabidopsis ft-1 mutants, suggesting that AcMFT functions similarly to FT in flowering plants. Interestingly, a similar partial compensation of the ft-1 late flowering phenotype was observed in Arabidopsis ectopically expressing only exon 4 of the C terminus of AcMFT and FT. This result indicated that the fourth exon of AcMFT and FT plays a similar and important role in promoting flowering. Further analysis indicated that exons 1-3 in the N terminus specifically enhanced the function of FT exon 4 in controlling flowering in Arabidopsis. Protein pull-down assays indicated that Arabidopsis FD proteins interact with full-length FT and AcMFT, as well as peptides encoded by 1-3 exon fragments or the 4th exon alone. Furthermore, similar FRET efficiencies for FT-FD and AcMFT-FD heterodimer in nucleus were observed. These results indicated that FD could form the similar complex with FT and AcMFT. Further analysis indicated that the expression of AP1, a gene downstream of FT, was up-regulated more strongly by FT than AcMFT in transgenic Arabidopsis. Our results revealed that AcMFT from a non-flowering plant could interact with FD to regulate the floral transition and that this function was reduced due to the weakened ability of AcMFT-FD to activate the downstream gene AP1.

The C-Terminal Sequence and PI motif of the Orchid (Oncidium Gower Ramsey) PISTILLATA (PI) Ortholog Determine its Ability to Bind AP3 Orthologs and Enter the Nucleus to Regulate Downstream Genes Controlling Petal and Stamen Formation.

This study focused on the investigation of the effects of the PI motif and C-terminus of the Oncidium Gower Ramsey MADS box gene 8 (OMADS8), a PISTILLATA (PI) ortholog, on floral organ formation. 35S::OMADS8 completely rescued and 35S::OMADS8-PI (with the PI motif deleted) partially rescued petal/stamen formation, whereas these deficiencies were not rescued by 35S::OMADS8-C (C-terminal 29 amino acids deleted) in pi-1 mutants. OMADS8 could interact with Arabidopsis APETALA3 (AP3) and enter the nucleus. The nuclear entry efficiency was reduced for OMADS8-PI/AP3 and OMADS8-C/AP3. OMADS8 could also interact with OMADS5/OMADS9 (the Oncidium AP3 ortholog) and enter the nucleus with an efficiency only slightly affected by the deletion of the C-terminal sequence or PI motif. However, the stability of the OMADS8/OMADS5 and OMADS8/OMADS9 complexes was significantly reduced by deletion of the C-terminal sequence or PI motif. Further analysis indicated that the expression of genes downstream of AP3/PI (BNQ1/BNQ2/GNC/At4g30270) was compensated by 35S::OMADS8 and 35S::OMADS8-PI to a level similar to wild-type plants but was not affected by 35S::OMADS8-C in the pi-1 mutants. A similar FRET (fluorescence resonance energy transfer) efficiency was observed for Arabidopsis AGAMOUS (AG) and the Oncidium AG ortholog OMADS4 for OMADS8, OMADS8-PI and OMADS8-C. These results indicated that the OMADS8 PI motif and C-terminus were valuable for the interaction of OMADS8 with the AP3 orthologs to form higher order heterotetrameric complexes that regulated petal/stamen formation in both Oncidium orchids and transgenic Arabidopsis. However, the C-terminal sequence and PI motif were dispensable for the interaction of OMADS8 with the AG orthologs.

FOREVER YOUNG FLOWER Negatively Regulates Ethylene Response DNA-Binding Factors by Activating an Ethylene-Responsive Factor to Control Arabidopsis Floral Organ Senescence and Abscission.

In this study of Arabidopsis (Arabidopsis thaliana), we investigated the relationship between FOREVER YOUNG FLOWER (FYF) and Ethylene Response DNA-binding Factors (EDFs) and functionally analyzed a key FYF target, an Ethylene-Responsive Factor (ERF), that controls flower senescence/abscission. Ectopic expression of EDF1/2/3/4 caused promotion of flower senescence/abscission and the activation of the senescence-associated genes. The presence of a repressor domain in EDFs and the enhancement of the promotion of senescence/abscission in EDF1/2/3/4+SRDX (converting EDFs to strong repressors by fusion with the ERF-associated amphiphilic repression motif repression domain SRDX) transgenic plants suggested that EDFs act as repressors. The significant reduction of ß-glucuronidase (GUS) expression by 35S:FYF in EDF1/2/3/4:GUS plants indicates that EDF1/2/3/4 functions downstream of FYF in regulating flower senescence/abscission. In this study, we also characterized an ERF gene, FOREVER YOUNG FLOWER UP-REGULATING FACTOR1 (FUF1), which is up-regulated by FYF during flower development. Ectopic expression of FUF1 caused similar delayed flower senescence/abscission as seen in 35S:FYF plants. This phenotype was correlated with deficient abscission zone formation, ethylene insensitivity, and down-regulation of EDF1/2/3/4 and abscission-associated genes in 35S:FUF1 flowers. In contrast, significant promotion of flower senescence/abscission and up-regulation of EDF1/2/3/4 were observed in 35S:FUF1+SRDX transgenic dominant-negative plants, in which FUF1 is converted to a potent repressor by fusion to an SRDX-suppressing motif. Thus, FUF1 acts as an activator in suppressing EDF1/2/3/4 function and senescence/abscission of the flowers. Our results reveal that FYF regulates flower senescence/abscission by negatively regulating EDF1/2/3/4, which is the downstream gene in the ethylene response, by activating FUF1 in Arabidopsis.

AGAMOUS-LIKE13, a putative ancestor for the E functional genes, specifies male and female gametophyte morphogenesis.

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant-negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet-like complexes (AGL13-AG)2 and interact with AG-AP3-PI to form a higher-order heterotetrameric complex (AGL13-AG-AP3-PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.

The NAC-like gene ANTHER INDEHISCENCE FACTOR acts as a repressor that controls anther dehiscence by regulating genes in the jasmonate biosynthesis pathway in Arabidopsis.

ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF:GUS flowers, ß-glucuronidase (GUS) activity was detected in the anther, the upper parts of the filaments, and in the pollen of stage 7-9 young flower buds; GUS activity was reduced in mature flowers. Yellow fluorescent protein (YFP)+AIF-C fusion proteins, which lacked a transmembrane domain, accumulated in the nuclei of the Arabidopsis cells, whereas the YFP+AIF fusion proteins accumulated in the membrane and were absent in the nuclei. Further detection of a cleaved AIF protein in flowers revealed that AIF needs to be processed and released from the endoplasmic reticulum in order to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX plants suggest that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/AOS/AOC3/OPR3/OPCL1. The external application of JA rescued the anther indehiscence in AIF-C and AIF-C+SRDX flowers. In AIF-C+VP16 plants, which are transgenic dominant-negative mutants in which AIF is converted to a potent activator via fusion to a VP16-AD motif, the anther dehiscence was promoted, and the expression of DAD1/AOS/AOC3/OPR3/OPCL1 was up-regulated. Furthermore, the suppression of AIF through an antisense strategy resulted in a mutant phenotype similar to that observed in the AIF-C+VP16 flowers. The present data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis.

A RING-type E3 ligase controls anther dehiscence by activating the jasmonate biosynthetic pathway gene DEFECTIVE IN ANTHER DEHISCENCE1 in Arabidopsis.

Suppression of expression of DAF [DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor], a gene that encodes a putative RING-finger E3 ligase protein, causes non-dehiscence of the anthers, alters pollen development and causes sterility in 35S:DAF RNAi/antisense Arabidopsis plants. This mutant phenotype correlates with the suppression of DAF but not with expression of the two most closely related genes, DAFL1/2. The expression of DAD1 was significantly reduced in 35S:DAF RNAi/antisense plants, and complementation with 35S:DAF did not rescue the dad1 mutant, indicating that DAF acts upstream of DAD1 in jasmonic acid biosynthesis. This assumption is supported by the finding that 35S:DAF RNAi/antisense plants showed a similar cellular basis for anther dehiscence to that found in dad1 mutants, and that external application of jasmonic acid rescued the anther non-dehiscence and pollen defects in 35S:DAF antisense flowers. We further demonstrate that DAF is an E3 ubiquitin ligase and that its activity is abolished by C132S and H137Y mutations in its RING motif. Furthermore, ectopic expression of the dominant-negative C132S or H137Y mutations causes similar indehiscence of anthers and reduction in DAD1 expression in transgenic Arabidopsis. This result not only confirms that DAF controls anther dehiscence by positively regulating the expression of DAD1 in the jasmonic acid biosynthesis pathway, but also supports the notion that DAF functions as an E3 ubiquitin ligase, and that the conserved RING-finger region is required for its activity.

Functional analysis reveals the possible role of the C-terminal sequences and PI motif in the function of lily (Lilium longiflorum) PISTILLATA (PI) orthologues.

Two lily (Lilium longiflorum) PISTILLATA (PI) genes, Lily MADS Box Gene 8 and 9 (LMADS8/9), were characterized. LMADS9 lacked 29 C-terminal amino acids including the PI motif that was present in LMADS8. Both LMADS8/9 mRNAs were prevalent in the first and second whorl tepals during all stages of development and were expressed in the stamen only in young flower buds. LMADS8/9 could both form homodimers, but the ability of LMADS8 homodimers to bind to CArG1 was relatively stronger than that of LMADS9 homodimers. 35S:LMADS8 completely, and 35S:LMADS9 only partially, rescued the second whorl petal formation and partially converted the first whorl sepal into a petal-like structure in Arabidopsis pi-1 mutants. Ectopic expression of LMADS8-C (with deletion of the 29 amino acids of the C-terminal sequence) or LMADS8-PI (with only the PI motif deleted) only partially rescued petal formation in pi mutants, which was similar to what was observed in 35S:LMADS9/pi plants. In contrast, 35:LMADS9+L8C (with the addition of the 29 amino acids of the LMADS8 C-terminal sequence) or 35S:LMADS9+L8PI (with the addition of the LMADS8 PI motif) demonstrated an increased ability to rescue petal formation in pi mutants, which was similar to what was observed in 35S:LMADS8/pi plants. Furthermore, ectopic expression of LMADS8-M (with the MADS domain truncated) generated more severe dominant negative phenotypes than those seen in 35S:LMADS9-M flowers. These results revealed that the 29 amino acids including the PI motif in the C-terminal region of the lily PI orthologue are valuable for its function in regulating perianth organ formation.

Delay of flower senescence and abscission in Arabidopsis transformed with an FOREVER YOUNG FLOWER homolog from Oncidium orchid.

The ectopic expression of FOREVER YOUNG FLOWER (FYF), a MADS box gene in Arabidopsis, caused significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. It was proposed that the function of the FYF gene was related to the regulation of senescence and abscission. This hypothesis was further supported by one line of evidence reported in this study. The evidence is the similar delay of flower senescence and abscission observed in transgenic Arabidopsis ectopically expressing OnFYF, an FYF homolog from the Oncidium orchid, a monocot. This data suggested that the function of FYF homologs in regulating flower senescence and abscission was highly conserved in both dicot and monocot plants.