Expression and Functional Analyses of Nymphaea caerulea MADS-Box Genes Contribute to Clarify the Complex Flower Patterning of Water Lilies

Nymphaeaceae are early diverging angiosperms with large flowers characterized by showy petals and stamens not clearly whorled but presenting a gradual morphological transition from the outer elements to the inner stamens. Such flower structure makes these plant species relevant for studying flower evolution. MADS-domain transcription factors are crucial components of the molecular network that controls flower development. We therefore isolated and characterized MADS-box genes from the water lily Nymphaea caerulea. RNA-seq experiments on floral buds have been performed to obtain the transcript sequences of floral organ identity MADS-box genes. Maximum Likelihood phylogenetic analyses confirmed their belonging to specific MADS-box gene subfamilies. Their expression was quantified by RT-qPCR in all floral organs at two stages of development. Protein interactions among these transcription factors were investigated by yeast-two-hybrid assays. We found especially interesting the involvement of two different AGAMOUS-like genes (NycAG1 and NycAG2) in the water lily floral components. They were therefore functionally characterized by complementing Arabidopsis ag and shp1 shp2 mutants. The expression analysis of MADS-box genes across flower development in N. caerulea described a complex scenario made of numerous genes in numerous floral components. Their expression profiles in some cases were in line with what was expected from the ABC model of flower development and its extensions, while in other cases presented new and interesting gene expression patterns, as for instance the involvement of NycAGL6 and NycFL. Although sharing a high level of sequence similarity, the two AGAMOUS-like genes NycAG1 and NycAG2 could have undergone subfunctionalization or neofunctionalization, as only one of them could partially restore the euAG function in Arabidopsis ag-3 mutants. The hereby illustrated N. caerulea MADS-box gene expression pattern might mirror the morphological transition from the outer to the inner floral organs, and the presence of transition organs such as the petaloid stamens. This study is intended to broaden knowledge on the role and evolution of floral organ identity genes and the genetic mechanisms causing biodiversity in angiosperm flowers.

MIR172d Is Required for Floral Organ Identity and Number in Tomato

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-dCR). The slmir172c-dCR plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172cCR loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172dCR alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets.

AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic and growth genes in young flowers

Arabidopsis flower primordia give rise to floral organ primordia in stereotypical positions within four concentric whorls. Floral organ primordia in each whorl undergo distinct developmental programs to become one of four organ types (sepals, petals, stamens, and carpels). The Arabidopsis transcription factors AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) play critical and partially overlapping roles during floral organogenesis. They are required for correct positioning of floral organ initiation, contribute to the specification of floral organ identity, and regulate the growth and morphogenesis of developing floral organs. To gain insight into the molecular means by which ANT and AIL6 contribute to floral organogenesis, we identified the genome-wide binding sites of both ANT and AIL6 in stage 3 flower primordia, the developmental stage at which sepal primordia become visible and class B and C floral homeotic genes are first expressed. AIL6 binds to a subset of ANT sites, suggesting that AIL6 regulates some but not all of the same target genes as ANT. ANT and AIL6 binding sites are associated with genes involved in many biological processes related to meristem and flower organ development. Comparison of genes associated with both ANT and AIL6 ChIP-Seq peaks and those differentially expressed after perturbation of ANT or AIL6 activity identified likely direct targets of ANT and AIL6 regulation. These include the floral homeotic genes APETALA3 (AP3) and AGAMOUS (AG) and four growth regulatory genes: BIG BROTHER (BB), ROTUNDIFOLIA3 (ROT3), ANGUSTIFOLIA3/GRF INTERACTING FACTOR (AN3/GIF1), and XYLOGLUCAN ENDOTRANSGLUCOLSYLASE/HYDROLASE9 (XTH9).One Sentence SummaryThe transcription factors ANT and AIL6 directly regulate genes involved in different aspects of flower development including genes that specify floral organ identity and those that regulate growth.

The movement of a leaf-derived mobile AGL24 mRNA specifies floral organ identity in Arabidopsis

Cell-to-cell and inter-organ communication play pivotal roles in synchronizing and coordinating plant development. In addition to serving as templates for protein translation within cells, many mRNAs can move and exert their function non-cell-autonomously. However, because the proteins encoded by some mobile mRNAs are also mobile, whether the systemic function of mobile mRNAs is attributed to proteins transported distally or translated locally remains controversial. Here, we show that Arabidopsis AGAMOUS-LIKE 24 (AGL24) mRNA acts as a leaf-derived signal to specify meristem identity. AGL24 is expressed in both apex and leaves. Upon floral meristem (FM) transition, apex-expressed AGL24 is transcriptionally inhibited by APETALA1 (AP1) to ensure FM differentiation. The leaf-expressed AGL24 can act as a mobile signal to bypass AP1 inhibition and revert FM differentiation. Although AGL24 mRNA is expressed in leaves, AGL24 protein is rapidly degraded in leaves. In contrast, AGL24 mRNA can move long distance from leaf to apex where the translocated AGL24 mRNAs can be used as templates to translate into proteins. Thus, the movement of AGL24 mRNA can provide the developmental plasticity to fit with environmental dynamics.