The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.

The development of an ideal model plant located at a key phylogenetic node is critically important to advance functional and regulatory studies of key regulatory genes in the evolutionary developmental (evo-devo) biology field. In this study, we selected Chirita pumila in the family Gesneriaceae, a basal group in Lamiales, as a model plant to optimize its genetic transformation system established previously by us through investigating a series of factors and further conduct functional test of the CYC-like floral symmetry gene CpCYC. By transforming a RNAi:CpCYC vector, we successfully achieved the desired phenotypes of upright actinomorphic flowers, which suggest that CpCYC actually determines the establishment of floral zygomorphy and the horizontal orientation of flowers in C. pumila. We also confirmed the activities of CpCYC promoter in dorsal petals, dorsal/lateral staminodes, as well as the pedicel by transferring a CpCYC promoter:GUS vector into C. pumila. Furthermore, we testified the availability of a transient gene expression system using C. pumila mesophyll protoplasts. The improved transformation system together with the inherent biological features would make C. pumila an attractive new model in functional and regulatory studies for a broad range of evo-devo issues.

SUMMARY: In many species, FLOWERING LOCUS T ( FT )‐like genes promote the floral transition by integrating environmental signals, in particular photoperiod, and internal cues. Here we show that Brassica napus contains six FT ‐like genes and two pseudogenes belonging to three orthogroups. All B. napus FT ‐like genes induce early flowering when expressed at the shoot apical meristems of Arabidopsis thaliana ft mutants; however, BnaFT.C6 and non‐orthologous FT ‐like genes do not encode fully functional mobile florigens. In the case of BnFT.C6 , the functional change is associated with a T to C amino acid change that is restricted to semi‐winter accessions. Expression of orthologs of FT is photoperiod‐dependent, and two distal enhancers are conserved; however, the homeologs BnaFT.A7 and BnaFT.C6 show rearrangements of DNA motifs binding NF‐Y/CO and NF‐Y transcriptional activator complexes between the promoter and downstream enhancers. Motif rearrangements correlate with differences in tissue‐specific expression. Furthermore, homeologs with rearranged motifs could not be transactivated by B. napus CO in transient assays, although they show LD photoperiod‐dependent expression. We propose that differential diurnal expression of NF‐Y genes contributes to the photoperiod‐dependent regulation of B. napus FT genes.

In many species, FLOWERING LOCUS T ( FT )-like genes promote the floral transition by integrating environmental signals, in particular photoperiod, and internal cues. Here we show that Brassica napus contains 6 FT -like genes and 2 pseudogenes belonging to 3 orthogroups. All B. napus FT -like genes induce early flowering when expressed at the shoot apical meristems of Arabidopsis thaliana ft mutants, but BnaFT.C6 and non-orthologous FT -like genes do not encode fully functional mobile florigens. In the case of BnFT.C6 , the functional change is associated with a T to C amino acid change that is restricted to semi-winter accessions. Expression of orthologs of FT is photoperiod-dependent and two distal enhancers are conserved; however, the homeologs BnaFT.A7 and BnaFT.C6 show rearrangements of DNA motifs binding NF-Y/CO and NF-Y transcriptional activator complexes between the promoter and downstream enhancers. Motif rearrangements correlate with differences in tissue-specific expression. Furthermore, homeologs with rearranged motifs could not be trans-activated by B. napus CO in transient assays although they genes show LD photoperiod-dependent expression. We propose that differential diurnal expression of NF-Y genes contributes to the photoperiod-dependent regulation of B. napus FT genes. Significance statement: B. napus FT -like genes are a case study for gene retention and retraction after multiple rounds of polyploidisation. Retained B. napus FT -like genes show preferred expression in different developmental stages and amino-acid changes that affect florigen function.