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 b-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.

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.

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.

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 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, early occurrence of greening according to their increased chlorophyll content and reduction of water content in flower organs. Our results support that PaFYF1/2 function as transcriptional repressors to prohibit flower senescence and abscission in Phalaenopsis.

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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.