New Results

PRESSED FLOWER works downstream of ASYMMETRIC LEAVES 2 to affect sepal flatness in Arabidopsis

Ruoyu Liu, Zeming Wang, Xi He, Heng Zhou, Yiru Xu, Lilan Hong
doi: https://doi.org/10.1101/2024.09.30.615753

ABSTRACT

The development of flattened organs such as leaves and sepals is essential for proper plant function. While much research has focused on leaf flatness, little is known about how sepals achieve flat organ morphology. Previous study has shown that in Arabidopsis an ASYMMETRIC LEAVES 2 (AS2) gene mutation as2-7D causes ectopic AS2 expression on the abaxial sepal epidermis, which leads to growth discoordination between the two sides of sepals, resulting in outgrowth formation on abaxial sepal epidermis and sepal flatness disruption. Here we report that the PRESSED FLOWER (PRS) works downstream of AS2 in affecting sepal flatness. Genetic analysis showed that PRS mutations suppressed the outgrowth formation on the abaxial sepal epidermis in as2-7D mutant. Through tracking the PRS expression dynamics at a cellular resolution throughout the early developmental stages in WT and as2-7D sepals, we found that on the abaxial epidermis of as2-7D sepals, ectopic AS2 expression up-regulated PRS expression, leading to the epidermal outgrowth initiation. AS2 affected PRS activity on multiple levels: AS2 activated PRS expression through direct binding to PRS promoter region; AS2 also physically interacted with PRS. Our study highlights the complex interplay between AS2 and PRS in modulating sepal flatness.

INTRODUTION

During development, plant organs display a myriad of complex shapes in three dimensions: cylindrical shoots, spherical or oblong fruits, lobed anthers, leaves and petals that can be flat, twisting, bending, waving, cup-shaped, tubular, etc (Tena, 2024). Flat morphology is a key structural form in plant organs, with flat leaves being the most common example (Tsukaya, 2005; Sandalio et al., 2016). The development of three asymmetrical axes, an adaxial-abaxial axis, a medio–lateral axis, and a proximal–distal axis, plays an crucial role in the transformation of the leaf blade from a radial primordium to a flattened structure during leaf growth and development (Ichihashi and Tsukaya, 2015; Nakayama et al., 2022). Extensive molecular genetic studies have identified a regulatory network for the establishment of adaxial-abaxial polarity in leaves, involving auxin and abaxial- and adaxial-promoting genes (Lin et al., 2007; Shi et al., 2017). How the proximal-distal polarity is established remains largely unknown (Du et al., 2018), while the establishment of the medio-lateral polarity (from the midrib to the margin) depends on the adaxial-abaxial polarity (Waites and Hudson, 1995).

The leaf blade exhibits anisotropic growth during morphogenesis, with cell division occurring primarily perpendicular to the medio-lateral axis, which results in flattened leaves (Du et al., 2018). Growth along the medio-lateral axis is contingent on the activity of leaf meristems (Nardmann and Werr, 2013; Ichihashi and Tsukaya, 2015; Guan et al., 2017). Although leaves exhibit determinate growth without typical anatomical features of meristematic tissue, there is transient leaf meristematic activity allowing the expansion of leaf blade (Alvarez et al., 2016). In Arabidopsis thaliana (Arabidopsis), the transient leaf meristematic activity, reflected in part by the expression of WUSCHEL-RELATED HOMEOBOX3 (WOX3) / PRESSED FLOWER (PRS), is progressively confined to the entire marginal region in young leaves and further confined to the proximal marginal region in older leaves (Alvarez et al., 2016). PRS and WUSCHEL-RELATED HOMEOBOX1 (WOX1) act redundantly in the marginal region (also called the middle domain) between the adaxial and abaxial domains and are critical for leaf blade outgrowth (Vandenbussche et al., 2009; Nakata et al., 2012). PRS and WOX1 work through coordinately regulating the proliferation of WOX-expressing cells and surrounding cells (Nakata et al., 2012). When ectopic expression of WOX1 and PRS occurs in the abaxial domain of leaf primordia, outgrowths form on the abaxial surface of leaves (Nakata et al., 2012).

Sepals, the exterior floral organs of most flowering plants, also exhibit a flattened morphology, which helps to protect the development of internal flower organs (Roeder, 2021). While many studies have focused on the flattened morphology of leaves, relatively few studies have explored on the morphogenesis of flattened organs based on sepals. In fact, Arabidopsis sepal serves as an excellent model for studying organ morphogenesis due to its simplicity, accessibility, and reproducibility of morphogenesis. Sepals are the most peripheral organ of the flower, making them easy to manipulate and observe (Zhu et al., 2020). The final size of the Arabidopsis sepal is only about 1 mm2, and the entire morphogenesis of living sepals can be imaged using high magnification microscopes (Hong et al., 2016). Plant growth, development, and morphogenesis are continuous processes that occur in both space and time. Dynamic observation of these biological processes has been a significant technical challenge in plant developmental biology. The use of time-lapse live imaging combined with data processing and analysis tools in Arabidopsis sepals allows for simultaneous temporal and spatial observation of sepal size, shape, and curvature, as well as tracking of sepal cell growth and division dynamics (Hong et al., 2017; Robinson et al., 2018). In addition, based on transcriptomic analysis of floral organs, only 13 genes were found to be specifically expressed in sepals; in other words, most genes expressed in sepals are also expressed in other organs (Wellmer et al., 2004). Therefore, insights gained from studying Arabidopsis sepals can be broadly applied to understanding the morphogenesis of other plant lateral organs.

In our previous work, we identified a mutant with abnormal sepal morphology, as2-7D, which has an unbalanced growth on the abaxial-adaxial surfaces of the sepals, resulting in numerous folds and outgrowths on the abaxial surface. The folds comprise ridges and invaginations, whereas the outgrowths, which are pointed epidermal projections, are typically located at the end of the folds. The as2-7D mutant sepal epidermis produces outgrowths due to conflicting growth directions and unequal epidermal stiffness. The mutant phenotype was found to be caused by a mutation in the AS2 gene by map-based cloning (Yadav et al., 2024). The AS2 gene encodes a transcription factor that contains a LATERAL ORGAN BOUNDARIES (LOB) domain (Iwakawa et al., 2002; Lin et al., 2003).The AS2 promoter region has a KANADI (KAN) transcription factor binding site, and the binding of KAN to the AS2 promoter represses AS2 expression (Wu et al., 2008). A point mutation occurred at the KAN binding site in as2-7D genome, which altered the expression pattern of the AS2 gene. The wild type (WT) AS2 promoter is active on the adaxial surface of the organ, while the mutant AS2 promoter drives expression on both the abaxial and adaxial surfaces of the sepals (Yadav et al., 2024). Although the effect of ectopic AS2 expression on sepal flatness have been explored in detail on the cellular level, the molecular mechanisms underlying AS2’s function in regulating sepal flatness remain not fully understood.

In this study, to further explore the genes involved in controlling sepal flatness, we did a suppressor screen of as2-7D’s sepal phenotype and obtained a suppressor mutant with reduced sepal epidermal outgrowths. The PRS gene was identified as the mutant gene of the suppressor. Using live imaging we provided a detailed depiction of the PRS expression pattern at a cellular resolution throughout the early developmental stages of WT and as2-7D sepals. We found that on the abaxial epidermis of as2-7D sepals, the ectopic expression of AS2 tends to retain PRS expression and keeps it to linger on the rising outgrowth, which initiates epidermal outgrowths initiation. Furthermore, AS2 interacts with PRS at the protein level and promotes PRS expression at the transcriptional level, contributing to the formation of outgrowths in as2-7D sepals. Our work highlights the complex interplay between AS2 and PRS in modulating sepal flatness.

METHOD

Plant materials and growth conditions Landsberg erecta (Ler) ecotype plants is the WT in this study. The as2-7D mutant was previously identified by our research group in a screen (Yadav et al., 2024). as2-7D has a G to A point mutation at 1484 bp upstream of the AS2 gene promoter, resulting in ectopic expression of the AS2 gene. 60Co-γ rays were used to mutagenize as2-7D seeds (radiation dose rate of 10 Gy·min-1, radiation dose of 600 Gy). In the M2 generation of the self-pollinated progeny from the M1 plants, a mutant showing significant suppression of the sepal outgrowth phenotype was identified and designated as as2-7D suppressors of as2-7D sepal phenotype 1 (as2-7D ssp1). Crossing as2-7D ssp1 with the WT resulted in the isolation of the single mutant ssp1 (prs-3) in the F2 generation. The prs-4 mutant (T-DNA insertion line SALK_127850) was obtained from the Arabidopsis Biological Resource Center (ABRC). The as2-5D mutant has been previously described (Wu et al., 2008) and was ordered from ABRC (CS67863). Plants were grown at 22°C under 16-hour light/8-hour dark conditions, with a light intensity of 12,000 Lux.

QTL-seq

In this study, as2-7D × as2-7D ssp1was hybridized, and individual plants with phenotypes of as2-7D and as2-7D ssp1 were selected from the F2 population to construct two mixed offspring pools with extreme phenotypes for QTL-seq analysis (Takagi et al., 2013). The Δ SNP index was obtained by subtracting the SNP index of as2-7D ssp1 phenotype pool with the SNP index of as2-7D phenotype pool. The Δ SNP index is plotted with the genome position as the horizontal axis and the Δ SNP index as the vertical axis to obtain the Δ SNP index map. In the plot, the region where the peak or valley is located in the map is the candidate region for the actual mutation site. QTL-seq analysis showed that there were differences in SNP index on chromosome 2 between the two separate populations, and the SSP1 suppression gene was preliminarily located in the genomic region of 9 Mb-16 Mb on chromosome 2.

Flower staging

Flower staging was based on Smyth et al (Smyth et al., 1990). The main observations in this study were on flowers at around stage 6 when outgrowth formation initiated in as2-7D sepals.

Sepal architecture

Flowers are enclosed by four sepals, of which the out-most sepal is located away from the meristematic tissue and is called the abaxial/outer sepal, while the sepal that is diametrically opposed to the outer sepal and oriented toward the meristematic tissue is called the adaxial/inner sepal. The remaining two sepals located on either side are the lateral sepals. For a detailed description, see Roeder (Roeder, 2021).

Scanning electron microscopy

Sepals at stage 14 were fixed overnight in 2.5% glutaraldehyde solution-0.2M sodium phosphate buffer (pH 7.0) at 4 □. The fixed samples were washed three times with 0.1 M sodium phosphate buffer (pH 7.0) for 15 minutes each time. The samples were washed with 0.1 M phosphate buffer solution (pH 7.0) and dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, and 95%). Next, the samples were placed in Hitachi HCP for critical point drying, coated with platinum, and inspected using a filed emission scanning electron microscope (Hitachi SU-8010).

Confocal microscopy and living image

For live imaging of sepal development, the main inflorescence was excised from six-to eight-week-old plants containing the pPRS::GFP-GUS, pAS2WT::GFP-GUS and pAS2as2-7D::GFP-GUS markers (pPRS::GFP-GUS in WT and as2-7D, pAS2WT::GFP-GUS and pAS2as2-7D::GFP-GUS in WT). Siliques were removed with surgical scissors. Flowers above stage 8 were dissected off using tweezers under a stereo microscope. The dissected inflorescences were inserted upright into plates containing live imaging medium (1/2 MS medium supplemented with a 1000-fold dilution of a plant preservative mixture). The plates were filled with autoclaved water and left for one to two hours to rehydrate the dissected inflorescences. Residual buds were further trimmed to early stages with a needle in the water. Recover the inflorescences from dissection by transferring them to fresh media plates for at least half an hour before live imaging. After recovery, the dissected tissue used for live imaging was positioned at an angle to ensure that the flower of the selected stage was directly facing the objective of the confocal microscope. Whole flowers were immersed in autoclaved water containing a 1000-fold diluted plant preservative mixture and imaged using a vertical confocal microscope with a 40x water dipping objective. The following settings were used: excitation laser at 488 nm, emission collection range of 500-550 nm, and a z-step of 0.7-1.0 µm. All confocal imaging were performed with a Nikon C2si confocal. Images were processed by ImageJ (https://imagej.net/software/imagej/) and MorphographX (de Reuille et al., 2015).

Vector construction and plant transformation

pPRS::GFP-GUS was constructed by inserting the PRS promoter sequence into the binary vector p35S::GUS-GFP (Chang et al., 2024) using the HindIII and SalI cutting sites. To generate p35S::PRS, the PRS coding sequence from the start codon to the stop codon was amplified from WT genomic DNA. The PCR product was cloned into pCAMBIA1300-35S (a binary vector derived from pCAMBIA1300, containing the 2× CaMV 35S promoter and the CaMV terminator) between the KpnI and PstI cutting sites. The 35S-PRS fragment from the resultant vector was then cut with HindIII and EcoRI and ligated into the binary vector pCAMBIA2300 (CAMBIA). pAS2WT::GFP-GUS and pAS2as2-7D::GFP-GUS have been described previously (Yadav et al., 2024). All primers used for were listed in Supplementary Table S1.

RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

Total RNA was extracted from inflorescences (with mature flowers removed) using the Easy Plant RNA Kit (Easy-Do, CAT DR0406050) according to the manufacturer’s instructions. First-strand of cDNA was synthesized using HiScript® II Reverse Transcriptase (Vazyme, CAT R333-01) and then used as a template for RT-qPCR. qPCR was performed in a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad) using Hieff qPCR SYBR Green Master Mix (Yeasen, CAT 11201ES08) according to the manufacturer’s instructions. Quantification of the UBQ gene was used as a control. All primers used for qRT-PCR are listed in Supplementary Table S1.

Yeast one-hybrid assay

To make the yeast one-hybrid assay bait constructs, the PRS promoter sequence were amplified from genomic DNA using specific primers (Supplementary Table S1). The PCR products were ligated into the pHIS2 vectorusing SacI and EcoRI cutting sites to construct the pPRS::HIS2 (PRS-HIS2). The AS2 coding sequence (CDS) was inserted into pGADT7 vector (AD) using XhoI and EcoRI cutting sites to construct the AS2-AD. The combinations of PRS-HIS2 and AS2-AD, as well as PRS-HIS2 and AD were transformed into yeast strain Y187. Yeast synthetic drop-out media (SD) lacking leucine (Leu) and tryptophan (Trp; Coolaber, PM2222) were used to identify transformed colonies. Three days after transformation, selective colonies were grown in -Leu and -Trp liquid media for 24 hours. The cell suspension was then pipetted onto yeast SD media lacking histidine (His), Leu, and Trp (Coolaber, PM2222) supplemented with 80 mM 3-amino-1,2,4-triazole (3-AT).

Yeast two-hybrid assay

To confirm the interaction between AS2 and PRS, the CDS of PRS gene was amplified and cloned into the pGBKT7 vector (BD) using PstI and EcoRI to construct the PRS-BD and the CDS of AS2 gene was inserted into pGADT7 vector using XhoI and EcoRI to construct the AS2-AD. The PCR primers used for plasmid construction are listed in Supplementary Table S1. The combinations of PRS-BD and AS2-AD, PRS-BD and AD, BD and AS2-AD, and AD and BD were transformed into yeast strain AH109. Yeast SD media lacking Leu and Trp (Coolaber, PM2222) was used to identify transformed colonies. Three days after transformation, selective colonies were grown in -Leu and -Trp liquid media for 24 hours. Then, cell suspension was pipetted onto yeast SD media lacking His, adenine (Ade), Leu, and Trp (Coolaber, PM2222).

Dual-luciferase assay

The dual-luciferase assays were performed in Nicotiana benthamiana (N. benthamiana) leaves as previously described (Yin et al., 2010). To generate the constructs for dual-luciferase assays, the promoter region of PRS was cloned into the pGreenII 0800-LUC vector using BamHI and HindIII to construct the pGreenII 0800-PRSpro-LUC (PRS-LUC), and the CDS of AS2 was cloned into the pGreenII 0029-62-SK (SK) (Yin et al., 2010) vector using BamHI and HindIII to construct pGreenII 0029-62-AS2-SK (AS2-SK). Both constructs were transferred into Agrobacterium tumefaciens strain GV3101 (pSoup) and used to infiltrate N. benthamiana leaves. Infiltrated plants were incubated at 22□ for 72 hours. For qualitative observations, the LUC images were captured using a low-light cooled CCD imaging apparatus (Tanon-5200 with AllDoc_x software). Transformed leaves were sprayed with 1 mM luciferin and placed in darkness for several minutes before luminescence detection. For quantitative analysis, firefly and renilla luciferase activities were monitored using the Dual-Luciferase Reporter Assay System (Promega, E1910) on the dual fluorescence detector (GloMax96).

Firefly luciferase complementation imaging assay

The transient assays were performed in N. benthamiana leaves as previously described (Zhao et al., 2019). The CDS of AS2 gene was PCR-amplified and cloned into the pCambia1300-35S-nLuc vector using SacI and SalI to construct the AS2-nLUC, and the CDS of PRS gene was PCR-amplified and cloned into the pCambia1300-35S-cLuc vector using SacI and SalI to construct the PRS-cLUC. Both constructs were transferred into Agrobacterium tumefaciens strain GV3101 and used to infiltrate N. benthamiana leaves. Infiltrated plants were incubated at 22°C for 72 hours. The transformed leaves were sprayed with 1 mM luciferin and placed in darkness for several minutes before luminescence detection. The LUC images were captured using a low-light cooled CCD imaging apparatus (Tanon-5200 with AllDoc_x software).

BiFC assay

The BiFC assays were performed in N. benthamiana leaves as previously described (Yang et al., 2007). To generate the constructs for BiFC assays, the coding regions of PRS and AS2 were cloned in frame into the BamHI and SalI digested p2YN and p2YC (Yang et al., 2007) vectors to generate PRS-cYFP and nYFP-AS2, respectively. Both constructs were transferred into Agrobacterium tumefaciens strain GV3101 and used to infiltrate N. benthamiana leaves. The fluorescence signals were examined by using a Nikon C2si confocal.

RESULTS

PRS gene deletion suppresses outgrowth formation on as2-7D sepal abaxial epidermis

To investigate the molecular mechanism involved in maintaining sepal flatness in Arabidopsis, we performed a suppressor screen on as2-7D and isolated a suppressor mutant of as2-7D termed as2-7D ssp1. Compared to as2-7D sepals, as2-7D ssp1 sepals are smoother on the outer (abaxial) epidermis, with significantly fewer outgrowths. Compared to WT sepals, as2-7D ssp1 sepals are narrower, exposing a part of the internal floral organs (Fig. 1A to 1C). Using the QTL-seq mapping strategy, the candidate region was narrowed down to 9-16 Mb on chromosome 2 (Supplementary Fig. S1). In this region, a large segment deletion of 25,189 bp closely linked to the ssp1 mutation was identified in the as2-7D ssp1 genome (Fig. 1D). This deletion disrupted the coding regions of six genes, among which only the PRS (At2G28610) gene has been reported to be involved in sepal morphogenesis (Matsumoto and Okada, 2001). Consequently, PRS was selected as the candidate gene of SSP1. Since two prs mutants have been previously reported, the as2-7D ssp1 mutant was renamed as as2-7D prs-3. The prs-3 single mutant was isolated in the F2 generation by crossing as2-7D prs-3 with WT. Sepals of prs-3 mutants have a smooth epidermis and are narrower compared to WT sepals (Fig. 1A to 1C; Supplementary Fig. S2), consistent with the phenotype of other prs alleles (Nakata et al., 2012). To further verify that the PRS gene is indeed the SSP1 gene, we crossed as2-5D (an AS2 mutant in the Col background with the same point mutation as as2-7D) with prs-4 (a prs mutant in the Col background) and isolated the as2-5D prs-4 double mutant. Phenotypic observations of Col-0, as2-5D, prs-4, and as2-5D prs-4 flowers revealed that Col-0 and prs-4 sepals had smooth surfaces. In contrast, as2-5D sepals formed outgrowths on the surface, though fewer than those on as2-7D sepals, while as2-5D prs-4 sepals were smooth and flat without obvious outgrowth formation (Figure. 1E). These results indicate that PRS mutation indeed suppresses the outgrowth formation on the abaxial sepal epidermis caused by ectopic AS2 expression.

Figure 1.
Figure 1. PRS gene deletion suppresses outgrowth formation on as2-7D sepal abaxial epidermis.

(A) Inflorescences of WT, as2-7D, prs-3 (ssp1) and as2-7D prs-3 (as2-7D ssp1). as2-7D sepals have many outgrowths on the surface; prs-3 (ssp1) sepals are narrower compared with WT sepals; the as2-7D prs-3 (as2-7D ssp1) sepals are smoother on the abaxial epidermis compared to as2-7D sepals. Scale bars: 400 μm. (B) Flowers of WT, as2-7D, prs-3 (ssp1), and as2-7D prs-3 (as2-7D ssp1). Sepals (green leaf-like organs) are marked with the letter “S” in white in WT. Scale bars: 200 μm. (C) Scanning electron microscope images of sepals of WT, as2-7D, prs-3 (ssp1) and as2-7D prs-3 (as2-7D ssp1). Scale bars: 100 μm. Arrowheads in A-C point to the outgrowths on as2-7D sepals. (D) The large fragment deletion in the genome of the as2-7D prs-3 (as2-7D ssp1) includes six genes, among which AT2G28610 (PRS) is the gene involved in the suppression of the as2-7D sepal phenotype. (E) Inflorescences of Col, as2-5D, prs-4, and as2-5D prs-4. as2-5D sepals have outgrowths on the surface (as arrowheads point at). Scale bars: 400 μm.

AS2 up-regulates PRS expression on as2-7D sepal abaxial epidermis

The abaxial epidermal outgrowths in as2-7D sepals are caused by ectopic AS2 expression on the abaxial epidermis. To investigate how the PRS deletion suppresses outgrowth formation on as2-7D sepal abaxial epidermis, we first examined whether PRS deletion affected AS2 expression pattern. We drove green fluorescent protein (GFP) expression under the WT AS2 promoter (pAS2WT::GFP-GUS) and the mutant as2-7D AS2 promoter (pAS2as2-7D::GFP-GUS) in prs-3, and obtained the pAS2WT::GFP-GUS prs-3 and pAS2as2-7D::GFP-GUS prs-3 reporter lines. Confocal microscopy showed that the WT AS2 promoter drives GFP expression specifically on the adaxial sepal epidermis of prs-3 (Fig. 2A,2C, and 2E), while the mutant AS2 promoter drives GFP expression on both the adaxial and abaxial epidermal layers of prs-3 (Fig. 2B,2D, and 2F). The expression patterns of pAS2WT::GFP-GUS and pAS2as2-7D::GFP-GUS in prs-3 sepals are similar to their expression patterns in WT sepals (Yadav et al., 2024), indicating that the prs-3 mutation does not alter the activity of WT and mutant AS2 promoters. Therefore, the suppression of outgrowth formation on the abaxial epidermis of as2-7D prs-3 sepals by prs-3 mutation is not achieved by suppressing the ectopic expression of AS2 on sepal abaxial epidermis. Instead, PRS is required for the ectopically expressed AS2 to induce outgrowths on the sepal abaxial epidermis.

Figure 2.
Figure 2. AS2 up-regulates PRS expression on as2-7D sepal abaxial epidermis

(A to F) Confocal images showing the WT AS2 promoter (pAS2WT::GFP-GUS) and the mutant AS2 promoter (pAS2as2-7D::GFP-GUS) activity in prs-3 flowers. The WT AS2 promoter drives GFP expression on the adaxial sepal epidermis of prs-3 (A, C, E). The mutant as2-7D promoter drives GFP expression on both the adaxial and abaxial epidermal layers of prs-3 (B, D, F). A and B are top views; C and D are the side views of clips at the white lines in the top views; E and F are sketches of GFP distribution patterns. GFP: green; Chlorophyll: gray. Scale bars: 400 μm. (G and H) Confocal images showing the PRS promoter (pPRS::GFP-GUS) activity in WT (G) and as2-7D (H) inflorescences. PRS is expressed at the margins of WT sepals, whereas at the margins and outgrowths (as arrowheads show in H) of as2-7D sepals. GFP: green; Chlorophyll: gray. Scale bars: 100 μm. (I and J) Confocal images of the PRS promoter (pPRS::GFP-GUS) activity in WT and as2-7D sepals at different developmental stages. PRS is widely expressed in all four sepals at stages 4 and 5 in both WT and as2-7D sepals. In WT sepals at stage 6 and later, PRS expression is restricted to the sepal margin, whereas PRS is expressed at both the margins and middle regions in as2-7D sepals, especially at outgrowths (as arrowheads point). GFP: green; Chlorophyll: gray. Scale bars: 50 μm. (K to R) Longitudinal sections of WT and as2-7D flowers at stage 5 and stage 8. PRS is widely expressed in WT (K and M) and as2-7D sepals at stage 5 (O and Q). In WT sepals at stage 8, PRS is expressed at the margins of the sepals (L and N). In as2-7D sepals at stage 8, PRS is expressed at the margins and the outgrowths (as arrowheads point at) (P and R). K and L are reproduced from I. O and P are reproduced from J. M, N, Q, and R are the side views of clips at the while lines in K, L, O, and P respectively. GFP: green; Chlorophyll: gray. Scale bars: 50 μm.

Our results suggest that PRS functions downstream of AS2 to influence sepal flatness. To further explore this hypothesis, we examined whether PRS expression was affected by the as2-7D mutation. Using RT-qPCR, we quantified the expression levels of PRS in WT and as2-7D sepals. The results showed that PRS expression level was significantly higher in as2-7D sepals than in WT sepals (Supplementary Fig. S3). PRS is a member of the WOX transcription factor family, whose genes exhibit specific expression patterns critical to their functional divergence, and previous study has shown that PRS displays a dynamic expression pattern throughout sepal development using RNA in situ hybridization (Matsumoto and Okada, 2001). To explore how ectopic AS2 expression up-regulates PRS, we generated PRS transcriptional reporter lines to compare PRS expression patterns in WT (pPRS::GFP-GUS) and as2-7D (pPRS::GFP-GUS as2-7D) sepals. Confocal microscopy showed that overall PRS was expressed at the margins of WT sepals (Fig. 2G) and at both the margins and outgrowths of as2-7D sepals (Fig. 2H). Observations of individual flowers at different developmental stages showed similar PRS expression patterns in WT and as2-7D sepals at stages 4 and 5, with broad expression in all four sepals (Fig. 2I, and 2J). However, starting from stage 6, as2-7D sepals exhibited divergent PRS expression patterns compared to WT sepals. In WT sepals at stage 6 and beyond, PRS expression was restricted to the sepal margins (Fig. 2I), consistent with the expression patterns observed in previous research through RNA in situ hybridization (Matsumoto and Okada, 2001). In contrast, as2-7D sepals at similar stages displayed broader PRS expression, with expression at both the margins and the outgrowths (Fig. 2J). Furthermore, longitudinal sections of sepals from stage 5 and stage 8 showed that PRS was widely expressed on both the adaxial and abaxial sides of sepals in WT and as2-7D at stage 5 (Fig. 2K,2M,2O, and 2Q). At stage 8, PRS expression was restricted at the sepal margins in WT (Fig. 2L and 2N), whereas as2-7D sepals exhibited prominent PRS expression at the margins and the outgrowths (Fig. 2P and 2R).

as2-7D sepals start to exhibit ectopic PRS expression at stage 6, which coincides with the developmental stage when the abaxial epidermis of as2-7D sepals starts to form outgrowths. This timing coincidence suggests a potential relationship between the altered PRS expression pattern and epidermal outgrowth formation. To investigate the connection between ectopic PRS expression and outgrowth formation on as2-7D sepals, we tracked PRS expression patterns before and after the outgrowth formation. Our observations revealed that ectopic PRS expression consistently preceded outgrowth formation. Before the formation of outgrowths (prior to stage 6), PRS was expressed throughout the sepal abaxial epidermis. As as2-7D sepals continued to develop and outgrowths began to form on the abaxial epidermis, PRS expression activity gradually receded toward the sepal margins but remained at the outgrowths (Fig. 3A and 3B).

Figure 3.
Figure 3. PRS promotes outgrowth formation on the sepal epidermis.

(A) Confocal images of the same flower from pPRS::GFP-GUS as2-7D at 0, 24, 48, and 72 hours, respectively. GFP: green; Chlorophyll: gray. Note the GFP expression at the outgrowths (arrowheads). (B) Gaussian curvature of the flowers in A. Warm colors denote out-of-plane (bulging) and cool colors represent inward (saddling) deformations. At 0 hour, PRS is widely expressed on the abaxial epidermis of sepals, and the sepal exhibits relatively uniform surface curvature. At 24 and 48 hours, PRS expression gradually retreats toward the sepal margin, and outgrowths initiate on the sepal abaxial epidermis. At 72 hour, as PRS expression retreats further to the margins, it lingers on the rising outgrowths (as arrowheads show). Scale bars:50 μm. (C to E) Phenotypes of p35S::PRS transgenic plants. (C) Outgrowths (arrowhead) on the peduncle. (D) Outgrowths (arrowhead) on the sepal. (E) White wrinkle structures (arrowhead) on the sepal. Scale bars: 400 μm.

In conclusion, our tracking of PRS expression during early sepal development supports a model in which PRS expression activity gradually recedes toward the sepal margins as sepals grow in WT, while in as2-7D sepals the ectopically expressed AS2 on the abaxial epidermis tends to detain PRS expression and keeps it to linger on the rising outgrowth. So we believe that AS2 up-regulates PRS expression on as2-7D sepal abaxial epidermis,

PRS promotes the formation of outgrowths on the sepal epidermis

To further investigate the relationship between up-regulated PRS expression and epidermal outgrowth formation, we generated transgenic lines that overexpressed PRS gene (p35S::PRS) in WT plants. In these transgenic plants, outgrowths were observed on the epidermis of the peduncles and sepals (Fig. 3C to 3E), showing that increased PRS expression can lead to outgrowth formation on sepals. Combined with the PRS expression pattern analysis, this result provides evidence that the ectopic PRS expression causes epidermal outgrowth formation in as2-7D sepals. Introducing p35S::PRS into as2-101, a loss-of-function mutant of AS2, also resulted in outgrowth formation on the sepal epidermis (Supplementary Fig. S4), indicating that PRS promotes epidermal outgrowth formation independently of functional AS2. This finding supports our previous conclusion that PRS functions downstream of AS2 to influence sepal flatness.

AS2 directly promotes PRS expression

Expression analysis demonstrated that ectopic AS2 expression up-regulates PRS in as2-7D sepals. A yeast one hybrid assay was performed to investigate whether AS2 up-regulates PRS directly. Results showed that AS2 can bind to the promoter region of PRS (Fig. 4A). Dual-luciferase assays in N. benthamiana leaves were further performed to test the effects of AS2 on the transcription of PRS-LUC reporter, which displayed that AS2 promoted the expression of PRS (Fig. 4B and 4C).

Figure 4.
Figure 4. AS2 promotes PRS expression directly

(A) AS2 binds to the PRS promoter region in the yeast one-hybrid assay. Transformed yeast cells were spotted on SD -Trp-Leu or SD -Trp-Leu-His medium supplemented with 80 mM 3AT in 10-, 100-, 1000- and 10000-fold dilutions. The pGADT7-T and pGBKT7-53 were used as positive controls, while the pGADT7-T and pGBKT7-lam vectors served as negative controls. (B) Transient expression assay showing that AS2 activates PRS. Representative image of a N. benthamiana leaf 72 hours after infiltration was shown. (C) Quantitative analysis of luminescence intensity in (B). Eleven independent replicates were performed. Error bars represent standard deviation (SD). Data are presented as mean ± SD for more than three independent experiments. Two-tailed Student’s t-test, **p <0.01.

Taken together, these results indicate that AS2 promotes PRS expression through direct binding to PRS promoter regions.

AS2 physically interacts with PRS

Previous studies have reported that AS2 physically interacts with transcription factor TEOSINTE BRANCHED 1, CYCLOIDEA, AND PCF FAMILY 4 (TCP4) (Li et al., 2012) and that PRS also interacts with TCP4 physically (Wanamaker et al., 2017). Therefore, we explored whether AS2 could interact with PRS at the protein level. In the yeast two-hybrid system, yeast AH109 colonies co-transformed with AS2-AD and PRS-BD grew well on selective medium (Fig. 5A), indicating that AS2 interacts directly with PRS in yeast. Luciferase complementation imaging assays further showed that AS2-nLUC interacted with PRS-cLUC (Fig.5B). Bimolecular fluorescence complementation (BiFC) assays displayed that YFP fluorescence was detected in the nuclei of N. benthamiana leaves co-transformed with AS2-nYFP and PRS-cYFP, but not in those of tobacco leaves transformed with the negative controls (Fig. 5C), further confirming that AS2 can physically interact with PRS in the nuclei.

Figure 5.
Figure 5. AS2 physically interacts with PRS

(A)AS2 interacted with PRS in the yeast two-hybrid assay. Transformed yeast cells were spotted on SD-Leu-Trp or SD-Leu-Trp-His-Ade medium in 10-, 100-, and 1000-fold dilutions. The pGBKT7-53 and pGADT7-T vector was used as a positive control. (B) Protein-protein interactions between AS2 and PRS were analyzed by dual-Luciferase reporter assay in N. benthamiana leaf. (C) BiFC assays in N. benthamiana leaves showing the interaction of PRS and AS2 in living cells. The nucleus was indicated by carrying a nuclear localization signal (H2B-RFP). Scale bars: 50 μm.

DISCUSSION

In this study, through suppressor screening, we demonstrated that loss-of-function of the PRS gene suppresses the outgrowth formation on as2-7D sepal abaxial epidermis. Using live imaging technology to track the PRS expression dynamics and sepal outgrowth formation on as2-7D sepal abaxial epidermis, we provided a detailed depiction of the PRS expression pattern at a cellular resolution throughout the early developmental stages of sepals, and found that on as2-7D sepal abaxial epidermis, AS2 expression up-regulated PRS expression, which resulted in the epidermal outgrowth initiation.

Previous studies have shown that ectopic AS2 expression disrupts sepal flatness through disturbing the balance of cellular growth and cell mechanics between the two epidermal layers of sepals (Yadav et al., 2024). Our study shows that PRS, a WOX family gene, affects sepal flatness downstream of AS2. The WOX family genes have been demonstrated to play important regulatory roles in critical developmental stages of plants, such as embryo formation, stem cell stability, and organ formation. Their multifaceted functions in plant development are closely related to their ability to promote cell division or prevent premature differentiation of cells (Deveaux et al., 2008; Baesso et al., 2020; Zhang et al., 2020; Bueno et al., 2021). The cellular mechanisms underlying PRS’s function in maintaining sepal flatness worth further exploration. Despite active research on the WOX family, detailed molecular mechanisms underlying and gene networks involved in the functions of these transcription factors still need to be deciphered (Lian et al., 2014; Tvorogova et al., 2021). Our study reveals that AS2 and PRS interact on multiple levels. AS2 and PRS interact physically, and AS2 also activate PRS expression through direct binding to PRS promoter region. These findings, combined with previous research on the versatile functions of WOX genes, suggest that complicated regulatory networks might be involved in role of AS2 and PRS in regulating sepal epidermal flatness. WOX family transcription factors, including PRS, contain WUS-box motifs that confer them transcriptional repressive activity and may act as transcriptional repressors in plants (Ikeda et al., 2009). Does PRS also directly or indirectly target AS2? Does PRS regulate its own transcription by forming a regulatory complex with AS2? What are the downstream genes co-regulated by PRS and AS2 to affect sepal morphology? These are interesting questions that will deepen our understanding of the working mechanisms of WOX family proteins and worth to be addressed in the future studies.

Research elucidating the mechanisms of abaxial-adaxial polarity establishment proposes that in Arabidopsis leaves, AS2 is specifically expressed on the adaxial epidermis, while PRS is expressed in the middle region (Du et al., 2018). However, our research shows that the AS2 expression region and the PRS expression domain overlap at the sepal margin throughout the sepal development. It is reasonable to hypothesize that the interaction between AS2 and PRS on as2-7D sepal abaxial epidermis, both on the transcription level and on the protein level, also works in WT sepal margins. However, when the pPRS::GFP-GUS vector was transferred into as2-101 mutant, it was observed that the expression pattern of PRS in as2-101 sepals was the same as that in WT ones (Supplementary Fig. S4), suggesting that the expression of PRS at the sepal margin does not require AS2 function. Therefore, probably divergent mechanisms are involved in PRS expression regulation in different tissues. It would be interesting to explore whether the interaction between AS2 and PRS also plays a role in sepal margin development.

The PRS deletion fails to completely suppress the as2-7D phenotype, suggesting that other factors or pathways are involved in modulating sepal epidermal flatness downstream of AS2. In addition, it has been reported that AUXIN RESPONSE FACTOR 3/ETTIN (ARF3/ETT) can suppress PRS expression by directly binding to its promoter region (Guan et al., 2017), while AS2 modulates the expression level of ETT/ARF3 by maintaining CpG methylation in specific exons of ETT/ARF3 (Vial-Pradel et al., 2018; Iwakawa et al., 2020). Hence, it is highly possible that that AS2 might also regulate PRS expression indirectly, probably through ARF3.

SUPPLEMENTARY DATA

The following supplementary data are available online.

Figure S1. QTL-seq mapping of the SSP1 gene.

Figure S2. The morphology of WT and prs-3 sepals.

Figure S3. RT-qPCR analysis of PRS expression in WT and as2-7D sepals.

Figure S4. The phenotype of p35S: PRS as2-101 transgenic plant.

Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Prof. Juan Xu for providing the confocal microscope, Prof. Songlin Bai for providing the pGreenII 0800-Luc and pGreenII 0029-62-SK plasmids, Prof. Xiaobo Zhao for providing the pCambia1300-35S-nLuc and pCambia1300-35S-cLuc plasmids, Prof. Dianxing Wu, Prof. Xiaoli Shu and Prof. Jingsong Bao for providing experimental facilities, Prof. Ming Zhou for the comments on the article.

AUTHOR CONTRIBUTIONS

Ruoyu Liu: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data curation, Writing – review & editing, Writing – original draft, Visualization, Validation. Zeming Wang: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data curation, Writing – Validation. Xi He: Methodology, Resources, Investigation. Heng Zhou: Investigation. Yiru Xu: Resources. Lilan Hong: Writing – review & editing, Supervision, Project administration, Funding acquisition, Data curation, Conceptualization.

CONFLICT OF INTEREST

No conflict of interest declared.

FUNDING

This research was supported by the National Natural Science Foundation of China (Grant no. 32270867) and Hundred-Talent Program of Zhejiang University.

REFERENCES

  1. ↵
    Alvarez JP, Furumizu C, Efroni I, Eshed Y, Bowman JL. 2016. Active suppression of a leaf meristem orchestrates determinate leaf growth. eLife 5.
  2. ↵
    Baesso B, Terzaghi M, Chiatante D, Scippa GS, Montagnoli A. 2020. WOX genes expression during the formation of new lateral roots from secondary structures in Populus nigra (L.) taproot. Scientific Reports 10, 6.
    OpenUrl
  3. ↵
    Bueno N, Cuesta C, Centeno ML, Ordás RJ, Alvarez JM. 2021. In vitro plant regeneration in tonifers: the role of WOX and KNOX gene families. Genes 12, 21.
    OpenUrl
  4. ↵
    Chang ZY, Zhao MZ, Qin BX, Hong LL. 2024. Polar-localized OsLTPG22 regulates rice leaf cuticle deposition and drought response. Plant Stress 14.
  5. ↵
    de Reuille PB, Routier-Kierzkowska AL, Kierzkowski D, et al. 2015. MorphoGraphX: A platform for quantifying morphogenesis in 4D. eLife 4, 20.
    OpenUrl
  6. ↵
    Deveaux Y, Toffano-Nioche C, Claisse G, Thareau V, Morin H, Laufs P, Moreau H, Kreis M, Lecharny A. 2008. Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis. Bmc Evolutionary Biology 8, 19.
    OpenUrl
  7. Du F, Guan C, Jiao Y. 2018. Molecular mechanisms of leaf morphogenesis. Molecular Plant 11, 1117–1134.
    OpenUrlCrossRef
  8. ↵
    Guan C, Wu B, Yu T, Wang Q, Krogan NT, Liu X, Jiao Y. 2017. Spatial auxin signaling controls leaf flattening in Arabidopsis. Current Biology 27, 2940–2950.
    OpenUrlCrossRef
  9. ↵
    Hong L, Dumond M, Tsugawa S, et al. 2016. Variable cell growth yields reproducible organ development through spatiotemporal averaging. Developmental Cell 38, 15–32.
    OpenUrlCrossRef
  10. ↵
    Hong LL, Brown J, Segerson NA, Rose JKC, Roeder AHK. 2017. CUTIN SYNTHASE 2 maintains progressively developing cuticular ridges in Arabidopsis sepals. Molecular Plant 10, 560–574.
    OpenUrlCrossRef
  11. ↵
    Ichihashi Y, Tsukaya H. 2015. Behavior of leaf meristems and their modification. Frontiers in Plant Science 6, 8.
    OpenUrl
  12. ↵
    Ikeda M, Mitsuda N, Ohme-Takagi M. 2009. Arabidopsis WUSCHEL is a bifunctional transcription factor that acts as a repressor in stem cell regulation and as an activator in floral patterning. Plant Cell 21, 3493–3505.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Iwakawa H, Takahashi H, Machida Y, Machida C. 2020. Roles of ASYMMETRIC LEAVES2 (AS2) and nucleolar proteins in the adaxial-abaxial polarity specification at the perinucleolar region in Arabidopsis. International Journal of Molecular Sciences 21, 19.
    OpenUrl
  14. ↵
    Iwakawa H, Ueno Y, Semiarti E, et al. 2002. The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana, required for formation of a symmetric flat leaf lamina, encodes a member of a novel family of proteins characterized by cysteine repeats and a leucine zipper. Plant and Cell Physiology 43, 467–478.
    OpenUrlCrossRefPubMedWeb of Science
  15. ↵
    Li Z, Li B, Shen WH, Huang H, Dong A. 2012. TCP transcription factors interact with AS2 in the repression of classLI KNOX genes in Arabidopsis thaliana. The Plant Journal 71, 99–107.
    OpenUrlCrossRefPubMedWeb of Science
  16. ↵
    Lian GB, Ding ZW, Wang Q, Zhang DB, Xu J. 2014. Origins and evolution of WUSCHEL-related homeobox protein family in plant kingdom. Scientific World Journal 2014, 12.
    OpenUrl
  17. ↵
    Lin WC, Shuai B, Springer PS. 2003. The Arabidopsis LATERAL ORGAN BOUNDARIES-domain gene ASYMMETRIC LEAVES2 functions in the repression of KNOX gene expression and in adaxial-abaxial patterning. Plant Cell 15, 2241–2252.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Lin X, Li Y, Hai H. 2007. Transcriptional, post-transcriptional and post-translational regulations of gene expression during leaf polarity formation. Cell Research 17, 512–519.
    OpenUrlCrossRefPubMedWeb of Science
  19. ↵
    Matsumoto N, Okada K. 2001. A homeobox gene, PRESSED FLOWER, regulates lateral axis-dependent development of Arabidopsis flowers. Genes & Development 15, 3355–3364.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Nakata M, Matsumoto N, Tsugeki R, Rikirsch E, Laux T, Okada K. 2012. Roles of the middle domain–specific WUSCHEL-related honebox genes in early development of leaves in Arabidopsis. The Plant Cell 24, 519–535.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Nakayama H, Leichty AR, Sinha NR. 2022. Molecular mechanisms underlying leaf development, morphological diversification, and beyond. Plant Cell 34, 2534–2548.
    OpenUrl
  22. ↵
    Nardmann J, Werr W. 2013. Symplesiomorphies in the WUSCHEL clade suggest that the last common ancestor of seed plants contained at least four independent stem cell niches. New Phytologist 199, 1081–1092.
    OpenUrlCrossRefPubMedWeb of Science
  23. ↵
    Robinson DO, Coate JE, Singh A, Hong LL, Bush M, Doyle JJ, Roeder AHK. 2018. Ploidy and size at multiple scales in the Arabidopsis sepal. Plant Cell 30, 2308–2329.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Roeder AHK. 2021. Arabidopsis sepals: A model system for the emergent process of morphogenesis. Quantitative Plant Biology 2, 1–15.
    OpenUrl
  25. ↵
    Sandalio LM, Rodríguez-Serrano M, Romero-Puertas MC. 2016. Leaf epinasty and auxin: A biochemical and molecular overview. Plant Science 253, 187–193.
    OpenUrl
  26. ↵
    Shi JM, Dong JQ, Xue JS, Wang H, Yang ZN, Jiao YL, Xu L, Huang H. 2017. Model for the role of auxin polar transport in patterning of the leaf adaxial-abaxial axis. Plant Journal 92, 469–480.
    OpenUrl
  27. ↵
    Smyth DR, Bowman JL, Meyerowitz EM. 1990. Early flower development in Arabidopsis. Plant Cell 2, 755–767.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Takagi H, Abe A, Yoshida K, et al. 2013. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant Journal 74, 174–183.
    OpenUrlCrossRefPubMedWeb of Science
  29. ↵
    Tena G. 2024. Flat sepals. Nature Plants.
  30. ↵
    Tsukaya H. 2005. Leaf shape: genetic controls and environmental factors. The International Journal of Developmental Biology 49, 547–555.
    OpenUrlCrossRefPubMedWeb of Science
  31. ↵
    Tvorogova VE, Krasnoperova EY, Potsenkovskaia EA, Kudriashov AA, Dodueva IE, Lutova LA. 2021. What does the WOX say? review of regulators, targets, partners. Molecular Biology 55, 311–337.
    OpenUrl
  32. ↵
    Vandenbussche M, Horstman A, Zethof J, Koes R, Rijpkema AS, Gerats T. 2009. Differential recruitment of WOX transcription factors for lateral development and organ fusion in Petunia and Arabidopsis. Plant Cell 21, 2269–2283.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Vial-Pradel S, Keta S, Nomoto M, et al. 2018. Arabidopsis Zinc-finger-like protein ASYMMETRIC LEAVES2 (AS2) and two nucleolar proteins maintain gene body DNA methylation in the leaf polarity gene ETTIN (ARF3). Plant and Cell Physiology 59, 1385–1397.
    OpenUrlCrossRef
  34. ↵
    Waites R, Hudson A. 1995. phantasticaL: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121, 2143–2154.
    OpenUrlAbstract
  35. ↵
    Wanamaker SA, Garza RM, MacWilliams A, et al. 2017. CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping. Nature Methods 14, 819–825.
    OpenUrl
  36. ↵
    Wellmer F, Riechmann JL, Alves-Ferreira M, Meyerowitz EM. 2004. Genome-wide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell 16, 1314–1326.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Wu G, Lin WC, Huang TB, Poethig RS, Springer PS, Kerstetter RA. 2008. KANADI1 regulates adaxial-abaxial polarity in Arabidopsis by directly repressing the transcription of ASYMMETRIC LEAVES2. Proceedings of the National Academy of Sciences of the United States of America 105, 16392–16397.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Yadav AS, Hong L, Klees PM, et al. 2024. Growth directions and stiffness across cell layers determine whether tissues stay smooth or buckle. bioRxiv: the preprint server for biology.
  39. ↵
    Yang X, Baliji S, Buchmann RC, Wang H, Lindbo JA, Sunter G, Bisaro DM. 2007. Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction. Journal of Virology 81, 11972–11981.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Yin XR, Allan AC, Chen KS, Ferguson IB. 2010. Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiology 153, 1280–1292.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Zhang ZJ, Runions A, Mentink RA, et al. 2020. A WOX/Auxin biosynthesis module controls growth to shape leaf form. Current Biology 30, 4857–4868.
    OpenUrl
  42. ↵
    Zhao XB, Huang JY, Chory J. 2019. GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling. Proceedings of the National Academy of Sciences of the United States of America 116, 10162–10167.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Zhu M, Chen W, Mirabet V, et al. 2020. Robust organ size requires robust timing of initiation orchestrated by focused auxin and cytokinin signalling. Nature Plants 6, 686–698.
    OpenUrl
Back to top
Posted October 01, 2024.
Download PDF

Supplementary Material

Email
PRESSED FLOWER works downstream of ASYMMETRIC LEAVES 2 to affect sepal flatness in Arabidopsis
Ruoyu Liu, Zeming Wang, Xi He, Heng Zhou, Yiru Xu, Lilan Hong
bioRxiv 2024.09.30.615753; doi: https://doi.org/10.1101/2024.09.30.615753
Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area