cell cycle

Abstract


Introduction
At the cellular level, plant growth is driven by cell proliferation and cell expansion.Cell proliferation, rather than cell expansion, determines the final size of organs, as shown by the meta-analysis performed by Gázquez and Beemster (2017).Abiotic stress often causes plants to grow at a slower rate by inhibiting cell division and expansion to varying degrees.For instance, under severe drought stress, maize leaf elongation rate was reduced by 63%, which could partially be explained by an increased cell cycle duration of 84% (Avramova et al., 2015a).Also, Kavanová et al. (2006) showed that phosphorus deficiency reduced leaf elongation rate by 39% due to decreases in the cell production rate and final cell length.In Arabidopsis, West et. al. (2004) showed that salt stress resulted in reduced growth of roots due to a decrease in cell production and mature cell size.
After cells have stopped proliferating, they grow in size, further increasing organ size.In roots, monocotyledonous leaves and hypocotyls, this elongation mainly occurs along the longitudinal axis due to the transverse orientation of the cellulose microfibrils (Green, 1962;Crowell et al., 2011).The increase in cell size is typically accompanied by endoreduplication (Sugimoto-Shirasu and Roberts, 2003).During endoreduplication, cells alternate between G1 and S-phases, skipping mitosis, doubling their genome with each completed S-phase (Sugimoto-Shirasu and Roberts, 2003).Endopolyploidy in plants can also be affected by abiotic stress, where plants typically increase endopolyploidy levels as an adaptive, plastic response to mitigate the effects of stress, as reviewed by Scholes and Paige (2015).
We use the maize leaf model system to study the impact of abiotic stress on organ growth because it allows to combine analyses at cellular, molecular and biochemical levels at high spatial resolution (Avramova et al., 2015b).Maize leaf growth is driven by linearly organized growth processes: cell division in the meristem (i.e. a pool of continuously dividing cells, occurring at the base of the leaf typically in the first 1 to 2 centimetres) and cell elongation in the elongation zone (occurring directly apical of the meristem and typically extending over 4 to 6 centimetres) (Avramova et al., 2015a).When cells have reached their mature cell length, they enter the mature zone and form the emerged part of the blade.The longitudinal separation of these developmental stages allows sampling of dividing and elongating cells from a single leaf (Nelissen et al., 2013).Moreover, the size of the maize leaf yields sufficient amounts of tissue for each of these developmental stages for biochemical and molecular analyses, making it an ideal plant system for these analyses (Avramova et al., 2015b).
Industrial activities and the use of phosphate fertilizers have caused cadmium (Cd) disposition and accumulation on large surfaces across the world (Nagajyoti et al., 2010).Though Cd is nonessential, plants take up this metal through transporters for essential bivalent cations such as calcium, iron and zinc (Verbruggen et al., 2009).Being a non-redox active metal, Cd may cause oxidative stress indirectly by perturbing the plants' reactive oxygen species (ROS) metabolism (e.g. by inhibiting enzymes which function in antioxidative defence mechanisms (Cuypers et al., 2010)).Despite the extensive antioxidant defence system of plants (Cuypers et al., 2012), Cd stress may inhibit growth by causing ROS induced DNA damage (Hendrix et al., 2018;Huybrechts et al., 2019), impaired cell wall metabolism (Loix et al., 2017), mitotic aberrations (Fusconi et al., 2007;Silva et al., 2013) and inhibited photosynthesis and respiration (Bi et al., 2009).
The impact of Cd on growth and more specifically the cell cycle is mostly studied in synchronised cell cultures and roots that are directly exposed to Cd treatments, as recently reviewed by Huybrechts et al. (2019).These studies mainly report a halted cell cycle at G1/S and G2/M transitions.However, studies on how Cd impacts the growth of plant organs that are not directly exposed, especially leaves, are limited.
Therefore, the aim of our research is to determine the mechanism(s) by which Cd inhibits leaf growth, using the maize leaf as a model system.Our 2 key research questions are: 1.Does Cd reach the leaf growth zone and hence directly affect dividing and elongating cells in the growing maize leaf and 2. What is the cellular basis of Cd inhibited leaf growth in maize (i.e.inhibition of cell division and/or cell elongation)?To tackle these research questions, we used a holistic approach, integrating data at the biochemical (i.e.mineral analysis), cellular (i.e.kinematic analysis and flow cytometry) and molecular level (i.e. gene expression analysis).
Through this approach we show that Cd accumulates in the division zone of the leaf, where it inhibits cell cycle progression.Cd deposition continues in the elongation zone, where cell elongation rates are reduced, possibly due to an inhibition of the endocycle.

Seeds, soil preparation and growth conditions
We grew maize plants (Zea mays L., B73 inbred line, obtained from the North Central Regional Plant Introduction Station) in a growth chamber under controlled conditions (16-h day/8-h night, 25°C/18°C day/night, 200 μmolm -2 s -1 photosynthetically active radiation, provided by high-pressure sodium lamps).
Peat potting medium (57% soil water content, Jiffy Products International B.V., The Netherlands) was spiked with 10 ml distilled water (control treatment) or 10 ml CdSO4 solutions (3CdSO48H2O, prepared in distilled water, Table 1).A fixed mass (650 grams) of potting medium was used for each individual pot (2.0L) to which the solutions were added dropwise under continuous mixing with a kitchen mixer (Kenwood kMix KMX50).Immediately after soil preparation, seeds were planted and the pots were placed in the growth room, covered with plastic wrap until germination.Pots were watered daily with tap water to maintain the original soil water content.

Dose-Response and treatment selection
We determined leaf elongation rate and final leaf length of the fifth leaf of plants exposed to 6 Cd concentrations and a control treatment.To this end, leaf length was measured daily with a ruler from its emergence from the whorl of older leaves until it reached maturity and stopped growing.Leaf elongation rate was determined using the first 4 leaf length measurements of each plant, when growth was approximately steady-state.
Based on the dose-response, 3 treatments were selected for use in the subsequent experiments: a control, a mild (46.5 mg Cd • kg -1 dry soil) and a severe treatment (372.1 mg Cd • kg -1 dry soil).At 24 days after sowing, plants subjected to these treatments show a clear difference in size (Supplementary Fig. S1).

Cadmium mineral analysis
We determined Cd concentrations in one-centimetre segments sampled along the maize leaf growth zone (i.e. 10 centimetres in total) and included a blade segment (middle of the remaining blade).Fresh weight of the sampled leaf segments was measured (AX124, Sartorius, Göttingen, Germany), after which they were oven-dried at 60 °C for 48 to 72 hours.
Hereafter, segments from the same position and treatment were pooled (2-3 segments per pool).Sample digestion was performed by an overnight predigestion in aqua regia (1:3 nitric acid and hydrochloric acid), followed by 20 minutes high pressure high temperature digestion (Discover SP-D, CEM, Matthews, NC, USA), allowing the samples to boil at 200 °C.The samples were then diluted 40 times with trace metal grade ultrapure water, after which the Cd concentration was measured with high resolution inductively coupled plasma mass spectrometry (Element XR, Thermo Scientific, Bremen, Germany).We used blanks to correct for background trace metals and Rye grass European Reference Material CD281 samples as a reference.

Kinematic analysis
We performed a kinematic analysis on the fifth leaf as described by Sprangers et al. (2016).
After the emergence of the fifth leaf from the whorl of older leaves, its length was measured daily with a ruler.Leaf elongation rate was determined using the first 3 leaf length measurements of each plant.Three days after emergence, 6 plants of each treatment (i.e.control, mild and severe, as determined in the dose-response experiment) were dissected for cell length and meristem size measurements, while the remaining plants (n = 4 to 5) were used to further measure growth until the final leaf length was reached.Cell length measurements (epidermal pavement cells directly adjacent to stomatal files) along the longitudinal axis of the leaf were performed on 1 centimetre sections that were fixed overnight in 70% ethanol and cleared, stored and mounted on slides in lactic acid.Cells were visualized using differential interference contrast microscopy (Zeiss Axio Scope.A1 microscope, Oberkochen, Germany) at 40x magnification and the length of abaxial epidermal cells adjacent to stomatal cell rows was determined using the online measurement module in the Axiovision software (Rel. 4.8,Zeiss).Leaf meristem size was determined using fluorescence microscopy of DAPI-stained (1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) staining solution) leaf sections at 20x magnification by locating the most distal mitotic figure in epidermal pavement cells.

Cadmium flux and deposition
To determine the uptake of Cd along the growth zone we calculated Cd deposition rates using a kinematic approach combining velocity profiles with Cd concentrations in function of position along the growth zone (Meiri et al., 1992).First, we determined cell flux, which is the number of cells passing by at a certain location per unit time.Cell flux outside the meristem was obtained by dividing leaf elongation rate by mature cell length.Inside the meristem, cell flux was set to zero at the base of the meristem, with a linear increase towards the end of the meristem, where cell flux equals the constant cell flux outside the meristem.Then, the velocity profile was obtained by multiplying local flux rates with local cell lengths and smoothed and interpolated using the locpoly function of the KernSmooth package according to Rymen et al. (2010).This fit also yields the derivative of the velocity profile that corresponds to local relative cell expansion rates.Finally, the velocity in the middle of each segment was multiplied by the Cd concentration of the same segment and corrected for segment length and number of plants in the pooled sample, yielding the Cd flux.To retain the variance in the velocity and Cd values from separate experiments, velocities from every replicate were multiplied with all corresponding Cd concentrations, yielding a minimum of 24 (6x4) combinations per treatment.Hereafter, the local rate of Cd deposition was obtained as the derivative of this Cd flux using the locpoly function of the KernSmooth package.

Flow cytometry
For each treatment (Table 1), we sampled 10 one-centimetre segments along the maize leaf growth zone (n = 6).Samples were processed as described before (Hendrix et al., 2018) using the CyStain PI Absolute P kit (Sysmex Partec).Using a CyFlow Cube 8 flow cytometer (Sysmex Partec), PI fluorescence intensity was determined using 488 nm excitation and 580 nm detection for a minimum of 7500 nuclei per sample.The number of 2C, 4C nuclei and S-phase nuclei were determined in R (v 3.6.1)using the flowCore package (v 1.50.0,Hahne et al. (2009) as described in supplementary Fig. S2).

Quantitative real-time PCR
We measured expression levels of 3 cell cycle genes: putative wee1-like protein kinase (further referred to as wee1), which controls S-phase progression in plants by phosphorylation of CDKs and arrests S-phase progression under DNA stress (Cools et al., 2011;Hu et al., 2016); mcm4, part of the prereplication complex that mediates unwinding the DNA during S-phase (Masai et al., 2010) and cyclin-B2-4, a member of the family of positive CDK regulators controlling G2-to-M transition (Scofield et al., 2014).Samples were obtained from the first 5 centimetres of the fifth leaf's growth zone, 3 days after emergence.These 5 centimetres were dissected in 6 half centimetre segments, followed by 2 one-centimetre segments in 3 biological replicates per treatment, each consisting of a pool of 4 plants.Sections were frozen in liquid nitrogen and stored at -80°C.We ground the leaf material with a ball mill grinder (Retsch MM400, Verder NV, Aartselaar, Belgium), using ceramic balls.Total RNA was extracted using the RNeasy Plant mini kit (Qiagen, Hilden, Germany) and diluted to 0.4 µgµL - 1 .First strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, Massachusetts, USA).The synthesised cDNA was used for quantitative real-time PCR using the SYBR Green Master Mix (Kaneka Eurogentec S.A., Seraing, Belgium).Expression values were normalised using Zm00001d036201 (hypothetical protein) as reference gene (Supplementary Table S1 for housekeeping gene selection (Lin et al., 2014)).Gene expressions values were calculated using the 2 -Cт method (Livak and Schmittgen, 2001), relative to the expression of the gene in the first segment of the control plants.Primers (Supplementary Table S2

Dose-Response
To determine the effect of Cd concentrations in the soil on the growth of maize leaves, we first performed a dose-response growth analysis.For studying the effect of abiotic stress on growth, we routinely study the 5 th leaf of maize seedlings because its growth is independent on seed reserves, approximately steady state for about 5 days after emergence and affected by environmental conditions (Avramova et al., 2017).
Leaf elongation rate (LER) was reduced by 25 to 57%, following a progressive, but non-linear decrease with increasing Cd concentrations (Fig. 1A).For all treatments the reduction in final leaf length was approximately half of that of the effect on LER (Fig. 1B), so that the highest dose only reduced final leaf length by 30%.The difference between the LER and final leaf length can be explained by a progressive increase of the duration of leaf elongation with increasing Cd levels, which partially compensates for the lower leaf elongation rate.Based on leaf elongation rate and final leaf length, we selected a mild (i.e.46.5 mg Cd  kg dry soil -1 ; inhibiting LER by 25%) and severe treatment (372.1 mg Cd  kg dry soil -1 ; inhibiting LER by 52%) for further detailed analyses (Table 1).

Cadmium accumulation
Next, we set out to determine whether the growth inhibition could be due to Cd accumulation in the leaf growth zone.Severe Cd stress significantly increased the dry to fresh weight ratio of the leaf material (Supplementary Fig. S3).On a fresh weight basis, Cd levels increased with increasing concentrations in the soil (treatment p < 0.001).On a dry weight basis, mild and severe Cd stress resulted in very similar values across the growth zone (Supplementary Fig. S4).However, in both cases Cd levels were highest at the base of the leaf, followed by a steep decline towards the mature tissues (segment p < 0.001; Fig. 2A).These findings indicate that dividing cells at the base of the leaf are exposed to higher amounts of Cd compared to later developmental stages.

Kinematic analysis
Cadmium accumulation in the leaf meristem suggested that, if the effect of Cd on leaf growth is caused by local accumulation in the growing tissues, cell division would be primarily responsible for the growth inhibition by Cd.To address this possibility, we performed a kinematic analysis to quantify the effects on cell division and cell elongation.We first determined the cell length profile for the epidermal cells directly adjacent to the stomatal files.In the first centimetre from the base of the leaf, cells were small and cell size decreased slightly, while their size steeply increased in the 2-4 centimetre region under severe Cd stress and in the 2-6 centimetre region under control conditions.Mature cell length was not affected by the treatments (Fig. 3A; Table 2).
The cell length data allowed us to calculate the velocity profile, which shows that the velocity at which cells move away from the base of the leaf gradually increases until it reaches a value equal to the leaf elongation rate at the end of the growth zone (Fig. 3B, Table 2).The derivative of the velocity curve yields relative cell expansion rates, which shows that increasing Cd levels progressively reduce the maximal expansion rates and the extent of the growth zone (Fig. 3C).
Because mature cell size is not affected, the decrease in leaf elongation rate (by 24 and 46% for mild and severe stress respectively in this experiment; p < 0.001; Table 2) was almost entirely caused by a reduced cell production rate (-21% and -43% for mild and severe stress, respectively; p < 0.001; Table 2).Cell production in turn, is determined by the number of dividing cells in the meristem and their cell division rate.Cadmium stress significantly reduced the number of cells in the meristem (by 17% in mild and 29% in severe stress; p < 0.001; Table 2) and cell division rate (by 5 and 19%, in mildly and severely stressed plants; p = 0.058; Table 2), which relates to an increased cell cycle duration (from 24 hours in control conditions to 26 and 30 hours in mild and severe stress, respectively; p = 0.0317; Table 2).Although mature cell length was not affected, the relative cell elongation rate was inhibited by Cd (by -13 and -33% for mild and severe stress, respectively; p < 0.001; Table 2).This reduction in cell elongation rate, however, was compensated for by an increased time cells spend in the elongation zone (12% and 45% for mildly and severely stressed plants, respectively, p < 0.001; Table 2).The reduced number of dividing cells was reflected by a significant decrease in the size of the meristem (p = 0.001; Table 2).As a consequence, the size of the growth zone as a whole decreased from 70 mm down to 64 and 59 mm for plants under mild and severe treatment, respectively (p = 0.054; Table 2).
In summary, Cd inhibits leaf growth primarily by reducing the meristem size and inhibiting cell division and expansion rates.

Cadmium flux and deposition
The decreasing Cd concentration with increasing distance from the leaf base (Fig. 2A) could be a consequence of dilution by cell growth, raising the possibility that all Cd is taken up by the dividing cells at the base of the leaf (Supplementary Fig. S5 illustrates Cd dilution by growth).To verify this possibility, we used kinematics to calculate Cd deposition rates along the leaf growth zone.
Based on Cd concentration and tissue velocity, we calculated Cd flux and deposition rates.
Cadmium flux, the bulk flow rate of Cd away from the leaf base, progressively increased in the first 6 to 7 centimetres, after which it became approximately constant (Fig. 2B) in both Cd treatments.Assuming steady state, the derivative of the flux curve yields the local rates of Cd deposition, which was highest at the base of the leaf where cells are actively dividing (Fig. 2C).
Towards the end of the growth zone, high velocity (Fig. 3B) in combination with only minor changes in Cd concentration, caused relatively large fluctuations in flux and even more in deposition rates.We consider this artifacts.Nevertheless, our data show that although Cd concentrations rapidly drop once cells leave the division zone, deposition continues in elongating cells and stops around the end of the elongation zone.

Flow cytometry
To analyse which phase of the cell cycle was affected by Cd, explaining the increased cell cycle duration (Table 2), and to assess if there was an effect on endoreduplication in expanding cells, we performed flow cytometry on one-centimetre sections along the leaf base.The fraction of 4C cells relative to cells with a 2C DNA content was highest in the second centimetre of the leaf (Fig. 4A), where cells exit the meristem (Table 2).After an initial drop, DNA contents increased towards the end of the elongation zone, suggesting a limited amount of endoreduplication (Fig. 4A).
Furthermore, this analysis suggests active proliferation in the first 3 centimetres of the leaf for all treatments.This result appears in contrast with our kinematic analysis that shows a meristem size of 1 to 1.5 centimetres for the severely stressed and control leaves, respectively.This difference may be due to flow cytometry being performed on a mix of all cell types, while kinematics is based on epidermal pavement cells.Nevertheless, the reduced meristem size is clearly reflected in the more rapid drop of the 4C/2C ratio in the Cd-treated leaves.Consistent with active proliferation at the base and limited endoreduplication in the elongation zone, cells in S-phase could be detected throughout the growth zone, with the highest levels in the second centimetre (Fig. 4B).
The 4C/2C ratio was reduced by severe Cd stress, whereas the mild treatment was very similar to the control treatment.Severe Cd stress reduced the fraction of S-phase cells throughout the growth zone, whereas at the leaf base mild stress was similar to the control treatment, but in the elongation zone resembled the severe stress.
In conclusion, the flow cytometry data support a reduced meristem size and a reduced 4C/2C ratio under Cd stress, suggesting an inhibition of the G1/S transition in both mitotic and endoreduplicating cells.

Quantitative real-time PCR
To better understand the molecular mechanism explaining the inhibition of cell division by Cd, we analysed the expression levels of 3 cell cycle regulatory genes: wee1, mcm4 and cyclin-B2-4.The overall expression pattern of these genes reflected the distribution of cell division activity and the inhibition by Cd (Table 2), with the highest expression levels around 1 centimetre from the base (Fig. 5).Severe Cd stress reduced the expression of these cell cycle regulators throughout the growth zone and caused a more rapid drop between 1.5 and 3.5 centimetres from the base, reflecting the reduced cell division rate and shortening of the meristem (Table 2), respectively.The response to mild stress was similar to the severe stress in the basal centimetre, whereas in more distal positions it appeared similar to the control condition.

Discussion
In order to answer our first research question, whether Cd could directly affect dividing and elongating cells in the growing maize leaf, we determined the Cd levels along the gradient of cell division and expansion at the base of the leaf into the mature blade tissue.Cd concentrations were highest at the base of the leaf, rapidly declined with increasing distance from the base and stabilised at around the 5 th centimetre (Fig. 2A).This closely relates to our kinematics data, showing that the growth inhibition exerted by Cd is primarily caused by a reduced cell production in the meristem, located in the base of the leaf.Noteworthy, these observations demonstrate that whole leaf sampling, typically used to evaluate leaf Cd concentrations (e.g.Khaliq et al., 2019;Masood et al., 2016;Nada et al., 2007;Shi et al., 2019;Ye et al., 2018;Zhou et al., 2018), underestimates the concentration in dividing and elongating cells .
Using kinematics, we were able to calculate Cd fluxes and Cd deposition rates.One possibility to account for the high levels of Cd in the meristem and their rapid decline in the elongation zone (Fig. 2A) could be that Cd is specifically deposited at the base of the leaf and diluted by cell expansion in the elongation zone.Under these circumstances, Cd flux in the elongation zone should remain constant, because the dilution of Cd and the increase in cellular velocity due to water uptake are directly proportional (Supplementary Fig. S5).However, we observed a steady increase in Cd flux until at least the 4 th centimetre (Fig. 2B), demonstrating that Cd deposition continues in the elongation zone.Cadmium deposition rates in the elongation zone are lower than those of water driving cell expansion, explaining the decreasing Cd concentrations from the leaf base towards the blade (Fig. 2A).Thus, while deposition rates are highest in the meristem of the growing maize leaf, Cd continues to be deposited while cells are expanding (Fig. 2C), suggesting that in elongating cells Cd is (passively) taken up with the influx of water required to drive cell growth.Interestingly, plants exposed to a mild Cd dose have a higher Cd flux compared to severely stressed plants (Fig. 2B), even though concentrations are higher in leaves exposed to the highest concentration (Fig. 2A).This is because both segment fresh weight and velocity are higher under mild stress compared to severe stress, resulting in more tissue passing per unit time.When the flux is expressed on a fresh weight basis, compensating for the amount of tissue passing by (Supplementary Fig. S6), severely stressed plants have a slightly higher Cd flux.
The past decade, Cd deposition in the meristem received attention in the shoot of eudicotyledonous and Graminae plants using a positron-emitting tracer imaging system together with positron-emitting Cd to trace the translocation and accumulation of Cd throughout the plant.This technique also showed that in rice Cd already accumulated at the base of the leaf after 1 hour of tracer exposure, whereafter the signal also increased in the rest of the sheet and in the blade (Fujimaki et al., 2010;Kobayashi et al., 2013).Radioactive Cd deposition was also studied in Arabidopsis thaliana, where Dauthieu et. al. (2009) showed that Cd was deposited throughout young leaves and that the zone of deposition retracted towards the base and petiole in older leaves.Young dicotyledonous leaves first consist entirely out of dividing cells, after which a cell cycle arrest front appears at the tip of the growing leaf which moves towards the petiole (Andriankaja et al., 2012).Therefore, the pattern of Cd deposition in these leaves also broadly coincides with cell proliferation.In addition to leaves, predominant accumulation of Cd in the meristem also occurs in roots of rice (Zhao et al., 2013;Zhan et al., 2017).Taken together, these results indicate that, during growth, Cd is mainly deposited and accumulated in dividing and elongating tissue.
Dividing and elongating tissue, acting as a Cd sink, is supported by the study performed by Kobayashi et al. (2013) on rice seedlings.They showed that the xylem transpiration stream facilitates Cd transport from the roots towards the shoot.However, once Cd reaches the base of the stem, it is loaded into the phloem at the nodes and mainly directed towards the young growing leaves.In the new leaves, Cd preferentially accumulated in the sheath (i.e.where the growth zone resides), whereas calcium was spread throughout the growing leaf.
To address our second research question, the cellular basis of Cd inhibited leaf growth in maize, we analysed the contribution of cell division and elongation to the growth inhibition by Cd.Our results indicate that Cd inhibited leaf growth by inhibiting cell production by up to 43 percent, while mature cell length remained largely unaffected.This is consistent with the meta-analysis performed by Gázquez and Beemster (2017), who showed that variations in meristematic cell number, rather than mature cell size, primarily determine organ size in plants.For Graminae leaves, they also showed that mature cell length is strictly controlled and does not contribute significantly to changes in leaf elongation rates, which matches the unaffected mature cell length in our analysis.
The main cause of a lower cell production rate in our study was a reduction in number of meristematic cells, resulting in shortening of the meristem size by up to 26 %.This reduced meristem size is consistent with Cd-induced meristem size reductions in roots of wheat, pea and Arabidopsis (Fusconi et al., 2007;Pena et al., 2012;Yuan and Huang, 2016;Bruno et al., 2017).Although we confirmed the reduction in meristem in 3 independent experiments (i.e. kinematics study, quantitative real-time PCR of cell cycle genes and a flow cytometry study), there was discrepancy in the apparent meristem sizes.Based on our kinematics results data, meristem sizes ranged from 1 to 1.5 centimetres for severe to control condition respectively (Table 2), whereas cell cycle gene expression patterns suggested it to be considerably longer (up to 2.5 centimetres under control conditions when interpreting cyclin-B2-4 expression data (Fig. 5)).In the flow cytometry results, the 4C/2C minimum at the meristem-elongation transition is reached 1 centimetre later by the control treatment (Fig. 4A), suggesting that cells are still dividing in the 2-to-3 centimetre segment under control conditions.This discrepancy between datasets can be related to the cell type studied by the different methodologies.In the kinematic analysis, epidermal pavement cells are studied, whereas in gene expression and flow cytometry study, whole leaf segments containing all cell types were used.Tardieu et al. (2000) showed that mesophyll cells can divide twice as long as epidermal cells, which could explain why the techniques which incorporate all cell types (i.e.quantitative real-time PCR and flow cytometry) result in longer meristems compared to kinematic analysis, which is based only on epidermal pavement cells.Nevertheless, all data consistently showed that Cd reduces maize leaf meristem size.
Besides a significant reduction in meristem cell number, cell cycle duration also increased from 24 hours under control conditions to 30 hours under severe stress conditions.This means that cells divided at a lower rate because they were halted at some point(s) in the cell division cycle.Inhibited cell cycle progression under Cd stress has previously been reported mainly in roots and synchronised cell culture experiments.In roots of Arabidopsis thaliana, Cd inhibited the cell cycle mainly at the G2/M transition, resulting in a relative increase in 4C nucleic content at the cost of 2C nuclei (Cui et al., 2017;Cao et al., 2018).No significant effect of Cd on the proportion of cells in the S-phase was reported by Cao et al. (2018).However, detrimental effects of Cd on the S-phase were shown in synchronised plant cell cultures, where Cd administration during S-phase delayed the mitosis by 2 hours in tobacco cells (Kuthanova et al., 2008) and Cd administration at the start of the cell cycle decreased the DNA-synthesis rate in soybean cells (Sobkowiak and Deckert, 2004).Also, in root apices of peas (Pisum sativum L.), Cd affected meristematic cells in the G1/S and G2/M transition, resulting in respectively less cells in the S-and M-phase.Inhibition of S-phase entry was also shown in a study on Cd stress in leaves of lettuce (Monteiro et al., 2012).Though insignificant, Monteiro et al. (2012) showed an increase in percentage of G0/G1 cells, followed by a decrease in cells in the S-phase and G2-phase when grown under mild to severe Cd conditions (respectively 10 and 50 µM Cd).Cd-inhibited G1/S transition is consistent with our flow cytometry data in the meristem of the growing maize leaf, where we show a lower proportion of cells in the S-phase, together with an accumulation of cells with a 2C nucleic content (Fig. 4).
In order to better understand why cells were progressing slower through the cell cycle, we selected 3 key cell cycle genes, i.e. wee1 and mcm4 which have a function during the S-phase and cyclin-B2-4, a B-type cyclin controlling G2/M transition.wee1, a kinase of which transcript abundance peaks during S-phase progression (Cools et al., 2011), controls cell cycle arrest upon DNA damage and is also important for meristem maintenance during replication stress (Hu et al., 2016).Since Cd is linked to DNA damage in multiple studies (as reviewed by Huybrechts et al., 2019), we expected wee1 transcript levels to increase under Cd stress.
Surprisingly, under severe Cd stress, expression levels of wee1 were consistently lower compared to the control treatment over the entire meristem (Fig. 5).However, these results do reflect those of Cao et al. (2018) and Cui et al. (2017), who also found wee1 downregulation under Cd stress in roots of Arabidopsis after 5 days of Cd exposure.Only low amounts of Cd caused a significant upregulation of wee1 transcription (Cui et al., 2017;Cao et al., 2018).
We also found similar expression profiles for mcm4 (helicase activity) and cyclin-B2-4 (controlling G2/M transition), i.e. lower expression under severe Cd stress over the entire meristem compared to the control condition.Downregulation of B-type cyclins was also shown in the experiments of Cao et al. (2018) and Cui et al. (2017) and also in soybean suspension-culture cells, Cd reduced in cyclin-B1 transcription (Sobkowiak and Deckert, 2003).Next, in Cd exposed shoots of wheat (Triticum urartu), 2 and 5 day Cd exposure reduced expression levels of multiple MCMs (Qiao et al., 2019), whereas 48 hour exposure to the same Cd dose decreased mcm2 transcript levels in roots of wheat seedlings (Pena et al., 2012).Downregulation of cell cycle-related genes by Cd seems to be common, as this was also supported by findings of Zhao et al. (2013) who reported that 12 out of 17 cell cyclerelated genes had severely reduced transcript levels in Cd exposed rice roots.
Taken together, exposure to of Cd appears to stops cells from entering the cell cycle (i.e. inhibited G1/S transition), which is supported by the lower proportion of cells in S-phase and with the 4C nuclei content found in our study.With less cells entering the cell cycle, transcript levels of cell cycle-related genes could be relatively less abundant.We therefore hypothesize that under severe Cd stress, cells are hindered in entering the cell cycle in general, which could lead to an overall downregulation of most cell cycle genes.
Next, although mature cell length was unaffected, Cd significantly reduced relative cell elongation rate under severe stress.Nevertheless, cells did achieve the same mature cell length due to an increased time spent in the elongation zone.The inhibited cell elongation rate could be related to lower endopolyploidy levels in the elongation zone under severe stress, since DNA content is often linked to cell growth (Melaragno et al., 1993;Sugimoto-Shirasu and Roberts, 2003) (Fig. 4).Based on our kinematics (Table 2) and cell cycle gene expression analysis (Fig. 5), we do not expect any cell division to occur further than 3 centimetres from the base of the leaf.Yet we do see a steady increase in 4C nuclei after this position, indicating a limited amount of endoreduplication to be present in the elongation zone.The endoreduplication process was negatively affected by our severe stress condition, where the 4C/2C ratio under severe stress stayed well below the one under control conditions over the entire elongation zone.However, the difference between control and severe Cd treatments on the 4C/2C ratio is quite constant from 1 to 7 centimetres, which could indicate that the process of endoreduplication itself is not really hampered, but the difference is there because a lower proportion of 4C nuclei was already present in the meristematic region under severe Cd stress.This difference in 4C/2C ratio is then retained throughout the elongation zone while the process of endoreduplication takes place at similar rates as in controls.
Because of the potential link between polyploidy level and cell growth, a reduced DNA content could negatively impact the process of cell elongation in the Cd exposed maize leaf growth zone.Similar to our results, Hendrix et al. (2018) related a decreased cell surface area to a lower extent of endoreduplication in leaves of Cd exposed Arabidopsis.However, in roots of Pisum sativum and Arabidopsis thaliana, Cd exposure resulted in increased polyploidy levels (Fusconi et al., 2006;Repetto et al., 2007;Cui et al., 2017;Cao et al., 2018).Therefore, in a recent review by Huybrechts et al. (2019), it was suggested that Cd exposure stimulates the endocycle in roots and inhibits it in leaves.
Lastly, it is remarkable that an eight-fold difference in Cd dose between mild and severe treatments resulted in a limited difference in Cd accumulation throughout the growth zone while the effects on growth, cellular and molecular processes were quite apparent.The relatively small differences in accumulation could potentially be explained by a saturated uptake and/or transport, to which the mild stress conditions might already get close.Related to this saturated uptake, Huang et al. (2019) have shown that Cd uptake in rice increased steeply under incremental low Cd concentrations, yet, at higher concentrations, Cd uptake was levelling off when Cd concentrations further increased.It is not clear how a relatively small difference in Cd accumulation (a maximal difference of 40% in the meristem between mild and severe stress, t-test p-value: 0.11) could result in drastic differences in growth response.Perhaps, a very tight threshold level is exceeded under severe stress conditions, where the plant is still able to cope with the mild treatment and succumbs under severe stress.Passing the threshold level might result in a different subcellular distribution, affecting more and potentially important processes.Also, the impact of Cd on roots was not studied in the research presented here.It is very well possible that, in addition to the effects of locally accumulating Cd in the leaf, signals originating from the roots inhibit leaf growth.Therefore, further research should be undertaken to explore whether potential long-distance signals and potential threshold levels of metabolic and regulatory processes become affected.Comparing the mild and severe Cd treatments may provide an interesting entry into this issue.

Conclusion
Our primary objective was to understand how Cd uptake by the roots inhibits leaf growth in maize.We found that Cd inhibits leaf growth through a reduction of the meristematic cell number and by impairing the cell cycle at the G1/S transition resulting in an increased cell cycle duration.In addition, Cd inhibited cell elongation, which might be related to lower ploidy levels under severe Cd stress.We also showed that Cd predominantly accumulates in the meristem and that deposition of Cd continues at lower rates throughout the elongation zone, which implies direct impact of Cd on the cell cycle and cell expansion in the maize leaf growth zone.
This study opens perspectives to further investigate the impact of Cd on the physiology of the leaf growth zone of a monocotyledonous leaf.We have shown in this study and earlier (Avramova et al., 2015b) that the maize leaf model allows sampling at subzonal resolution for a wide range of analyses.This will allow us to determine how and to what extent changes in micro-and macronutrient levels, phytohormone profiles, energy metabolism, cell wall metabolism, etc. in the leaf growth zone further explain the regulatory mechanisms by which Cd inhibits leaf growth.

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