RETINOBLASTOMA-RELATED Has Both Canonical and Noncanonical Regulatory Functions During Thermo-Morphogenic Responses in Arabidopsis Seedlings

Abstract

Warm temperatures accelerate plant growth, but the underlying molecular mechanism is not fully understood. Here, we show that increasing the temperature from 22°C to 28°C rapidly activates proliferation in the apical shoot and root meristems of wild-type Arabidopsis seedlings. We found that one of the central regulators of cell proliferation, the cell cycle inhibitor RETINOBLASTOMA-RELATED (RBR), is suppressed by warm temperatures. RBR became hyper-phosphorylated at a conserved CYCLIN-DEPENDENT KINASE (CDK) site in young seedlings growing at 28°C, in parallel with the stimulation of the expressions of the regulatory CYCLIN D/A subunits of CDK(s). Interestingly, while under warm temperatures ectopic RBR slowed down the acceleration of cell proliferation, it triggered elongation growth of post-mitotic cells in the hypocotyl. In agreement, the central regulatory genes of thermomorphogenic response, including PIF4 and PIF7, as well as their downstream auxin biosynthetic YUCCA genes (YUC1-2 and YUC8-9) were all up-regulated in the ectopic RBR expressing line but down-regulated in a mutant line with reduced RBR level. We suggest that RBR has both canonical and non-canonical functions under warm temperatures to control proliferative and elongation growth, respectively.

Keywords: cell cycle; elongation growth; retinoblastoma‐related; thermomorphogenesis.

Figure 1

Warm temperatures increase proliferation activity in both the shoot and root meristems of Arabidopsis seedlings. Wild‐type seedlings were grown continuously at 22°C (control) or transferred from 22°C to 28°C (22 + 28°C) for the number of days indicated in the parentheses. (A) Leaf primordia of seedlings that were grown at a continuous 22°C for 6 days after germination (DAG) 22°C (6) or 2 days after transfer of 4‐day‐old seedlings from 22°C to 28°C (22 + 28°C [4 + 2]) are pictured under a scanning electron microscope. Positions and orders of cotyledons (Cot), leaves (L), and primordia (P) are indicated by numbers. Scale bar is 100 µm. (B) The epidermal cell size of the third leaf primordium was determined by using ImageJ software. (C) The developmental order of cotyledons (C) and leaves (L) is marked with numbers on seedlings grown at 22°C (9) and 22 + 28°C (4 + 5). (D) Representative pictures of the first leaf pairs of seedlings grown at 22°C(9) or at 22 + 28°C (4 + 5). (E–G) Leaf area (E), palisade cell area (F), and palisade cell number (G) were determined for the first leaf of seedlings grown at the indicated temperature regimes. (H) The seedlings from left to right were grown at 22°C (8), 22 + 28°C (4 + 4), or 28°C (8), respectively. Scale bar is 1 cm. (I) Representative pictures of propidium iodide‐stained roots of seedlings grown at a continuous 22°C (7) or 22 + 28°C (4 + 3) obtained by confocal laser scanning microscopy. Arrowheads indicate the quiescent centres, and arrows point to the first elongated cells in the cortical cell file, reflecting meristem size. Scale bar is 100 µm. (J) Spatial distribution of cell length across root meristems in seedlings grown at continuous 22°C (7) or 22 + 28°C (4 + 3). Mean cell length was calculated at each position along the cortical cell file. The positions were defined by counts of cortical cells from the quiescent centre. Values are averages from the data obtained by the analysis of eight roots from different plants (±SD). (K) Cortex cell number in the same roots as in (J). (L) The root tips of transgenic seedlings expressing CYCB1; 2‐YFP^NLS, a G2 and M‐phase marker, under the control of its own promoter. The seedlings were grown at 22°C (6) or 22 + 28°C (4 + 2), respectively, before being stained with propidium iodide and imaged using a confocal laser scanning microscope. Arrowheads indicate the quiescent centres. Scale bar is 100 µm. (M, N) The expression of the S‐ and the G2‐M‐phase regulatory genes, ORC2 (M) and CDKB1;1 (N), is elevated in seedlings transferred at four DAG from 22°C to 28°C compared to seedlings grown continuously at 22°C. Expressions were analysed at 8, 12, 24 and 48 h after the time of transfer in seedlings at both 22°C and 28°C, respectively. Values represent fold change normalised to the value of the relevant transcript of the seedlings at T0 (4DAG), which was set arbitrarily at 1. Data are means ± SD of three biological replicates. Different letters indicate significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test (B, E–K, M, N).

Figure 2

Ectopic RBR inhibits the warm temperature‐induced root growth acceleration by repressing cell proliferation. Wilde type (WT), ectopic RBR‐GFP expressing, and rbr1‐2 mutant seedlings were grown continuously at 22°C (control) or transferred from 22°C to 28°C (22 + 28°C) for the number of days indicated in the parentheses. (A) Images of seedlings from the investigated lines grown on vertical plates at either 22°C (7) (a) or 22°C + 28°C (4 + 3) (b). The scale bar is shown on the right side. (B) Comparison of root length increase between the 5th and 7th days in WT, RBR‐GFP and rbr1‐2 seedlings grown continuously at 22°C or transferred on the fourth day to 28°C (n = 15). Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test. (C) Representative images of root tips of WT, RBR‐GFP and rbr1‐2 seedlings grown at 22°C (7) for 22 + 28°C (4 + 3). The roots were stained with propidium iodide, and imaged under a confocal laser scanning microscope. Arrowheads indicate quiescent centres; arrows show the boundary of the root meristem in the cortex cell file. (D) Cortex cell number in the root meristem of 7‐day‐old WT, RBR‐GFP and rbr1‐2 seedlings grown at 22°C (7) or 22 + 28°C (4 + 3) (n = 8 roots). Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analysis with Tukey's HSD post‐hoc test. (E) Spatial distribution of cell length across root meristems in WT, RBR‐GFP and rbr1‐2 seedlings grown either at 22°C (7) or 22 + 28°C (4 + 3). Mean cell length was calculated at each position along the cortical cell file, which is defined by counts of cortical cells from the quiescent centre. Values are averages from the data analysed in eight roots from different plants (±SD). (F) EdU (light blue‐green) and Hoechst (blue) co‐staining of nuclei in root tips of WT, RBR‐GFP and rbr1‐2 lines grown at 22°C for 4 days and transferred to 28°C (+28°C) or left at 22°C for additional 8 h before a 30 min. incubation with 5 mM EDU labelling cells passing the S‐phase of the cell cycle. (G) The number of EdU‐positive root cells was counted at the indicated time points from eight roots for each genotype at each time point. Data are average ± standard deviation (n = 3 biological replicates, N = 8 samples in each). Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analysis with Tukey's HSD post‐hoc test. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Warm temperatures suppress the cell proliferation inhibitor function of RBR through activating G1 cyclins and RBR‐phosphorylation. Wilde type (WT), ectopic RBR‐GFP expressing and rbr1‐2 mutant seedlings were grown continuously at 22°C (control) or transferred from 22°C to 28°C (22 + 28°C) for the number of days indicated in the parentheses. (A–D) Warm temperatures increase leaf size in an RBR‐dependent manner. Size of the first leaf (A), images of palisade cells of the first leaf (B), the measured palisade cell area (n = 5 leaf ≥ 40 cells) (C) and the calculated cell number (leaf area/palisade cell area) (D). (E, F) The S‐ and the G2‐M‐phase‐specific OR2 (E) and CDKB1;1 (F) genes were induced by warm temperatures, depending on RBR. (G, H) Transcript levels of the G1 cyclins, CYCLIN D3;1 and CYCLIN A3;1, were elevated by warm temperatures, and are oppositely regulated in the RBR‐GFP and rbr1‐2 lines. Expression of all the four genes (E–H) was monitored by qRT‐PCR. Seedlings of WT, RBR‐GFP and rbr1‐2 lines were grown for 96 h at 22°C after germination and the samples were collected at the indicated time points at 22°C or 28°C, respectively. Values represent fold change normalised to the value of the relevant transcript of the seedlings at T0 (96 h), which was set arbitrarily at 1. Data are means ± SD. N = 3 biological replicates. Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test. (I) Western blot shows RBR and phospho‐RBR (P‐RBR at the 911 serine site) levels in WT seedlings grown as indicated. Sampling was made as in (E–H). The membrane was stained with comassie brilliant blue to indicate equal loading. Molecular weight marker (kDa) is shown at the left side. Arrow marks the position of the RBR protein. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

At warm temperatures, RBR stimulates the elongation growth of the hypocotyl, inducing the expression of PIF4 and PIF7 transcription factors. Wild type (WT), ectopic RBR‐GFP expressing, and rbr1‐2 mutant seedlings were grown continuously at 22°C (control) or transferred from 22°C to 28°C (22 + 28°C) for the number of days indicated in the parentheses. (A) Ectopic RBR increases hypocotyl length following the transfer of seedlings to 28°C. (B) Hypocotyl length of the three genotypes with different RBR levels was measured both at continuous 22°C or after transfer to 28°C at the indicated time points. N = 10; Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test. (C) Hypocotyl epidermis was analysed under a scanning electron microscope at both temperature regimes at the indicated time points. Scale bar is 100 µm. (D) Cell length of hypocotyl epidermis in the three lines was measurred by using the ImageJ software (N = 5). Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test. (E, F) Expression of PIF4 (E) and PIF7 (F) in WT, RBR‐GFP and rbr1‐2 seedlings at the indicated temperatures and times. T0 represents 96‐h‐old seedlings grown at 22°C. These were either kept growing at 22°C or after transfered to 28°C, and samples were taken at 8, 12, 24 and 48 h afterwards. Values represent fold change normalised to the value of the relevant transcript of the seedlings at T0 (96 h), which was set arbitrarily at 1. Data are means ± SD. N = 3 biological replicates. Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

RBR positively regulates the auxin synthesis regulatory YUCCA genes. (A–D) Expression of four YUCCA genes were followed by qRT‐PCR as indicated. T0 represents 96‐h‐old seedlings, and they were either kept growing at 22°C or transferred to 28°C, and the samples were taken at 8, 12, 24 and 48 h afterwards. Values represent fold change normalised to the value of the relevant transcript of the seedlings at T0 (96 h), which was set arbitrarily at 1. Data are means ± SD. N = 3 biological replicates. Different letters mean statistically significant differences (p < 0.05) based on one‐way ANOVA analyses with Tukey's HSD post‐hoc test. (E, F) Schematic model how temperature regulates cell proliferation and elongation growth through modifying RBR activity. (E) Warm temperatures activate G1 cyclins like CYCD3;1 and CYCA3;1. This might be mediated by the increased sucrose (suc) level due to the temperature‐enhanced metabolic activity. The cell cycle inhibitor RBR is phosphorylated and repressed by an RBR‐kinase complex, such as CDKA;1‐CYCD/A. This results in RBR‐free E2Fs, which drive cell cycle entry and accelerate meristematic function in both the root and shoot apices. (F) Warm temperatures might cause post‐translational modifications on RBR, such as phosphorylation at unknown site(s) in post‐mitotic cells. This could result in the stimulation, either directly or indirectly, of the expression of non‐canonical targets, like YUCCAs. [Color figure can be viewed at wileyonlinelibrary.com]