A comprehensive fluorescent sensor for spatiotemporal cell cycle analysis in Arabidopsis

Abstract

Assessing cell proliferation dynamics is crucial to understand the spatiotemporal control of organogenesis. Here we have generated a versatile fluorescent sensor, PlaCCI (plant cell cycle indicator) on the basis of the expression of CDT1a-CFP, H3.1-mCherry and CYCB1;1-YFP, that identifies cell cycle phases in Arabidopsis thaliana. This tool works in a variety of organs, and all markers and the antibiotic resistance are expressed from a single cassette, facilitating the selection in mutant backgrounds. We also show the robustness of PlaCCI line in live-imaging experiments to follow and quantify cell cycle phase progression.Development in multicellular organisms depends on the highly regulated formation of new organs. Organogenesis requires the production of a sufficient number of cells in a temporally and spatially regulated manner, that eventually will constitute the cohort of different cell types present in the organ. Therefore, understanding morphogenesis requires the availability of confident markers to assess cell proliferation and identify unequivocally the different cell cycle phases in a living cell environment.Efforts in several animal models have led to the development of the FUCCI (fluorescent, ubiquitination-based cell cycle indicator) systems that identify each cell cycle phase [1][2][3][4] . Comparable advances for live-imaging of cell proliferation studies in plants lag behind. One reason is that plants lack the CDT1a-interacting protein, geminin, a key component of the FUCCI system, although they contain a CDT1a-interacting protein (GEM) that is structurally unrelated to animal geminin 5,6 . Attempts to circumvent this have been reported. Thus, in addition to the tags used for G2 + M (refs. 7,8 ), the expression of a truncated version of CDT1a, a DNA replication initiator protein, from a heterologous promoter has been reported as an S + G2 marker 9 . In another approach, the fluorescent protein Venus fused to the destruction box of CYCB1;1 was expressed under the control of H4 promoter and labelled cells in S + G2 + M (ref. 10 ). However, a comprehensive cell cycle marker line should ideally (1) be able to distinguish in an unequivocal manner all cell cycle phases to follow individual cells in a growing organ and (2) offer the possibility of using advanced live-imaging strategies. This is precluded when using nucleoside analogues for labelling the S-phase, since they require specific detection protocols incompatible with live imaging.Still, one major drawback of cell cycle marker lines developed so far is the lack of appropriate markers to identify unequivocally cells in G1. We have recently analysed in detail CDT1a dynamics in proliferating cells and demonstrated that CDT1a starts to be loaded into chromatin shortly after mitosis, reaches a maximum during G1 and is rapidly degraded only minutes after S-phase initiation 11 , expanding previous claims of its proteasome-dependent degradation 12 . Importantly, we demonstrated that CDT1a accumulates rapidly after the S-phase of endocycling cells and is...

ThePolycombgroup protein MEDEA controls cell proliferation and embryonic patterning inArabidopsis

“…We focused on the latter since mea embryos have more cells compared to the wild type (Grossniklaus et al, 1998). In order to measure the rate at which cells divide in mea embryos, we crossed mea/MEA plants with a triple cell cycle marker line (Desvoyes et al, 2020), enabling the simultaneous visualization of G1, S+early G2, and late G2+M phases. Two distinct classes of embryonic expression patterns were identified ( Figure 4B-F, Figure S4): in the first, many nuclei were in G1 (high G1/G2 ratio, i.e.…”

Estimation of cell cycle kinetics in higher plant root meristem links organ position with cellular fate and chromatin structure

“…This protocol requires a living cell image scan, which may affect cell cycle progression (Tosheva, K. L., et al, 2020). Moreover, the Desvoyes et al (2020) method’s serious disadvantage is lacking positional information (cell file, distance from QC, periclinal division). As you have seen from our results, cell cycle kinetics were dependent on cell fate and cell position and required precise 3D analysis.…”

The phosphate starvation response recruits the TOR pathway to regulate growth in Arabidopsis cell cultures

“…They are induced as early as after 6 hours of nutrient resupply but not affected or repressed in response to phosphorus deficiency or TOR inactivation. Desvoyes et al (2020) have recently shown that three genes are particularly important in idetifying the stage of the cell cycle in a given cell. Based on the expression profile of these three genes, it appears that the cell cycle is re-initiated as early as 6 hours after nutrient resupply (even in the absence of sugar in the medium) (supplemental figure 9).…”

“…Expression profile of cell cycle marker genes (Desvoyes et al, 2020): CDT1a was shown to be specifically expressed in cells in G1, CycB1;1 was shown to be specifically expressed in cells in late G2 phase and early in mitosis while the histone HTR13 is expressed predominantly during S phase and early G2 phase.…”