Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy

Abstract

CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping-by-sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein-protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel-inside-carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

Keywords: CRABS CLAW (CRC); WUSCHEL (WUS); carpel development; floral meristem determinacy; fruit formation; incomplete penetrance; tomato (Solanum lycopersicum); variable expressivity.

Fig. 1

Phenotypic characterization of the tomato (Solanum lycopersicum) fig mutant. (a–c) Representative fig flowers, pistils, closed fruits and longitudinal open fruits (from left to right) showing WT‐like (identical to wild‐type, WT) (a), weak (b) and severe (c) phenotypes. (d) Percentage of different types of fruits (WT‐like, weak or severe) produced by WT (cv P73) and fig plants. N, number of plants evaluated; n, number of fruits harvested. (e–g) Scanning electron microscopy images of flowers at stages 5 and 8 of floral development, and histological sections of flowers at anthesis day (AD) stage exhibiting WT‐like (e), weak (f) and severe (g) phenotypes. Sepals were removed in samples at stage 5, whereas only the carpels were maintained in developing flowers at stage 8. Petals are coloured in yellow, stamens in orange and carpels in blue.

Fig. 2

Tomato FIG gene encodes a homologue of the Arabidopsis CRC gene. (a) Distribution of the average allele frequency of wild‐type (WT; blue line) and fig (red line) pools grouped by chromosomes. (b) Schematic representation of the SlCRCa gene (introns are represented by black lines, and coding and UTRs are in black and grey boxes, respectively) and the 367‐bp sequence inserted at the fourth intron in fig mutants. (c) Relative expression of SlCRCa in WT and fig flowers at different stages of floral development. FB0‐6, floral buds from stages 0 to 6; FB7‐12, floral buds from stages 7 to 12; PA, flowers at pre‐anthesis stage; AD, flowers at anthesis stage; AD+10, flowers 10 d after anthesis stage. (d) SlCRCa transcripts quantification of flowers at stage FB0‐6 from RNAi SlCRCa and WT lines. (e) Representative RNAi SlCRCa flowers, pistils and fruits displaying WT‐like, weak and severe mutant phenotypes. (f) Percentage of different types of fruits harvested from WT and T₀ RNAi SlCRCa plants. (g) CRISPR/Cas9‐slcrca (CR‐slcrca) alleles identified by cloning and sequencing PCR products from the SlCRCa‐targeted region from four T₀ CRISPR/Cas9 plants. Black bold and underlined letters indicate protospacer adjacent motif (PAM) sequences, blue dashed lines show InDel mutations, and blue letter and arrow indicate an insertion sequence. (h) Representative CR‐slcrca flowers, pistils and fruits exhibiting WT‐like, weak and severe phenotypes. (i) Percentage of different types of fruits harvested from WT and T₀ CR‐slcrca plants. In (f, i), n value indicates the number of fruits harvested per plant. In (c, d), data are means ± SD of three biological and two technical replicates. A two‐tailed, two‐sample Student t‐test was performed, and significant differences are represented by asterisks: *, P < 0.01; **, P < 0.001; ns, no statistically significant differences.

Fig. 3

Dynamic expression of tomato SlCRCa and SlCRCb genes. (a) Relative expression of SlCRCa determined by qRT‐PCR in different developmental tissues and stages of wild‐type (WT) flowers. (b–e) In situ mRNA hybridization of SlCRCa using antisense or sense probes in histological sections of WT flowers at different developmental stages: stage 3 (b), stage 6 (c), stage 8 (d) and stage 9 (e). (f) Relative expression of SlCRCb determined by qRT‐PCR in different developmental tissues and stages of WT flowers. (g–j) In situ mRNA hybridization of SlCRCb using antisense or sense probes in histological sections of WT flowers at different developmental stages: stage 3 (g), stage 6 (h), stage 8 (i) and stage 9 (j). In (a, f), data are means ± SD of three biological and two technical replicates. FB0‐6, floral buds from stages 0 to 6; FB7‐12, floral buds from stages 7 to 12; PA, flowers at pre‐anthesis stage; AD, flowers at anthesis stages; AD+10, flowers 10 d after anthesis stage; IG; immature green fruit; MG, mature green fruit; BR, breaker fruit; and RR, mature red fruit. Bars, 100 μm; the floral organ primordia of sepal (se), petal (pe), stamen (st) and carpel (ca), as well as ovules (ov) in the carpel cavities, are indicated.

Fig. 4

Characterization of tomato CRISPR/Cas9‐slcrcb (CR‐slcrcb) and double‐mutant CR‐slcrca:slcrcb lines. (a) CR‐slcrcb alleles identified by cloning and sequencing PCR products from the SlCRCb‐targeted region from four T₀ CRISPR plants. Black bold and underlined letters indicate protospacer adjacent motif (PAM) sequences, blue dashed lines show InDel mutations, and blue letter and arrow indicate an insertion sequence. (b) Representative CR‐slcrcb flowers, pistils and fruits exhibiting WT‐like (identical to wild‐type, WT), weak and severe phenotypes. (c) Percentage of different types of fruits harvested from WT and T₀ CR‐slcrcb plants. (d) Relative expression of SlCRCa and SlCRCb in CR‐slcrcb and CR‐slcrca lines, respectively, at different floral developmental stages. FB0‐6, floral buds from stages 0 to 6; FB7‐12, floral buds from stages 7 to 12; PA, flowers at pre‐anthesis stage; AD, flowers at anthesis stage; and AD+10, flowers 10 d after anthesis stage. Data are means ± SD of three biological and two technical replicates. A two‐tailed, two‐sample Student t‐test was performed, and significant differences are represented by asterisks: *, P < 0.01; **, P < 0.001; ns, no statistically significant differences. (e, f) Representative flower, pistil and fruits developed by CR‐slcrca:slcrcb double mutants. (e) Detail of the fourth floral whorl organs and morphological features of their epidermal cells in a flower at anthesis stage. (f) Immature green and mature red fruits. (g) Percentage of different types of fruits harvested from WT and T₀ CR‐slcrca:slcrcb plants. In (c, g), n value indicates the number of fruits harvested per plant.

Fig. 5

Tomato CRC paralogues interact with the chromatin remodelling complex members repressing SlWUS expression. (a) In situ mRNA hybridization of SlWUS in histological sections of wild‐type (WT), CR‐slcrca, CR‐slcrcb and CR‐slcrca:slcrcb flowers at developmental stages 4, 6 and 8. (b) Subcellular localization of SlCRCa and SlCRCb. The entire SlCRCa and SlCRCb coding sequences were N‐terminally fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana leaves. (c) Bimolecular fluorescence complementation confocal images showing in vivo interactions in N. benthamiana leaves between either the yellow fluorescent protein (YFP) C‐terminal region fused to SlCRCa (SlCRCa‐cYFP) or SlCRCb (SlCRCb‐cYFP), and fusions of the YFP N‐terminal region fused to SlKNU (nYFP‐SlKNU), SlIMA (nYFP‐SlIMA), SlHDA1 (nYFP‐SlHDA1) or SlTPL1 (nYFP‐SlTPL1). The SlCRCa‐cYFP fusion was also examined with SlCRCb fused to the YFP N‐terminal region (nYFP‐SlCRCb). As negative control, each protein under study was co‐infiltrated with the nonplant β‐glucuronidase (GUS) enzyme fused to cYFP or nYFP. No YFP signal was observed in negative controls (Supporting Information Fig. S6). (d) Co‐immunoprecipitation studies of N. benthamiana leaves expressing either GFP‐tagged SlCRCa (SlCRCa^GFP) or SlCRCb (SlCRCb^GFP) and the different hemagglutinin (HA)‐tagged members of the chromatin remodelling complex (SlKUN^HA, SlIMA^HA, SlHDA1^HA or SlTPL1^HA). SlCRCb^GFP was also tested with HA‐tagged SlCRCa (SlCRCa^HA). The input total protein extracts were immunoprecipitated with anti‐GFP beads, and the unbound and recovered fractions (CoIP) were incubated with anti‐GFP (Ab^GFP) and anti‐HA (Ab^HA) antibodies to detect precipitated and co‐purified proteins, respectively. In (a), the floral organ primordia of sepal (se), petal (pe), stamen (st) and carpel (ca) are indicated. Bars, 100 μm.

Fig. 6

Complementation of the Arabidopsis crc‐1 mutation by transformation with tomato CRC paralogues. (a–d) Fully elongated siliques (a), silique length (b), silique apices showing different degrees of carpel fusion (c) and development of nectaries (arrows) at the base of the third floral whorl in the Ler wild‐type, which are absent in crc‐1 mutant and transgenic pCRC::SlCRCa and pCRC::SlCRCb plants (d). In (b), pairwise comparisons of means using the least significant difference test were performed. Values followed by the same lower‐case letter are not statistically different (P < 0.05). Error bars represent the SD of the mean values.

Fig. 7

Proposed model for the function of tomato CRC paralogues in SlWUS repression and the floral meristem determinacy. Tomato CRC paralogues (SlCRCa and SlCRCb), SlIMA and SlKNU interact with SlHDA1 and SlTPL1 to form a chromatin remodelling complex that represses SlWUS expression to terminate floral stem cell activity once carpel primordia are initiated. The blunt‐ended arrow indicates regulatory repression of gene expression. se, sepal; pe, petal; st, stamen; and ca, carpel.