Characterization of Two AGAMOUS-like Genes and Their Promoters from the Cymbidium faberi (Orchidaceae).

Arabidopsis AGAMOUS (AG) play roles in determining stamens' and carpels' identities, floral meristem determinacy, and repression of the A-function. Gynostemium fused by stamens and carpels is a characteristic reproductive structure in orchid flowers, which shows a considerable difference from the reproductive organs of eudicots and other monocot species. The molecular basis of orchid gynostemium development remains largely unknown. Here, we report the identification and functional characterization of two AG-like genes, CyfaAG1 and CyfaAG2, and their promoters from C. faberi. Both CyfaAG1 and CyfaAG2 are highly expressed in the anther cap, gynostemium, and ovary. Ectopic expression of CyfaAG1 and CyfaAG2 promotes early flowering of wild-type Arabidopsis. Moreover, ectopic expression of CyfaAG1 completely rescues floral defects in the Arabidopsis ag-1 mutant, while ectopic expression of CyfaAG2 only completes filament and carpel development. Our findings suggest that CyfaAG1 acts as an evolutionarily conserved C-function gene in determining reproductive organ identity and mediating floral meristem determinacy. CyfaAG2 redundantly mediates the C-function in floral meristem determinacy and gynostemium development. Our results provided more details to understand how the C-class function has been partitioned in orchids, and the roles of two AG orthologs in regulating gynostemium development in C. faberi.

FaesAP3_1 Regulates the FaesELF3 Gene Involved in Filament-Length Determination of Long-Homostyle Fagopyrum esculentum.

The identification downstream genes of floral organ identity regulators are critical to revealing the molecular mechanisms underlying floral morphogenesis. However, a general regulatory pathway between floral organ identity genes and their downstream targets is still unclear because of the lack of studies in nonmodel species. Here, we screened a direct downstream target gene, FaesELF3, of a stamen identity transcription factor, FaesAP3_1, in long-homostyle (LH) Fagopyrum esculentum moench by using yeast one-hybrid (Y1H) and dual-luciferase reporter (DR) assays. Furthermore, FaesAP3_1-silenced LH plants that produced flowers with part stamens or anthers homeotically converted into a tepaloid structure, and FaesELF3-silenced plants that had flowers with part stamens consisting of a short filament and empty anther (male sterile anther). All these suggested that transcription factor (TF) FaesAP3_1 directly activates FaesELF3 in order to regulate filament elongation and pollen grain development in LH buckwheat. Our data also suggested that other stamen development pathways independent of FaesAP3_1 remain in F. esculentum.

Characterization of PISTILLATA-like Genes and Their Promoters from the Distyly Fagopyrum esculentum.

Arabidopsis PISTILLATA (PI) encodes B-class MADS-box transcription factor (TF), and works together with APETALA3 (AP3) to specify petal and stamen identity. However, a small-scale gene duplication event of PI ortholog was observed in common buckwheat and resulted in FaesPI_1 and FaesPI_2. FaesPI_1 and FaesPI_2 were expressed only in the stamen of dimorphic flower (thrum and pin) of Fagopyrum esculentum. Moreover, intense beta-glucuronidase (GUS) staining was found in the entire stamen (filament and anther) in pFaesPI_1::GUS transgenic Arabidopsis, while GUS was expressed only in the filament of pFaesPI_2::GUS transgenic Arabidopsis. In addition, phenotype complementation analysis suggested that pFaesPI_1::FaesPI_1/pFaesPI_2::FaesPI_2 transgenic pi-1 Arabidopsis showed similar a flower structure with stamen-like organs or filament-like organs in the third whorl. This suggested that FaesPI_2 only specified filament development, but FaesPI_1 specified stamen development. Meanwhile, FaesPI_1 and FaesPI_2 were shown to function redundantly in regulating filament development, and both genes work together to require a proper stamen identity. The data also provide a clue to understanding the roles of PI-like genes involved in floral organ development during the early evolution of core eudicots and also suggested that FaesPI_1 and FaesPI_2 hold the potential application in bioengineering to develop a common buckwheat male sterile line.

Isolation and Characterization of APETALA3 Orthologs and Promoters from the Distylous Fagopyrum esculentum.

Common buckwheat (Fagopyrum esculentum) produces distylous flowers with undifferentiated petaloid tepals, which makes it obviously different from flowers of model species. In model species Arabidopsis, APETALA3 (AP3) is expressed in petal and stamen and specifies petal and stamen identities during flower development. Combining with our previous studies, we found that small-scale gene duplication (GD) event and alternative splicing (AS) of common buckwheat AP3 orthologs resulted in FaesAP3_1, FaesAP3_2 and FaesAP3_2a. FaesAP3_2 and FaesAP3_2a were mainly expressed in the stamen of thrum and pin flower. Promoters functional analysis suggested that intense GUS staining was observed in the whole stamen in pFaesAP3_2::GUS transgenic Arabidopsis, while intense GUS staining was observed only in the filament of stamen in pFaesAP3_1::GUS transgenic Arabidopsis. These suggested that FaesAP3_1 and FaesAP3_2 had overlapping functions in specifying stamen filament identity and work together to determine normal stamen development. Additionally, FaesAP3_2 and FaesAP3_2a owned the similar ability to rescue stamen development of Arabidopsis ap3-3 mutant, although AS resulted in a frameshift mutation and consequent omission of the complete PI-derived motif and euAP3 motif of FaesAP3_2a. These suggested that the MIK region of AP3-like proteins was crucial for determining stamen identity, while the function of AP3-like proteins in specifying petal identity was gradually obtained after AP3 Orthologs acquiring a novel C-terminal euAP3 motif during the evolution of core eudicots. Our results also provide a clue to understanding the early evolution of the functional specificity of euAP3-type proteins involving in floral organ development in core eudicots, and also suggested that FaesAP3_2 holds the potential application for biotechnical engineering to develop a sterile male line of F. esculentum.

Instantaneous Phase Coherence Imaging for Near-Field Defects by Ultrasonic Phased Array Inspection.

This paper describes an imaging method for near-field defect detection in aluminum plates based on Green's function recovery and application of instantaneous phase coherence weighting factors. The directly acquired acoustic information of near-field defects is usually obscured by the nonlinear effects due to the physical limitation of the acquisition system. Using the diffuse field to recover the Green's function can effectively retrieve the early time information. However, averaging operations of finite number in this process produces an imperfect imaging result. In order to improve the image quality, two kinds of instantaneous phased coherence weighting factors are used to weight the Green's function to reduce the background noise and improve the signal-to-noise ratio: the instantaneous phase coherence factor (IPCF), and the instantaneous phase weighting factor (IPWF). Experiments are conducted on two aluminum plates with two and four near-field defects, respectively. As a result, the background noise of amplitude images weighted by IPCF and IPWF is less than that of the conventional total focusing method (TFM). In addition, the IPCF image achieves a better signal-to-noise ratio (SNR) than that of IPWF, and the phase discontinuity in an IPWF image is suppressed through the IPCF.