K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis

Abstract

Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.

Fig 1. PEP regulates flower reproductive organ identity and determinacy.

A) Scanning Electron Microscopy micrograph (SEM) of a post-anthesis wild-type flower. The different parts of the pistil have been artificially colored: stigma (yellow), style (purple), ovary (green), gynophore (g, orange). pt, petal; st, stamen; s, sepal. B-F) Post-anthesis flowers of different mutant backgrounds. Some outer organs were removed to better show petaloid stamens in the third whorl (arrows). G-I) SEM micrographs. Petal (G) and anther (I) adaxial surface in wild-type. Adaxial side in third whorl organs in hua1 hua2 pep/+ flowers (I). J) SEM apical portion of wild-type pistil. K-N) Top portion (SEM micrographs) of gynoecia/fruits from different mutant combinations. O) hua1 hua2 pep/+ pistils developed extra valves (asterisks) topped by white tissue that resembled that of sepal tips (white arrows). No style and very rudimentary stigmatic tissue were observed (yellow arrow). P) Medial view (SEM micrograph) of stage 17 wild-type fruit (according to [106]). Q) Abaxial surface of hua1 hua2 pep/+ gynoecia. R) Abaxial side of a wild-type sepal. S) A hua1 pep flower after outer organ abscission. Sepaloid carpels (white arrows) formed the fourth whorl that developed on a long gynophore. Similar structures were seen developing inside the sepaloid gynoecia which also contained rudiments of stigmatic tissue (yellow arrow) and further additional floral organs (red arrow). T) hua1 hua2 pep/+ fourth whorl. U-W) hen4 hua1 hua2/+ pep/+ flowers. Fourth whorl organs were removed to observe inner flowers (U, V). X) mRNA expression levels of AG in the wild type (WT) and diverse hua-pep mutant backgrounds, monitored by quantitative RT-PCR (qPCR). Error bars, standard deviation (SD). Asterisks indicate statistically significant differences from hua1 hua2 plants (*P < 0.05). On panels (K, L, N, Q) and (R) some giant cells appear false-colored. Scale bars: 1 mm (B, C, D, E, F, S, T, U, V, W), 500 μm (O), 200 μm (A), 100 μm (J, K, L, M, N, P, Q, R) and 10 μm (G, H, I). nc, nectaries; r, replum; sy, style; v, valve.

Fig 2. The loss of HUA-PEP activity is epistatic over the ful phenotype.

A) SEM images of the top portion of a ful fruit. The typical long style and wide zig-zag replum were suppressed in ful hua1 hua2 and ful hua1 hua2 pep/+ pistils, and sepaloid giant cells were observed on the valve surface. Simple or branched trichomes (white and yellow arrows, respectively) were occasionally observed on the surface of ful hua1 hua2 pep/+ pistils. B) SEM images of the abaxial ovary surface in wild-type (WT) and different mutant backgrounds. Observe interspersed stomata (arrows) in a ful hua1 hua2 pep/+ panel. C-F) GUS reporter whole-mount staining (ful-1) in ful (C), ful hua1 hua2 (D), ful hua1 hua2 pep/+ (E) pistils and wild-type sepal (F). Observe long gynophores and full petaloid conversion of stamens in (E). Scale bars: 100 μm (A, B), except 10 μm in the last B panel (ful hua1 hua2 pep/+ genotype), and 1 mm (C-F). r, replum; v, valve.

Fig 3. Detection of the AP1-GFP protein in hua1 pep gynoecia.

A) Apical region of a mildly affected gynoecium with recognizable pistil morphology. Specific AP1-GFP signal is detected in some style cells. B) Fourth whorl organs of a pre-anthesis flower displaying a severe sepaloid phenotype. C) Adaxial (inner) view of a manually open pistil with severe sepaloid transformations, but containing some developing ovules (do). D) Detail of a fourth whorl organ from panel D showing nuclear-localized AP1-GFP. A cell has been outlined with a dotted line and the nucleus marked with an arrow. Scale bars: 25 μm (A), 50 μm (B and C) and 10 μm (D).

Fig 4. PEP overexpression impairs flower morphogenesis and AG pre-mRNA processing.

A-D) hua1 hua2 35S::PEP flowers. E) hen4 hua1 35S::PEP flower. In both genotypes, loss of determinacy was frequent. All flowers displayed severely transformed petaloid stamens and sepaloid carpels. F-H) SEM micrograph of a hua1 hua2 35S::PEP flower (G), and close-up views of the fourth whorl organ abaxial surface (F) and inner additional whorl organ (H), respectively. Sepaloid traits were found in these gynoecia. I, J) Relative expression levels of AG mRNA (I) and AG transcripts including intron 2 sequences (J), in wild-type plants (WT) and diverse hua-pep mutant backgrounds, monitored by qPCR. In (J), a diagram of the AG gene is shown below. Purple boxes denote exons whereas intronic regions are colored in orange. Relative positions of forward (black arrow) and reverse (red arrow) primers are indicated. To increase annealing specificity, the forward primer sequence was split between exons 1 and 2. Error bars, SD. Asterisks indicate statistically significant differences from hua1 hua2 plants (**P < 0.01). Scale bars are 1 mm (A-E, G) and 100 μm (F, H).

Fig 5. Loss of FLK dramatically enhances the floral phenotypes of hua1 hua2 plants.

A) flk hua1 hua2 flower with all stamens converted into petals. B) Gynoecium with a long gynophore. A sepaloid valve was manually removed to better observe a new flower developing inside. C) SEM image of a sepaloid carpel with giant cells and epicuticular wax ridges. D) SEM magnification of the inner flower shown in (B). E) Close-up view of the sepaloid organ shown in (D). F, G) qPCR relative expression levels of AG mRNA (F), and AG transcripts including intron 2 sequences (G) in wild type (WT) and mutant backgrounds. Error bars, SD. Asterisks indicate statistically significant differences from hua1 hua2 plants (**P < 0.01). Scale bars: 1 mm (A, B), 20 μm (C) 500 μm (D) and 50 μm (E).

Fig 6. The hnRNPs PEP and FLK physically interact with HUA1 and HEN4.

BiFC visualization of protein dimerization (yellow fluorescence) in Nicotiana benthamiana leaf cells agroinfiltrated with plasmids encoding fusion proteins. In each test, the first protein was fused to the C-terminal fragment of the YFP (YFPct), and the second protein to the N-terminal portion (YFPnt), respectively (see Materials and Methods section).

Fig 7. The HUA-PEP activity facilitates pre-mRNA processing of target genes.

A) As the RNA polymerase (RNA pol II) activity progresses, the HUA-PEP hnRNP complex coats the nascent transcript, still chromatin-associated, thus sequestering intronic cryptic sites (ICS) from cleavage and polyadenylation. B) The elongation complex reaches the distal terminal cleavage and polyadenylation site (CPS), where correct termination occurs. C) Adequate intron excision and 3’ maturation take place. D) Conversely, an altered HUA-PEP activity does not prevent the RNA 3’ processing machinery to access cryptic motifs in the elongating transcript, producing thereby a prematurely terminated and ineffective RNA.