Axillary bud development is a major factor that impacts plant architecture. A runner is an elongated shoot that develops from axillary buds and is frequently used for clonal propagation of strawberry. However, the genetic control underlying runner production is largely unknown. Here, we identified and characterized loss of axillary meristems (lam), an EMS-induced mutant of the diploid woodland strawberry (Fragaria vesca) that lacked stamens in flowers and had reduced numbers of branch crowns and runners. The reduced branch crown and runner phenotypes were caused by a failure of axillary meristem initiation. The causative mutation of lam was located in FvH4_3g41310, which encodes a GRAS transcription factor, and was validated by a complementation test. lamCR mutants generated by CRISPR/Cas9 produced flowers without stamens and had fewer runners than the wild type. LAM was broadly expressed in meristematic tissues. Gibberellic acid (GA) application induced runner outgrowth from the remaining buds in lam, but failed to do so at the empty axils of lam. In contrast, treatment with the GA biosynthesis inhibitor paclobutrazol (PBZ) converted the runners into branch crowns. Moreover, genetic studies indicated that lam is epistatic to suppressor of runnerless (srl), a mutant of FveRGA1 in the gibberellic acid pathway, during runner formation. Our results demonstrate that LAM is required for stamen and runner formation and acts sequentially with GA from bud initiation to runner outgrowth, providing insights into the molecular regulation of these economically important organs in strawberry.
Flower and fruit development are two key steps for plant reproduction. The ABCE model for flower development has been well established in model plant species; however, the functions of ABCE genes in fruit crops are less understood. In this work, we identified an EMS mutant named R27 in woodland strawberry (Fragaria vesca), showing the conversion of petals, stamens, and carpels to sepaloid organs in a semidominant inheritance fashion. Mapping by sequencing revealed that the class E gene homolog FveSEP3 (FvH4_4g23530) possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain. Additional fvesep3CR mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27. Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect. Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry. Moreover, both R27 and fvesep3CR mutants exhibited parthenocarpic fruit growth and delayed fruit ripening. Transcriptome analysis revealed that both common and specific differentially expressed genes were identified in young fruit at 6–7 days post anthesis (DPA) of fvesep3 and pollinated wild type when compared to unpollinated wild type, especially those in the auxin pathway, a key hormone regulating fruit set in strawberry. Together, we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.
Fragaria vesca, a wild diploid strawberry, has recently emerged as a model for the cultivated strawberry and other members of the Rosaceae. Differentiation and maintenance of meristems largely determines plant architecture, flower development and ultimately fruit yield. However, in strawberry, our knowledge of molecular regulation of meristems in different developmental context is limited. In this study, we hand dissected three types of tissues than contain meristematic tissues corresponding to shoot apical meristem (SAM), flower meristem (FM), and receptacle meristem (REM), in F. vesca for RNA-seq analyses. A total of 3,009 differentially expressed genes (DEGs) were identified through pairwise comparisons. These DEGs were grouped into nine clusters with dynamic and distinct expression patterns. In these nine clusters, 336 transcription factor genes belong to 46 families were identified; some of which were significantly enriched in FM and REM such as the MADS-box family or in REM such as the B3 family. We found conserved and distinctive expression patterns of totally 149 genes whose homologs regulate flowering time or SAM, leaf, and flower development in other plant species. In addition to the ABCE genes in flower development, new MADS box genes were identified to exhibit differential expression in these different tissues. Additionally, the cytokinin and auxin pathway genes also exhibited distinct expression patterns. The Arabidopsis homeobox gene WUSCHEL (WUS), essential for stem cell maintenance, is expressed in organizing center of meristems. The F. vesca homolog FvWUS1 exhibited a broader expression domain in young strawberry flowers than its Arabidopsis counterpart. Altogether, this work provides a valuable data resource for dissecting gene regulatory networks operating in different meristematic tissues in strawberry.
The plant-specific transcription factor LEAFY (LFY), generally maintained as a single copy gene in most angiosperm species, plays critical roles in flower and leaf development. However, wild strawberry Fragaria vesca possesses four LFY homologues in the genome, their respective functions and evolution remain unknown. Through chemical mutagenesis screen, we identified two allelic mutations in one of the four LFY homologues, FveLFYa, in F. vesca, causing homeotic conversion of floral organs and reiterative outgrowth of ectopic florets. Both CRISPR-knockout and transgenic rescue confirmed the identity of FveLFYa. Ectopic expression of FveLFY homologues in Arabidopsis lfy-5 mutant revealed that only FveLFYa and FveLFYb can rescue the flower defects and induce solitary flowers in leaf axils. Disruption of FveLFYc, the second most abundantly expressed LFY homologue, caused no obvious morphology phenotypes in F. vesca. FveLFYb and FveLFYd are barely expressed. Expression of FveAP1, homologue of the well-known LFY target AtAP1, is not changed in the fvelfya flowers, possibly caused by an absence of any FveLFYa binding site in its promoter. Loss of Axillary Meristems encodes a GRAS transcription factor essential for stamen initiation. The ectopic florets are eliminated in fvelfya lam, suggesting that LAM is required for floret production. Moreover, approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Among these homologues, only FveLFYa is syntenic to the homologues in other species. Overall, the detailed analyses of the four LFY homologues in woodland strawberry demonstrate that only FveLFYa plays crucial roles in floral patterning with rewired gene network.
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