SUMMARY The transition from the juvenile to the adult phase of shoot development in plants is accompanied by changes in vegetative morphology and an increase in reproductive potential. Here we describe the regulatory mechanism of this transition. We show that miR156 is necessary and sufficient for the expression of the juvenile phase, and regulates the timing of the juvenile-to-adult transition by coordinating the expression of several pathways that control different aspects of this process. miR156 acts by repressing the expression of functionally distinct SPL transcription factors. miR172 acts downstream of miR156 to promote adult epidermal identity. miR156 regulates the expression of miR172 via SPL9 which, redundantly with SPL10, directly promotes the transcription of miR172b. Thus, like the larval-to-adult transition in Caenorhabditis elegans, the juvenile-to-adult transition in Arabidopsis is mediated by sequentially operating miRNAs. miR156 and miR172 are positively regulated by the transcription factors they target, suggesting that negative feedback loops contribute to the stability of the juvenile and adult phases.

The FT gene integrates several external and endogenous cues controlling flowering, including information on day length. A complex of the mobile FT protein and the bZIP transcription factor FD in turn has a central role in activating genes that execute the switch from vegetative to reproductive development. Here we reveal that microRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes not only act downstream of FT/FD, but also define a separate endogenous flowering pathway. High levels of miR156 in young plants prevent precocious flowering. A subsequent day length-independent decline in miR156 abundance provides a permissive environment for flowering and is paralleled by a rise in SPL levels. At the shoot apex, FT/FD and SPLs converge on an overlapping set of targets, with SPLs directly activating flower-promoting MADS box genes, providing a molecular substrate for both the redundant activities and the feed-forward action of the miR156/SPL and FT/FD modules in flowering control.

Flavonoids are synthesized through an important metabolic pathway that leads to the production of diverse secondary metabolites, including anthocyanins, flavonols, flavones, and proanthocyanidins. Anthocyanins and flavonols are derived from Phe and share common precursors, dihydroflavonols, which are substrates for both flavonol synthase and dihydroflavonol 4-reductase. In the stems of Arabidopsis thaliana, anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. We show here that this accumulation pattern is under the regulation of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are deeply conserved and known to have important roles in regulating phase change and flowering. Increased miR156 activity promotes accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols. We further provide evidence that at least one of the miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Our results reveal a direct link between the transition to flowering and secondary metabolism and provide a potential target for manipulation of anthocyanin and flavonol content in plants.

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.

Cells of eukaryotic multicellular organisms have inherent heterogeneity. Recent advances in single-cell gene expression studies enable us to explore transcriptional regulation in dynamic development processes and highly heterogeneous cell populations. In this study, using a high-throughput single-cell RNAsequencing assay, we found that the cells in Arabidopsis root are highly heterogeneous in their transcriptomes. A total of 24 putative cell clusters and the cluster-specific marker genes were identified. The spatial distribution and temporal ordering of the individual cells at different developmental stages illustrate their hierarchical structures and enable the reconstruction of continuous differentiation trajectory of root development. Moreover, we found that each root cell cluster exhibits distinct patterns of ion assimilation and hormonal responses. Collectively, our study reveals a high degree of heterogeneity of root cells and identifies the expression signatures of intermediate states during root cell differentiation at single-cell resolution. We also established a web server (http://wanglab.sippe.ac.cn/rootatlas/) to facilitate the use of the datasets generated in this study.