Regulation of early seedling establishment and root development in Arabidopsis thaliana by light and carbohydrates

Abstract

Root development is regulated by sucrose and light during early seedling establishment through changes in the auxin response and chromatin topology. Light is a key environmental signal that regulates plant growth and development. The impact of light on development is primarily analyzed in the above-ground tissues, but little is known about the mechanisms by which light shapes the architecture of underground roots. Our study shows that carbohydrate starvation during skotomorphogenesis is accompanied by compaction of nuclei in the root apical meristem, which prevents cell cycle progression and leads to irreversible root differentiation in the absence of external carbohydrates, as evidenced by the lack of DNA replication and increased numbers of nuclei with specific chromatin characteristics. In these conditions, induction of photomorphogenesis was unable to restore seedling growth, as overall root growth was compromised. The addition of carbohydrates, either locally or systemically by transferring seedlings to sugar-containing medium, led to the induction of adventitious root formation with rapid recovery of seedling growth. Conversely, transferring in vitro carbohydrate-grown seedlings from light to dark transiently promoted cell elongation and significantly reduced root meristem size, but did not primarily affect cell cycle kinetics. We show that, in the presence of sucrose, dark incubation does not affect zonation in the root apical meristem but leads to shortening of the proliferative and transition zones. Sugar starvation led to a rapid increase in lysine demethylation of histone H3 at position K9, which preceded a rapid decline in cell cycle activity and activation of cell differentiation. In conclusion, carbohydrates are required for cell cycle activity, epigenetics reprogramming and for postmitotic cell elongation and auxin-regulated response in the root apical meristem.

Keywords: Arabidopsis thaliana; Carbohydrates; Cell differentiation; Cell division; Histone modifications; Root apical meristem.

Fig. 1

Sugar is required for PR growth during early seedling establishment. a, b Root and RAM length were measured in seedlings grown for 5 days in the medium with and without 1% sucrose, under 0 (Dark, D) or 100 µmol m⁻² s⁻¹ continuous light (Light, L). c Expression of the CycB1;1-GUS marker (shown in blue) in the same conditions as indicated above. The purple line indicates the region of the PZ of the RAM. d Number of LR primordia in the same conditions as indicated above. e Quantitative analysis of the RAM structure under dark and light conditions without sucrose. Seedlings were grown 3 days under dark or light conditions, fixed, segmented and a cellular map of the outer cell layers was obtained as described elsewhere (Pasternak and Pérez-Pérez 2021). Orange: cortex cells, blue: atrichoblasts, gray: trichoblasts. f Quantitative analysis of the cortex layer in a representative sample across spatial coordinates. g Length of cortex cells in the mature zone. The bars in a, b, d and g show the mean values ± standard deviation (SD), n = 10. Letters indicate significant differences between treatments (P-value < 0.01; Least Significant Difference [LSD]). Scale bars = 40 μm

Fig. 2

Sugar is required to maintain functional chromatin in the RAM. a Details of nuclei structure in the proximal RAM as stained by DAPI after 5, 6 and 8 days in light or in dark conditions without 1% sucrose. b–d Quantitative analysis of nuclei parameters. Volume (b), number of chromocenters (c) and mean distance from barycenter to border (d) were estimated as using the NucleusJ 2.0 plugging (Dubos et al. 2020); n = 600. e, f Chromatin features in the RAM (e, f) and the differentiation zone (e′, f′) in response to sugar. Seedlings were cultured in the dark in the absence (e, e’) and presence (f, f’) of 1% sucrose. DAPI staining is shown in white, H3K4me2 in purple and H3K9me2 in green. Letters in b–d indicate significant differences between treatments (P-value < 0.01; LSD). Scale bars = 50 μm

Fig. 3

a–c Effect of light and sucrose on auxin homeostasis in the RAM during early seedling establishment. DR5::GFP (green, a), expression of the auxin biosynthesis genes (b, pYUC::GUS, blue), and overall TAA1 expression levels (c) in the RAM estimated from fluorescence. RU, relative units. d–f Abundance of the PIN1 (d) and PIN2 (e) auxin efflux facilitators shown in green. H⁺-ATPase in f as a RAM integrity marker. DAPI staining of nuclei is shown in blue. To quantify PIN1 and PIN2, GFP fluorescence was measured in relative units (RU) in a given area (shown in red). g Overall DII-VENUS expression in the RAM shown in green. Seedlings have been grown for 5 days under dark or light conditions in the presence or absence of 1% sucrose, as indicated. Scale bars = 40 μm

Fig. 4

Post-germinative carbon starvation affects PR growth. a–c PR growth (a, b) and RAM length (c) in seedlings grown on 1% sucrose medium for 4.5 d in the light and that have been transferred to liquid medium under dark (D) or light (L) conditions with 0% or 1% sucrose. Arrow in b indicates the end of the PZ of the RAM estimated by the increased volume of cortical cells; asterisks mark the QC cells. d DNA replication and mitosis events of cortical cells in a representative root sample. 4.5-day-old seedlings were cultured for 24 h in the dark/light conditions, EdU/colchicine were added for the last 90 min. EdU (green) and mitotic figures (orange) were detected and analyzed as previously described (Pasternak et al. 2022). Blue lines and orange lines indicate the limit of PZ and EZ, respectively. e Expression of the CycB1;1-GUS marker (blue) in the same conditions as indicated in (a). f Overall DII-VENUS expression levels in the RAM estimated from fluorescence. RU, relative units. The bars in a, c and f show the mean values ± SD (n = 10). Letters indicate significant differences between treatments (P-value < 0.01; LSD). Scale bars = 40 μm

Fig. 5

Sugar-induced recovery of seedling growth after carbon starvation. a, b Seedlings grown for 4.5 days in the dark without sucrose were transferred to plates supplemented with 0% (a) or 1% (b) sucrose. Images of whole seedlings were obtained after 10 days of growth in continuous light (10dL). c Number of ARs and LRs of seedlings in (a, b) at 10 dL. d Local application of sucrose (white arrowheads) after 4.5 days of growth in the dark without sucrose. Images in d were scanned after 14 days under continuous light (14 dL). e Sugar-mediated root growth recovery after carbon starvation. Seedlings were grown on the surface of agar medium without carbon and thereafter were transferred to the medium containing 1% of filter-sterilized glucose (Glc), fructose (Fru), or sucrose (Suc). New root growth has been measured after 90 h. The bars in c, e show the mean values ± SD (n = 10). Letters indicate significant differences between treatments (P-value < 0.01; LSD). Scale bars = 10 mm

Fig. 6

Sucrose dynamically alters heterochromatin features and cell cycle progression in the RAM of carbon-starved seedlings. a Changes in chromatin and cell cycle progression in response to sucrose were detected by EdU incorporation (green, arrows), DAPI staining (blue) and H3K9me2 immunolocalization (magenta). Seedlings in a have been grown in the dark for 4.5 days in the absence of sucrose and thereafter were transferred to liquid medium in light conditions with or without sucrose, as indicated. b Changes in chromatin and cell cycle progression were detected by EdU incorporation (red, arrows), DAPI staining (blue) and H3K9me2 immunolocalization (green). Seedlings have been grown in the light for 4.5 days with sucrose and thereafter were transferred to liquid medium for 48 h in dark or light conditions without sucrose. Seedlings in (a, b) were incubated in the presence of EdU and colchicine for 90 min prior fixation. c Nucleolus volume from seedlings in b. Scale bars = 40 μm

Fig. 7

Effect of sucrose and auxin in seedling growth restoration after sugar starvation. a Seedlings were subjected to sugar starvation by growing them in the dark during 4.5 days without sucrose. Thereafter seedlings (a–d) or isolated root explants (e–j) were transferred to 0% or 1% sucrose medium supplemented with 150 nM NAA and grown in continuous light for several days. b Number of ARs after 10 days of growth in continuous light (10dL). c, d PR morphology of Col-0 (c) and of CycB1::GUS marker lines (d). Seedlings in c and d were incubated for 24 h in the dark on 0% or 1% sucrose medium with 150 nM NAA. e, f PR length (e) and number of LR primordia (f). g, h Localization of (g) PIN1 and (h) PIN4 in the RAM; green: PIN proteins and blue: DAPI. i Overall PIN1 levels in the RAM estimated from fluorescence. RU, relative units. PIN immunolocalization and quantification was done according to standard procedures (see Materials and methods). The bars in b, e, f and i show the mean ± SD values (n = 10). Asterisks indicate significant differences between treatments (P-value < 0.01; LSD). Scale bars = 2 mm (a, c, d) and 40 μm (h, i)