Heterotrimeric Gα-subunit regulates flower and fruit development in CLAVATA signaling pathway in cucumber

Abstract

Flowers and fruits are the reproductive organs in plants and play essential roles in natural beauty and the human diet. CLAVATA (CLV) signaling has been well characterized as regulating floral organ development by modulating shoot apical meristem (SAM) size; however, the signaling molecules downstream of the CLV pathway remain largely unknown in crops. Here, we found that functional disruption of CsCLV3 peptide and its receptor CsCLV1 both resulted in flowers with extra organs and stumpy fruits in cucumber. A heterotrimeric G protein α-subunit (CsGPA1) was shown to interact with CsCLV1. Csgpa1 mutant plants derived from gene editing displayed significantly increased floral organ numbers and shorter and wider fruits, a phenotype resembling that of Csclv mutants in cucumber. Moreover, the SAM size was enlarged and the longitudinal cell size of fruit was decreased in Csgpa1 mutants. The expression of the classical stem cell regulator WUSCHEL (WUS) was elevated in the SAM, while the expression of the fruit length stimulator CRABS CLAW (CRC) was reduced in the fruit of Csgpa1 mutants. Therefore, the Gα-subunit CsGPA1 protein interacts with CsCLV1 to inhibit floral organ numbers but promote fruit elongation, via repressing CsWUS expression and activating CsCRC transcription in cucumber. Our findings identified a new player in the CLV signaling pathway during flower and fruit development in dicots, increasing the number of target genes for precise manipulation of fruit shape during crop breeding.

Figure 1

Phenotype identification of Csclv3 mutants in cucumber. A, B Two mutation sites in CsCLV3 generated by CRISPR/Cas9. A Genotype identification of Csclv3 mutants indicated the Csclv3#1 mutant with a 1-bp insertion and the Csclv3#2 mutant with a 1-bp deletion, leading to premature termination of protein translation and a frameshift mutation, respectively. B Schematic illustration of the two mutation forms in CsCLV3. Red arrows represent the two target sites and the red star indicates where the protein's translation is prematurely terminated. Orange horizontal bars represent amino acids translated in Csclv3 mutants, different from WT. C–E Representative phenotypes of petals (C), stamens (D), and carpel numbers (E) in Csclv3#1 mutants and WT. The numbers in the pictures represent floral organ numbers. Scale bars = 1 cm in (C) and 100 μm in (D and E). F–I Quantification for percentages of sepals (H), petals (I), stamens (J), and carpel numbers (I) in WT and Csclv3 lines (n = 25). J Representative images of cucumber ovaries at anthesis from WT and Csclv3 mutants (scale bars, 1 cm). K–M Ovary length (K), ovary diameter (L), and ovary shape index at anthesis (M) in WT and Csclv3 mutants. Values are means ± standard deviation (n = 8). ^**P < 0.01 (two-tailed Student’s t-test).

Figure 2

Expression analysis of CsCLV1 and phenotype identification of floral organ numbers in Csclv1 mutants in cucumber. A–C  In situ hybridization analysis of CsCLV1 in SAM and FM of different stages. le, leaf or leaf primordium; fm, floral meristem; se, sepal primordium; pe, petal primordium; st, stamen primordium; ca, carpel primordium. Scale bars, 100 μm. D Two mutation sites in CsCLV1 generated by CRISPR/Cas9, both of which result in premature termination of protein translation, with red arrows representing the two target sites and red stars indicating where the protein's translation is prematurely terminated. E–G Representative phenotypes of petals (E), stamens (F), and carpels (G) in Csclv1#1 mutants and WT. Scale bars = 1 cm in (E) and 100 μm in (F and G). H–K Percentages of sepals (H), petals (I), stamens (J), and carpels (K) in WT and Csclv1 lines (n = 50).

Figure 3

Functional disruption of CsCLV1 led to stumpy fruit in cucumber.  A, B Cucumber ovaries at anthesis (A) and fruits at 40 DAA (B) in WT and Csclv1 mutants, Scale bars, 1 cm. C, D Quantification of ovary length at anthesis (C) and fruit length at 40 DAA (D) in WT and Csclv1 mutants. Values are means ± standard deviation (n = 8). E, F Quantification of fruit diameter (E) and fruit shape index (F) in WT and Csclv1 mutants at 40 DAA. Values are means ± standard deviation (n = 8). G Representative images of longitudinal sections of cucumber fruit at 40 DAA in WT and Csclv1 mutants (scale bars, 100 μm). H Number of cells per unit area in longitudinal sections of fruit at 40 DAA in WT and Csclv1 mutants. Values are means ± standard deviation (n = 15). ^**P < 0.01 (two-tailed Student’s t-test).

Figure 4

CsCLV1 physically interacts with CsGPA1 at protein level. A BiFC assay. CsGPA1-YFP^N, CsCLV1-YFP^C, and PM-mCherry were co-infiltrated into N. benthamiana leaves. The other combination was used as a control. Green fluorescence represents the interaction signal, red fluorescence represents the plasma membrane marker signal, and orange fluorescence represents the merged outcome. B Firefly LCI assay. CsCLV1-nluc and CsGPA1-cluc were co-infiltrated into N. benthamiana leaves and the remainder of the combinations were used as controls. Imaging in red represents strong interactions and blue indicates weak interactions. C Co-IP assay. The constructs specified were expressed in leaves of N. benthamiana, and Co-IP was performed using anti-GFP antibody. The band indicated by the red arrow represents the in vivo interaction between CsCLV1 and CsGPA1.

Figure 5

Expression analysis of CsGPA1 and phenotypic analysis of floral organ numbers in Csgpa1 mutants. A–C  In situ hybridization analysis of CsGPA1 in SAM and FM at different stages. le, leaf or leaf primordium; fm, floral meristem; se, sepal primordium; pe, petal primordium; st, stamen primordium; ca, carpel primordium; vb, vascular bundle. Scale bars, 250 μm. D Mutant alleles generated in Csgpa1 mutants by CRISPR/Cas9 in cucumber. The red inverted triangles represent the two targets located in the fourth and sixth exons of CsGPA1. The fourth exon resulted in a 92-bp deletion including the intron in both mutants, leading to loss of the fourth exon. A base was added in the sixth exon (+G and +A, respectively) and resulted in translation into different amino acids, D (aspartic acid) and N (asparagine) at the 111th position, respectively. Both forms led to frameshift mutation. Blue bars represent amino acids translated in Csgpa1 mutants, different from WT. E–G Representative phenotypes of petals (E) and stamens (F) and carpel numbers (G) in WT and Csgpa1#2 mutants. Scale bars = 1 cm in (E) and 100 μm in (F and G). H–K Percentages of sepals (H), petals (I), stamens (J), and carpels (K) in WT and Csgpa1 lines (n = 50).

Figure 6

Phenotypic analysis of fruits in Csgpa1 mutants. A, B Cucumber ovaries at anthesis (A) and fruits at 40 DAA (B) in WT and Csgpa1 mutants, Scale bars, 1 cm. C, D Quantification of cucumber ovary length at anthesis (C) and fruit length at 40 DAA (D) in WT and Csgpa1 mutants. Values are means ± standard deviation (n = 8). E–H Fruit diameter (E, F) and fruit shape index (G, H) in WT and Csgpa1 mutants at anthesis (E, G) and 40 DAA (F, H). Values are means ± standard deviation (n = 8). I Representative images of longitudinal sections of cucumber fruit at 40 DAA in WT and Csgpa1 mutants (scale bars, 100 μm). J Number of cells per unit area in longitudinal sections of fruit at 40 DAA in WT and Csgpa1 mutants. Values are means ± standard deviation (n = 15). ^**P < 0.01 (two-tailed Student’s t-test).

Figure 7

CsGPA1 affects the expression of CsWUS and CsCRC in cucumber. A Stereoscopic images of SAM of 20-day-old cucumber seedlings from WT and Csgpa1 mutants (scale bars, 0.25 mm). lp, leaf primordium. B SAM diameter in stereoscopic images (scale bars, 100 μm). C Longitudinal sections of shoot apexes from 20-day-old WT and Csgpa1 cucumber seedlings. D SAM diameter in longitudinal sections from shoot apexes of WT and Csgpa1 mutants. E, F  CsWUS expression was detected in the shoot apex of 20-day-old cucumber seedlings from WT and Csgpa1 mutants by in situ hybridization (E) and qRT–PCR (F) (scale bars, 100 μm). G Heat map of the related DEGs of transcription factors in WT and Csgpa1 lines. Members of different gene families are separated by vertical lines. H, I Gene expression analysis of CsCRC in Csgpa1 mutants (H) and Csclv1 mutants (I). ^*P < 0.05, ^**P < 0.01 (two-tailed Student’s t-test).

Figure 8

A model for the regulation of floral organ number and fruit length by CsGPA1 in the CLV signaling pathway. The CsCLV3 peptide is received by receptor kinase CsCLV1. Gα-subunit CsGPA1 protein interacts with CsCLV1 to inhibit floral organ number but promote fruit elongation, by repressing CsWUS expression and activating CsCRC transcription in cucumber.