Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 2;185(3):1059-1075.
doi: 10.1093/plphys/kiaa087.

Gibberellin and auxin signaling genes RGA1 and ARF8 repress accessory fruit initiation in diploid strawberry

Junhui Zhou  1 , John Sittmann  1 , Lei Guo  1 , Yuwei Xiao  1 , Xiaolong Huang  1 , Anuhya Pulapaka  1 , Zhongchi Liu  1
Affiliations

Gibberellin and auxin signaling genes RGA1 and ARF8 repress accessory fruit initiation in diploid strawberry

Junhui Zhou et al. Plant Physiol. .

Abstract

Unlike ovary-derived botanical fruits, strawberry (Fragaria x ananassa) is an accessory fruit derived from the receptacle, the stem tip subtending floral organs. Although both botanical and accessory fruits initiate development in response to auxin and gibberellic acid (GA) released from seeds, the downstream auxin and GA signaling mechanisms underlying accessory fruit development are presently unknown. We characterized GA and auxin signaling mutants in wild strawberry (Fragaria vesca) during early stage fruit development. While mutations in FveRGA1 and FveARF8 both led to the development of larger fruit, only mutations in FveRGA1 caused parthenocarpic fruit formation, suggesting FveRGA1 is a key regulator of fruit set. FveRGA1 mediated fertilization-induced GA signaling during accessory fruit initiation by repressing the expression of cell division and expansion genes and showed direct protein-protein interaction with FveARF8. Further, fvearf8 mutant fruits exhibited an enhanced response to auxin or GA application, and the increased response to GA was due to increased expression of FveGID1c coding for a putative GA receptor. The work reveals a crosstalk mechanism between FveARF8 in auxin signaling and FveGID1c in GA signaling. Together, our work provides functional insights into hormone signaling in an accessory fruit, broadens our understanding of fruit initiation in different fruit types, and lays the groundwork for future improvement of strawberry fruit productivity and quality.

PubMed Disclaimer

Figures

Figure 1
Figure 1
srl-1 mutants develop larger and parthenocarpic fruit. A, Images of bisected WT and srl-1 receptacles at stages 1, 4, and 5 without emasculation. Bars = 1 mm. B, Quantitative measurement of WT and srl-1 receptacles at stages 1 and 4. C, Images of emasculated WT and srl-1 receptacles at stages 1, 3, and 5. Bars = 1 mm. D, Quantitative measurements of emasculated WT and srl-1 receptacles at stages 1 and 3. n indicates number of fruits measured. **Indicates significant difference (two-tailed Student’s t test: P < 0.01) between genotypes or stages.
Figure 2
Figure 2
Relative transcript level of fruit-development genes in WT and srl-1 mutant receptacles. After removing achenes, developing receptacles at stages 1 and 2 were collected for RNA extraction. Three biological replicates were collected and tested. The y-axis indicates relative transcript level compared to the reference gene PP2a using the formula 2−ΔCt. **Indicates significant difference (two-tailed Student’s t test: P < 0.01) between WT and srl-1.
Figure 3
Figure 3
Network analysis identifies FveARF8, FveARF6, and FveIAA4 in the same co-expression module. A, Consensus Network analysis identifies FveARF8, FveARF6, and FveIAA4 in module 80 of Consensus 90 Fruit Network. The five-digit ID numbers for all other genes in the same module are shown and listed in Supplemental Data S1. This module correlates with young receptacle fruit. The consensus score and correlation score between genes are indicated by color and thickness of lines, respectively. B, RT-qPCR of FveARF8, FveARF6, and FveIAA4 in developing receptacle at stages 1–3. Data represent mean ± se of gene expression from three biological replicates of WT (YW5AF7) receptacle tissues. The relative transcript level (y-axis) was derived by comparing to the same reference gene PP2a using the formula 2−ΔCt. C, BiFC in N. benthamiana cells testing interaction between FveIAA4 and FveARF8 or FveARF6. D, Y2H assays testing the interaction between FveIAA4 and FveARF8 or FveARF6. E, Different domains of FveARF8 were tested against FveIAA4 in Y2H. A diagram of FveARF8 protein domains is shown. The numbers beneath the diagrams are amino acid numbers.
Figure 4
Figure 4
The C-terminal domain of FveRGA1 interacts with FveARF8. A, BiFC in N. benthamiana leaf testing for interactions between FveRGA1 and FveARF8. B, A diagram of FveRGA1 protein domains and truncations used in Y2H. The numbers beneath the diagrams are amino acid numbers. C, Y2H assays testing the interactions between FveRGA1-C or FveRGA1-C (-SAW) and FveARF8 or different truncations of FveARF8. D, Co-immunoprecipitation assay to confirm FveRGA1-FveARF8 interaction. Immunoprecipitation was performed with the anti-MYC antibody, and the pull-down products in the immunoblots were detected with the anti-HA antibody. AD and AD* both indicate AD-HA peptides derived from the pGADT7 control vector (with AD a read through product).
Figure 5
Figure 5
fvearf8 mutants develop larger and rounder fruit. A, Images of bisected receptacles at stage 1 (before fertilization), stages 3 and 5 (post-fertilization), and ripe stage in WT and fvearf8-1. Bars = 1 mm. B, Average size of receptacle fruit at stages 1 and 3. The height and width of fruits were normalized, respectively, to the height and width of petals of the same flower to minimize plant to plant variation due to environmental impact. C, Relative receptacle fruit size (height and width, normalized to petal size) in WT and additional fvearf8 mutant alleles at stage 3. D, WT and fvearf8-1 receptacle size at ripe stage. Since the petals have senesced, the absolute fruit size was measured. **Significant difference (two-tailed Student’s t test: P < 0.01) between WT and fvearf8-1.
Figure 6
Figure 6
fvearf8-1 mutants do not develop parthenocarpy but exhibit enhanced response or sensitivity to auxin and GA. A, Photos of bisected receptacles of WT and fvearf8-1 at stages 1 and 3. The flowers were emasculated at 0 DPA. Bars = 1 mm. B, Quantitative measurement of receptacle size in emasculated fruit shown in (A). C, Photos of bisected stage 3 receptacles of WT and fvearf8-1. The flowers were first emasculated and then treated with NAA, GA3, or mock solution until 7 DPA (stage 3). Bars = 1 mm. D, Relative receptacle size of WT and fvearf8-1 at stage 3 after GA (top graph) or NAA (bottom graph) treatment of emasculated flowers. The height and width of hormone-treated receptacles are divided, respectively, by the height and width of mock-treated receptacles of the same genotype. E, RT-qPCR data of FveSAUR1 and FveEXPL_B1 in WT and fvearf8-1 receptacles treated with different concentrations of GA. Stage 3 receptacles with achenes removed were collected and used for RNA extraction. Three biological replicates gave similar results; one replicate is shown here. Relative transcript level was derived by comparing to the same reference gene PP2a. **Significant difference (two-tailed Student’s t test: P < 0.01) between WT and fvearf8-1
Figure 7
Figure 7
Characterization of FveGID1c encoding a putative GA receptor. A, Y2H assay testing the interaction between FveGID1c and FveRGA1 in the presence of GA3. The DELLA domain was deleted in FveRGA1-C. B, BIFC assay in N. benthamiana leaf cells confirming positive interaction between FveRGA1 and FveGID1c. C, A CRISPR/CAS9-induced fvegid1c-1 mutant exhibiting severely retarded growth when compared with a WT plant (left) of the same age. The red arrows indicate the yellow strawberry fruits born on the WT plant. A close-up of the fvegid1c-1 mutant inside the red box is shown.
Figure 8
Figure 8
FveARF8 directly represses the expression of FveGID1c. A, RT-qPCR showing relative transcript level of FveGID1c in WT (YW5AF7) and fvearf8-1 stages 1–3 receptacle fruit and young leaves. B, RT-qPCR showing relative transcript level of FveGID1c in WT (H4) and srl-1 stages 1–3 receptacle fruit. C, Y1H assay testing activation of FveGID1c promoter fragment 2 (P2) when FveARF8 (DBD) fused to GAL4 AD is introduced into the yeast. On the right, three different promoter fragments (P1, P2, and P3) were tested for self-activation at two different concentrations of antibiotics Abi. D, Diagram of the LUC reporter system, and the expression of LUC driven by the FveGID1c promoter in the presence of 35S::FveARF8. Robust self-activation in the absence of transacting factor (with YFP serving as a negative control) is shown when 1 µm GA3 is added. Y-axis shows relative LUC expression normalized against 35S::REN expressed from the same vector. **Significant difference (two-tailed Student’s t test: P < 0.01) in respective comparisons.
Figure 9
Figure 9
Strawberry fruit structure and a model illustrating regulatory pathways during fruit initiation and growth. A, A diagram of strawberry fruit illustrates fertilization-induced auxin/GA synthesis in the achene (see enlarged) and subsequent signaling in the receptacle. Transport is necessary for the auxin and GA to travel from the achenes to the receptacle. Each achene is an ovary containing a single seed. B, FveRGA1 is shown as a central player that regulates fruit set. The red lines indicate regulatory actions post-fertilization. Positive (arrows) or negative (bar) regulations are indicated.
See this image and copyright information in PMC

References

    1. Ariza MT, Soria C, Medina JJ, Martínez‐Ferri E (2011) Fruit misshapen in strawberry cultivars (Fragaria×ananassa) is related to achenes functionality. Ann Appl Biol 158:130–138.
    1. Boer DR, Freire-Rios A, van den Berg WAM, Saaki T, Manfield IW, Kepinski S, López-Vidrieo I, Franco-Zorrilla JM, de Vries SC, Solano R, et al. (2014) Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors. Cell 156:577–589. - PubMed
    1. Carrera E, Ruiz-Rivero O, Peres LEP, Atares A, Garcia-Martinez JL (2012) Characterization of the procera tomato mutant shows novel functions of the SlDELLA protein in the control of flower morphology, cell division and expansion, and the auxin-signaling pathway during fruit-set and development. Plant Physiol 160:1581–1596. - PMC - PubMed
    1. Caruana JC, Sittmann JW, Wang W, Liu Z (2018) Suppressor of Runnerless encodes a DELLA protein that controls runner formation for asexual reproduction in strawberry. Mol Plant 11:230–233. - PubMed
    1. Chandler JW (2016) Auxin response factors. Plant Cell Environ 39:1014–1028 - PubMed

Publication types

MeSH terms

LinkOut - more resources