As revealed in the previous chapter, CEP3 affects cell cycle activity in the RAM, potentially by regulating entry into the S-phase (Chapter 4 Section 5). Given that this severely affected root growth and that CEP3 was found to be induced in response to nutritional cues (Delay et al., 2013; Chapter 3 Section 4), it was hypothesised that CEP3 affected the ability of the plant to effectively sustain photoautotrophic growth.
Therefore, to explore the dynamics of CEP3 under C depletion and to examine whether it played a role in the heterotrophic/ photoautotrophic transition, a time course was performed. WT, cep3-1a or WT + 1 µM CEP3 (continuous exposure to CEP3 from imbibition) were grown in liquid ½ MS medium with minimal lighting for up to 7 days post germination (dpg). Sample roots were treated with EdU, harvested and the number of EdU positive cells per root was recorded (Fig. 2).
Figure 2. CEP3 promotes entry into mitotic quiescence under carbon limitation. (A) Time course showing the number of meristematic cells undergoing DNA synthesis. Plants were gown in standard ½ MS liquid medium with restricted light (≤ 50 µmol/m2/s1) to prevent photoautotrophic growth. Red box indicates age of plants used in (B). (B) Number EdU positive cells after 24 hours of incubation with glucose. Dpg = days post germination Black shading = WT, no shading = cep3-1a, grey shading = WT + 1 µM CEP3 peptide. n ≥ 5 for each time point. Letters indicate statistically significant differences within each time point (p < 0.05, ANOVA using Bonferroni multiple comparisons test (Genstat)).
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The number of EdU positive cells was similar in WT and cep3-1a under ½ MS
conditions, with a steady decline until day 6 (Fig. 2A). In CEP3 peptide treated plants however, the number of EdU positive cells was significantly reduced even at day 2 and cells reached mitotic quiescence by 3-4 days (Fig. 2A). After 5-6 days, when the
majority of cells were no longer undergoing cell cycle in WT and cep3 (Fig. 2A, red box), glucose was added to all samples to examine re-initiation of the cell cycle. After 24 h of incubation with glucose, meristem cells from all three treatments had re-entered the cell cycle. CEP3 treated meristems had around 50% fewer cells undergoing DNA synthesis, as expected based on previous results showing that CEP3 reduces
meristematic activity (Chapter 4 Section 5). These data indicated that CEP3 peptide caused cells in the RAM to enter quiescence much more rapidly. However, CEP3 did not affect the ability of the root meristem to switch form heterotrophic to
photoautotrophic growth as meristems in all treatments could be reactivated by glucose addition.
Previous results showed that CEP3 was strongly induced in roots by nitrogen starvation (Delay et al., 2013; Chapter 3 Section 4). Furthermore, treatment with CEP3 peptide resulted in a slowing of root growth (Delay et al., 2013; Chapter 4 Section 5) and reduced cell cycle activity (Chapter 4 Section 5). It was therefore hypothesised that CEP3 imparts a specific, “conservative-growth-rate” strategy by affecting nutrient allocation or balance in response to nitrogen limitation. To examine this, a novel experiment was designed to determine the effects of nitrogen starvation on meristematic growth. It is known that there is a strong interplay between carbon availability and the response to nitrogen (Stitt and Krapp, 1999; Malamy and Ryan, 2001; Takatani et al., 2014). Therefore, in order to minimise any confounding effects caused by carbon availability, plants were grown under the same carbon-limited conditions as in the previous experiment (Fig. 2) and a nitrogen limitation was also imposed.
Nitrogen was withheld by growing plants in N-free liquid medium (Fig. 3). To examine the effects of nitrogen starvation on WT, CEP3 peptide treated and cep3 plants, a time course was performed. As the previous results in ½ MS medium indicated that CEP3 (or lack thereof) primarily affected mitotic activity or quiescence over time, results were
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measured as the percentage of plants with at least one mitotically active RAM cell (marked by EdU fluorescence indicating DNA synthesis had occurred).
Figure 3. CEP3 levels affect mitotic activity under nitrogen starvation. (A) Time course showing the percent of roots with at least 1 EdU positive cell. Plants were grown in modified N-free liquid medium with restricted light to prevent photoautotrophic growth. (B) Percent of roots with at least 1 EdU positive cell after 24 h of incubation with 1 mM glucose. Black shading = WT, no shading = cep3-1a, grey shading = WT + 1 µM CEP3 peptide. n ≥ 10 roots. Letters indicate statistically significant differences within each time point (p <0.05, ANOVA using Bonferroni multiple comparisons test (Genstat)).
These results showed that CEP3 clearly affects the developmental response to nitrogen starvation. Few CEP3 treated root cells showed EdU incorporation at day 5 and none had meristematic activity by 6 dpg, 2-3 days earlier than untreated plants (Fig. 3A). This is similar to the results seen in the previous experiment (Fig. 2), where CEP
treatment induced mitotic quiescence earlier than in WT, perhaps as a result of altered nutrient allocation/use. Once meristematic activity had ceased in CEP3 peptide treated roots, glucose was not able re-activate CEP3 treated meristems, indicating they may have become permanently inactive rather than entering quiescence (Fig. 3B). The comparative responses of the WT and the cep3 mutant results provide support for this hypothesis. A much higher percentage of cep3 plants (90-100% compared with only 50-70% of WT) remained mitotically active for a longer period of time (Fig. 3A). Surprisingly, glucose addition could still re-activate meristematic activity in 75% of
cep3 roots compared to only 14% of WT roots, even after all samples in the population
had ceased mitotic activity (compare day 9 and 10 results in Fig. 3A and B). This shows that cep3 could use nitrogen more efficiently under starvation conditions to sustain mitotic activity for a longer period. Unlike in the WT and CEP3 peptide treated plants, where meristems became permanently inactive, the cep3 meristems entered
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before nitrogen did, supporting the assertion that CEP3 affects nutrient use or allocation.
These nutrient depletion assays were then used to determine how quickly CEP3 peptide was able to elicit a reduction in mitotic activity. Root meristems were nutritionally induced into mitotic quiescence by withholding carbon before being reactivated by glucose addition and assayed using EdU assays. Plants were grown for 5 days in liquid ½ MS medium under minimal lighting to synchronise cells in the G0/G1 phase (Xiong et al., 2013). Glucose or glucose + 1 µM CEP3 was then added at various time points to gauge how quickly the number of cells entering S phase was reduced by CEP3 peptide addition (Fig. 4).
Figure 4. CEP3 peptide treatment reduces the number of root meristem cells in the S phase of the cell cycle within 12 h. Plants were grown in standard ½ MS liquid medium to synchronise meristematic cells. Glucose or 1 µM CEP3 + glucose was added at time 0 (after synchronisation) and plants were assayed at the specified times. ** p ≤ 0.01; *** p ≤ 0.001 (t-test, compared to no CEP treatment at the same time point). n = 5 roots per time point.
After 12 h of treatment, CEP3 had significantly reduced the number of cells undergoing DNA synthesis (Fig. 4). The number of cells in S-phase continued to drop, reducing to around 50% after 24 h. This indicated that CEP peptide was perceived and subsequent signal transduction elicited a demonstrable phenotype within 12 h. To determine if this response was similar under N- free conditions, a similar experiment was undertaken using N-free liquid medium (Fig. 5). Similar results were seen, with CEP3 significantly reducing the number of cell in S-phase after 12 hours.
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Figure 5. Under nitrogen starvation, CEP3 peptide treatment reduces the number of root meristem cells in the S phase of the cell cycle within 12 hours. Plants were grown in modified N-free liquid medium to synchronise meristematic cells. Glucose or 1 µM CEP3 +glucose was added at time 0 (after
synchronisation) and plants were assayed at the specified times. No CEP results presented are at 12 hours post glucose treatment. *** p ≤ 0.001 (t-test, compared to No CEP). n = 8 roots per time point.
It was clear from the nutrient depletion assays presented that increasing levels of CEP3 peptide affects root growth as a result of diminishing or inhibition cell cycle
progression and that diminishing CEP levels in cep3 has the opposite effect. Two likely scenarios may explain this: CEP could be affecting cell cycle directly or it could be altering nutrient allocation. As the N starvation assay produced a specific response to CEP3 perturbation in both the long (Fig. 3) and short term (Figs. 4 and 5), it was used to explore the downstream output of CEP3 in further detail using a transcriptomic
approach.