6) Apical samples, contained pooled root tips (8-10 mm) of the same expert root class

(see section 1.4). Longitudinal samples, contained pools of 3 mm lateral root segments from the apex up to 30 mm in the shootward direction. The number of pooled roots depended on the sample type but in all cases aimed at reaching 10 mg per sample, thus ~ 10 lateral roots for apical samples and 30 lateral roots for longitudinal samples.

Figure IV-6. Schema describing the two types of samples collected for sugar analysis. Left: Pooling of

roots for apical samples. One sample contained about ten pooled 10 mm root apical segments. Right: Pooling of roots for longitudinal samples. One sample contained about thirty pooled 3 mm root segments. Indicative scale-bar: 10 mm.

1.5.2 Sugar content quantification

Sugar content was measured using spectrophotometric analysis of the soluble fraction of ethanol-water extracts as described by (Cross et al., 2006). In all cases, the sucrose content was very close to the detection limit and was thus omitted. Fructose was present, at lower concentration than glucose and most often in a 1:2 proportion of glucose. In the results, we only considered glucose for simplicity and because its measurement was more accurate.

1.6 Gene expression

1.6.1 Total RNA extraction

Apical (8-10 mm long) lateral root samples from each expert class (see 1.4 section) were pooled (~ 10 mg FW) and stored under -80°C. Pooled roots were then ground in liquid nitrogen and RNA was extracted using TRIzol reagent (ThermoFisher) using a modified protocol, and purified using RNase-Free DNase Set (Qiagen) according to the manufacturer’s protocol.

1.6.2 Quantitative real-time PCR (qRT-PCR) analysis

Quantitative real-time PCR was performed in a LightCycler 480 (Roche) using the gene- specific oligonucleotides specified in Table IV-1. The cDNA for qRT-PCR analysis was synthesized from 1 μg total RNA using Moloney murine leukemia virus reverse transcriptase (Promega) and oligo(dT)15 (Promega) primers. Each qRT-PCR reaction contained 1 μl cDNA sample and 9 μl LightCycler 480 SYBR Green I master kit for PCR (Roche). PCR conditions were as follows: 10 min at 95°C followed by 45 cycles of 5 s at 95°C, 7 s at 65°C, and 8 s at 72°C; the melting curve cycle was 10 s at 95°C, 30 s at 73°C, 0.11°C s-1 increase to 95°C, and then 10 s at 40°C. The primer efficiency of each pair of oligonucleotides was calculated using the following dilution series: 1/5, 1/10, 1/20 and 1/100. The relative expression levels of the transcripts were calculated with reference to the housekeeping gene eukaryotic initiation factor 4 (ZmEIF-4).

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Table IV-1 List of oligonucleotide primers used for qRT-PCR experiments and their corresponding PCR

2 Results of multi-scale analysis

2.1 Early lateral root development: analysis of the variations in lateral

root primordium development

2.1.1 Longitudinal development of lateral root primordia

Development of lateral root primordia was observed on cleared primary root segments of 15- d plants imaged after Schiff staining (see section 1.1 for a detailed description of methods).

The position of each detectable primordium and lateral root within the rootward 20 cm of the primary root was recorded. To describe more accurately the development of lateral root primordia, the primary root was divided into three zones (Figure IV-8). Zone 1 was defined as the root segment from the root tip to the youngest primordium detected (closest to the root tip). This zone is supposed to contain the primordia at stages under the limit of detection of our method. Zone 2 extended from the youngest visible lateral root primordium to the most rootward emerged lateral root (usually around 0.4 mm long). Zone 3 was defined as the root segment shootward from the first emerged lateral root. This zone was considered a part of the branched zone) containing all emerged lateral roots. In contrast, zones 1 and 2, containing exclusively non-emerged primordia, actually correspond to the unbranched zone).

In order to estimate the development of primordia in terms of change in size, from their initiation to their emergence, we looked at the profile of primordium basal diameters within zone 2 (Figure IV-7). Since we did not perform any temporal tracking of lateral root primordium development, it is mostly a conjectural analysis, but it seems reasonable to assume that spatial trends contain some information on the developmental history of lateral root primordia. From preliminary observations, the basal diameter appeared to increase linearly with the distance to root tip, which was well confirmed by the linear regressions estimated separately on each plant (Table IV-2). The only exception was plant C1 for which the R2 was particularly low. This plant presented a 90° bend at the root extremity. We thus chose to discard this plant for further analyses. Average rate of diameter increase with distance was 14 µm cm-1 (n=3). The linear model could account for 50% of total variance only (R2 ranging from 0.42 to 0.5), in accordance with a high dispersion of lateral root primordia diameters within this zone.

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Figure IV-8. Zones defined along the rootward 20 cm region of the maize primary root for the analysis of

LRP development. (A) Zone 1 extends from the primary root tip (green arrowhead) to the to the youngest primordium detected (blue arrowhead). Zone 2 extends from this point to the earliest emerged lateral root imaged visible in imaged roots (red arrowhead). Both Zone 1 and 2 form the unbranched zone (UBZ) of the root. Zone 3 situates shootward to the first emerged lateral root, considered a part of the branched zone (BZ). Note the presence of a mix of lateral root primordia and LRs within this zone. (B) Zoom on each of the 5-cm segments used to build the composite image in A. Scale-bar: 1 cm.

Figure IV-7. Longitudinal

profile of basal diameters of lateral roots primordia (LRPs) and emerged lateral roots (LRs) along the rootward 20 cm of the primary root in a control plant (C4). Lines indicate the 50% (solid), 10% (dashed bottomost) and 90% (dashed uppermost) quantile; estimated by the linear quantile regression method. Zones are the ones defined in Figure IV-8.

We used these linear models to extrapolate the behaviour of a canonical lateral root primordium from its early initiation (considered to occur nearly at the root apex (Dubrovsky et al., 2000, 2006) to its emergence and exit of the zone of lateral root primordium development. Considering the average intercept of linear models, the size of a primordium at its initiation is predicted to be 88 µm. The earliest emerged lateral root in wild-type plants was found at an average distance of 13.22 cm from the root tip, which we can consider to be the average unbranched zone length . At this distance, the predicted diameter value for an emerging primordium was 271 µm. Based on the average unbranched zone length and on the average rate of root growth between days 14 and 15 (4 cm day-1), the residence time of an average lateral root primordium would be approximately 3.3 days. The canonical developmental profile of a primodium in our growing conditions would then consist of a gradual 3-fold increase in diameter from its initiation at the root tip to its emergence a bit more than 3 days later.

However, this is just a simplistic view as it clearly exists a high degree of individual variability in the development of lateral root primordia. Indeed, if we look at the spread of diameter values for a given plant, as the one shown in Figure IV-7, it suggests) marked differences in the rate of development among individual primordia. In order to estimate the lower and higher rates of lateral root primordia development, we applied the quantile regression method to the lateral root primordium basal diameter as a function of the distance to the root tip (package ‘quantreg’ in R software (Koenker, 2015)), using respectively the 0.1 and 0.9 quantiles (Table IV-2). We found respective quantile slopes of 7.82 and 18.82 µm cm-1 and a median slope (0.5 quantile) of 10.46 µm cm-1. The 0.1-quantile regression line (44% inferior to the median rate) suggests that there exists slowly developing lateral root primordia while other fast growing lateral root primordia could well develop following the upper trend (30% greater to the median rate). Another clue conforting this view comes from the qualitative comparison of the distribution of organ diameters in Zones 2 and 3 (Figure