Circadian, carbon, and light control of expansion growth and leaf movement

We used Phytotyping4D to investigate the contribution of clock and light signaling to the diurnal regulation of rosette expansion growth and leaf movement in Arabidopsis (Arabidopsis thaliana). Wild-type plants and clock mutants with a short (lhycca1) and long (prr7prr9) period were analyzed in a T24 cycle and in T-cycles that were closer to the mutants’ period. Wild types also were analyzed in various photoperiods and after transfer to free-running light or darkness. Rosette expansion and leaf movement exhibited a circadian oscillation, with superimposed transients after dawn and dusk. Diurnal responses were modified in clock mutants. lhycca1 exhibited an inhibition of growth at the end of night and growth rose earlier after dawn, whereas prr7prr9 showed decreased growth for the first part of the light period. Some features were partly rescued by a matching T-cycle, like the inhibition in lhycca1 at the end of the night, indicating that it is due to premature exhaustion of starch. Other features were not rescued, revealing that the clock also regulates expansion growth more directly. Expansion growth was faster at night than in the daytime, whereas published work has shown that the synthesis of cellular components is faster in the day than at nighttime. This temporal uncoupling became larger in short photoperiods and may reflect the differing dependence of expansion and biosynthesis on energy, carbon, and water. While it has been proposed that leaf expansion and movement are causally linked, we did not observe a consistent temporal relationship between expansion and leaf movement.This work was supported by the Max Planck Society, the European Union (collaborative project TiMet under contract no. 245143), and an International Max Planck Research School stipend (to D.B.)

GROWTH-REGULATING FACTOR 9 negatively regulates arabidopsis leaf growth by controlling <i>ORG3</i> and restricting cell proliferation in leaf primordia

<div><p>Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the <i>Arabidopsis thaliana</i> TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF <i>OBP3-RESPONSIVE GENE 3</i> (<i>ORG3</i>). While <i>grf9</i> knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing <i>GRF9</i> (<i>GRF9ox</i>). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that <i>GRF9</i> restricts cell proliferation in the early developing leaf. Performing <i>in vitro</i> binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified <i>ORG3</i> as a direct downstream, and positively regulated target of GRF9. Genetic analysis of <i>grf9 org3</i> and <i>GRF9ox org3</i> double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf.</p></div

Time-course analysis of leaf growth.

<p>(A) Number of cells along the basal-apical axis of the first set of developing leaves in WT, <i>grf9</i> knockout and <i>GRF9ox</i> lines. The curves show fitting to the experimental data with a polynomial function. R² values are > 0.99 in all cases. At days 3 and 4 after germination, cell numbers are significantly higher in the two <i>grf9</i> mutants than in WT (Student's <i>t</i>-test; day 3, <i>p</i> ≤ 0.01; day 4, <i>p</i> ≤ 0.001). At days 5 to 14, cell numbers are significantly higher in the two <i>grf9</i> mutants than in WT, and significantly lower in the two <i>GRF9ox</i> lines (Student's <i>t</i>-test; <i>P</i> values between <i>p</i> ≤ 0.05 and <i>p</i> ≤ 0.001). Data are means of 6–14 leaves for the different genotypes and time points ± SD. The full data are given in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007484#pgen.1007484.s004" target="_blank">S4 Table</a></b>). (B) Increment of the number of cells in the adaxial subepidermal layer along the basal-apical leaf axis. Left: increment (Δ cell number) between day 3 and day 4 (D4-D3); right: increment between day 5 and day 10 (D10-D5) in WT and <i>GRF9</i> transgenic plants. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007484#pgen.1007484.s004" target="_blank">S4 Table</a></b> for the full data. (C) Relative expression levels of cell cycle-related genes in the leaf primordia from 7-day-old and 14-day-old WT, <i>grf9-2</i> and <i>GRF9ox1</i> plants. Gene expression in the WT is set to 1. Data are means ± SD (n > 8 seedlings).</p

Model of GRF9 action.

<p>(A) GRF9 restricts the size of the incipient leaf primordium at the shoot apex, whitout affecting cell size or the size of the SAM. While leaf primordium size is increased in <i>grf9</i> mutants, compared to wild type (WT), it trends to be smaller in <i>GRF9</i> overexpressor (<i>GRF9ox</i>) plants. Similarly, cell numbers in young developing leaves are bigger in <i>grf9</i>, but smaller in <i>GRF9ox</i> plants, in accordance with a higher number of cells in the developing <i>grf9</i> leaves, potentially contributing to the position of the arrest front. (B) Gene regulatory network by which GRF9 and ORG3 influence leaf size. <i>MiR396</i> targets <i>GRF9</i> transcript and negatively regulates its abundance. GRF9 interacts with GIF1 which, similar to other GRFs, influences leaf size determination. <i>ORG3</i> expression is positively regulated by GRF9 and OBP3, but repressed by the TCP20 transcription factor [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007484#pgen.1007484.ref007" target="_blank">7</a>]. Finally, ORG3 negatively regulates cell proliferation thereby directly influencing leaf size. For more details, see text.</p

Determination of actively proliferating cells in WT, <i>grf9</i> and <i>GRF9ox</i> plants using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay.

<p>The first true leaves of 5-day-old seedlings were analysed. (A) Example of an EdU-stained Arabidopsis leaf. Green signals indicate cells undergoing mitosis. (B) EdU signal distribution in leaves, and (C) relative occupancy of proliferating cells within the developing leaves in <i>grf9-2</i>, <i>GRF9ox1</i> and WT plants. Data represent average signals from at least eight seedlings.</p

Characterization of the <i>grf9 org3</i> and <i>GRF9ox org3</i> double mutants.

<p>(A) Scans of representative first-pair leaves, (B) leaf area, (C) number of palisade cells, and (D) cell area of palisade cells (n > 240 cells) in WT, <i>grf9-2</i>, <i>org3-1</i>, <i>org3-1 grf9-2</i> (line 7), <i>GRF9ox1</i>, and <i>GRF9ox1 org3-1</i> (line 34) mutants. Data are expressed as a percentage of WT ± SD. First leaves from 21-day-old plants were analyzed (n > 8 in all cases). Asterisks in panels (B) and (C) indicate significant difference (Student's <i>t</i>-test; <i>p</i> < 0.05). In (A), bar = 10 mm.</p

Characterization of <i>org3</i> mutants and <i>ORG3ox</i> plants.

<p>(A) Scans of representative first-pair leaves, (B) leaf size, (C) number of palisade cells, and (D) size of palisade cells (n > 240 cells) in WT, <i>org3-1</i>, <i>org3-2</i>, <i>ORG3ox1</i> and <i>ORG3ox2</i> plants. Results are expressed as percentage of WT ± SD. First-pair leaves from 21-day-old plants were analyzed (n > 8 in all cases). Asterisks indicate significant difference from WT (Student's <i>t</i>-test; <i>p</i> < 0.05). In (A), bar = 10 mm.</p