LATERAL ROOT GROWTH PATTERN IN MAIZE 

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Components of phenotypic variation: Phenotypic plasticity and developmental instability

The ability of a plant root system to acquire soil resources depends on its architecture, i.e. the spatial location and structure of the root axes within the soil (Fitter et al., 1991; Pagès et al., 2013a) and on its uptake properties, referring to the ability of each root segment to take up resources from soil (Clarkson, 1985).
The reasons why root architecture is important for resource uptake efficiency are multiple.
First, resources are not uniformly distributed in the soil and cannot be reached with equal facility by the root system. For instance, phosphorus is more present in upper soil layers while water can be both present at depth through water table or at the surface following rains and nitrate is more associated with nitrification patches (Hodge, 2004). Moreover, nitrate can move rapidly in the soil thanks to its high diffusion coefficient while phosphate (the common form of inorganic phosphorus in soils) is poorly diffusive and demands that roots come very close to it to absorb (Nye and Tinker 1977; Fitter et al., 2002). Second, root systems are costly in terms of carbon assimilates. Root mass is classically 1/10 to 1/2 of total plant mass (Gregory et al., 1997) and it is estimated that every gram of C present in the root mass has to be accompanied by 0.2 to 1 g of respired C (Nielsen et al., 1994). Any investment into the root system is at the expense of other part of the plant, in particular reproductive organs and yield. Thus, optimal resource uptake efficiencies would be achieved by absorbing the necessary resources with as little root mass as possible.
Root system architecture is known to change in situations where plants are challenged by the external or internal environment (e.g. nutrient availability (Zhang and Forde, 2000) or carbon status (Farrar and Jones, 1986)), a response that could affect the fitness of the plant in a resource-limited environment (Fitter et al., 1991). The modulation of root architecture in response to the environment is usually termed ‘phenotypic plasticity’ and is recognized to be a valuable adaptive trait (Crick and Grime, 1987; Drew, 1975; Giehl et al., 2014; Hodge, 2004, 2006). Since the root system is responsible for the acquisition of a large number of mineral resources, being able to adjust to their space-varying availability in the soil is a guarantee of survival for the plant. For instance, barley plants grown in a soil with a heterogeneous supply of phosphorus or nitrate showed a preferential development of their roots in rich areas (Figure I-3). This way, the plant is likely to obtain a given amount of the resource with a lower “cost” than if the root system had developed with no spatial preference.
Phenotypic plasticity also has a genetic component because the response of an individual to the environment depends on its genotype (Forde, 2009).
However, there is an additional component of phenotypic variations that cannot be related to environmental or genetic causes. Maybe the clearest manifestation of this phenomenon is the large variation of lateral root lengths observed in genetically identical plants, showing up to 10-fold differences for neighboring roots, even in homogeneous conditions. These apparently unpredictable variations in root growth trajectories have been described in a diverse range of species either annuals or perennials, dicots or monocots (Freixes et al., 2002; Pagès, 1995).
Unpredictable phenotypic variations also appear in other stages of root development such as lateral root initiation and growth duration, and have a significant impact on the final architecture of the root system.
These variations in root development have first been referred as ‘developmental instability’ (Forde, 2009; Mather, 1953) and have often been neglected or treated as an unwanted source of ‘noise’ in studies of root development. Yet, this so-called instability is an important component when building a root system including root system models, where it is impossible to achieve realistic shapes without introducing a stochastic component in root angles or growth rates of lateral roots (Pagès, 2011), as illustrates Figure I-4. Hereafter, we will use the term ‘stochastic’ as synonymous of ‘unpredictable’, as it is employed in (Forde, 2009) to designate a process that cause a developing trait to deviate from its expected path under a given genotypic and environmental conditions.
Figure I-3 Illustration of root environmental plasticity. (A) Control plants of barley (HHH) received a high nutrient solution to all parts of the root system. (B, C) The other plants (HLH) received the high nutrient solution only in the middle zone, the top and the bottom being supplied with a solution deficient in the specified nutrient. Adapted from (Drew, 1975).
Figure I-4 Illustration of root developmental instability. Simulated root systems obtained by varying a
parameter controlling the variance of the distribution of lateral root diameters, linearly related to growth rates in the model described in (Pagès, 2011). Example in (A) has a low variance (V=0.5) compared to example in (B) (V=3). Adapted from (Pagès, 2011).

Root developmental instability as a foraging strategy to optimize efficiency of resource uptake

The importance of developmental instability for the function of the root system is still poorly understood. The principal function of a root system is to acquire resources in a heterogeneous soil environment, with an unforeseeable distribution in both space and time. Since it is impossible for the growing root system to know beforehand where the resources are located, it is necessary to sample the soil for the detection of resource-rich patches: a foraging strategy is therefore required (Forde, 2009). Just as the random-walk strategy used by ant colonies searching for food patches, an indiscriminate exploration of the soil appears to be the preferred strategy to locate the unseen target. Once it is found, appropriate responses can be adopted for its exploitation, such as root proliferation in the rich zone for roots, or “telling others” by lying down pheromones in the case of ants (Forde, 2009).
The benefits obtained by exploring more soil volume must be balanced by the metabolic costs associated with the construction and maintenance of new root segments. The total cost is essentially depending on the root mass, approximately proportional to the root volume (when root tissue density is constant) (Pagès, 2014). An efficient exploration strategy should consequently explore a volume of soil with as little total root mass as possible. In this respect, it has been shown that variations in growth among lateral roots contribute to improve the efficiency of soil exploration. Lateral roots of variable lengths allow exploring a larger volume of soil than if the same cumulative root length had been produced in a deterministic way. A major reason for that is the minimization of the overlap between rhizosphere volumes of root axes so that they do not compete for the same resources (Pagès, 2011). It thus appears that some instability in root development is required for an efficient exploration of the soil.
The existence of an “optimal” degree of instability, and the mechanisms at its origin are still to be determined.

Origins of root developmental instability

Developmental instability can manifest at different stages of lateral root development: (i) the initiation of the lateral root primordium, (ii) the development of this primordium (from initiation to emergence) and (iii) the elongation of the lateral root. This section aims at identifying the specific events where experimental evidence of developmental variations exists by an analysis of the existing literature on lateral root development (especially on Arabidopsis plant model) and to eventually propose mechanisms that could be at its origin.

Initiation of lateral root primordia

Lateral roots typically arise from pairs of pericycle founder cells (Malamy, 2005). In the radial plane, only pericycle cells adjacent to protoxylem poles can become lateral root founder cells. However, founder cell fate affects only a limited number of these cells, and their exact location in the vertical axis is difficult to predict. The analysis of lateral root spacing in Arabidopsis revealed 25-fold variations in the distance between successive founder cells for the col-0 accession (Dubrovsky et al., 2006), showing no regular pattern in lateral root spacing for this species. Significantly, no correlation was observed between this distance and the growth rate of adjacent lateral roots (Dubrovsky et al., 2006), suggesting that lateral root spacing have no influence on lateral root growth. The timing between two initiation events was also highly variable, ranging from 2 to 14 hours in the same experiment, indicating that neither the time elapsed from the preceding lateral root initiation nor between-lateral root distance were determinant for specifying the site of new initiations.
Despite the highly variable behavior of lateral root initiation, a certain level of structuration could be identified. For instance, the average distance between lateral roots appears to be constant for each Arabidopsis accession, suggesting a genetic component in the regulation of root branching (Dubrovsky et al., 2006; Forde, 2009). The average between-lateral distance was also found to be significantly dependent on the species in the study of (Pagès, 2014), consisting of an analysis of the branching pattern in a large panel of dicotyledonous species.
In addition, the variation in parental root structure (e.g. stele diameter and number of protoxylem poles) seemed to affect the average density of lateral roots in Banana (Draye, 2002), indicating some predictability of the branching pattern in function of the structure of the parent root. Adding a layer of complexity, the branching pattern has also been observed to be correlated with the internal nutritional status of the plant (e.g. carbon availability of the primary root (Freixes et al., 2002)) and to respond locally to a variety of external stimuli (Forde and Lorenzo, 2001).
At the cellular level, the initiation of lateral root primordia has been proposed to be regulated by local auxin maxima. The DR5 auxin reporter expression shows roughly periodic peaks in the protoxylem cells along the elongation zone of the primary root (in a region called the ‘oscillation zone’) that have been proposed to provide the competence to the adjacent pericycle cells to become founder cells of lateral root primordia (Moreno-Risueno et al., 2010; De Smet et al., 2007). Remarkably, the frequency of DR5 signal followed a Gaussian distribution with a mean period similar to that of lateral root initiation sites (Laskowski, 2013; Moreno-Risueno et al., 2010). Thus, the pattern of DR5 expression appears to evidence a determinant role of auxin in the selection of founder cells and the definition of the sites of future lateral roots, at least in the Arabidopsis plant model.
Remarkably, the cellular patterns of lateral root initiation can substantially vary between individual primordia. Typically, two longitudinally adjacent pericycle cells undergo a first asymmetric division giving rise to two central short cells flanked by two longer cells (Lucas et al., 2013). The resulting figure is referred as the longitudinal bi-cellular type of lateral root initiation (Dubrovsky et al., 2000), illustrated in Figure I-5A. However, there is evidence that a longitudinal uni-cellular type of lateral root initiation also occur (even if rarely) in Arabidopsis (Dubrovsky et al., 2000); see Figure I-5B. Concerning the number of adjacent pericycle files involved in the formation of the lateral root primordium (i.e. those in direct contact with the protoxylem), it can range from one to three (see Figure I-5C for illustration), indicating that two nascent lateral organs may already differ in the number of pericycle cells recruited both longitudinally and radially to form the original group of founder cells. It is possible that cellular differences in the perception or sensitivity to auxin are at the origin of the different patterns of lateral root initiation.
Figure I-5 Longitudinal and radial variants in the number of pericycle founder cells involved in lateral root initiation (LRI) in Arabidopsis. (A) The longitudinal bi-cellular type of LRI is characterized by synchronous asymmetrical cell divisions in two adjacent cells of the same file. (B) The longitudinal unicellular type of LRI occurs when only one pericycle cell becomes a founder cell for the entire longitudinal extent of the primordium. (A, B) Arrowheads indicate end cell walls of pericycle founder cells (convex); arrows indicate position of cell walls resulting from the anticlinal division of founder cells (not-convex). (C) The number of pericycle files that are in direct contact with the protoxylem (asterisks) can be one (not shown), two, or three. pp protophloem, px protoxylem. Bars= 20 μm (A, B), 10 μm (C). Adapted from (Dubrovsky et al., 2001).

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Development of lateral root primordia

The morphogenesis of lateral root primordia has been extensively studied in Arabidopsis (Dubrovsky et al., 2001; Lucas et al., 2013). After founder cell specification, a coordinated sequence of periclinal and anticlinal divisions follows leading to the formation of a small dome-shaped organ. The activation of the cell division program seems to be genetically separable from the acquisition of founder cell identity (Dubrovsky et al., 2008).
Several developmental stages (from I to VII) are defined in function of the increasing number of cell layers of the lateral root primordium (Malamy and Benfey, 1997). Interestingly, the pattern of cell divisions in lateral root primordium development is not unique as revealed by the variable number of cells among different primordia at the same stage (Lucas et al., 2013), showed in Figure I-6. The overlap observed in the ranges of cell number between two consecutive developmental stages strongly suggests that cell divisions in lateral root primordia do not follow a stereotypical sequence.
Figure I-6 The pattern of cell divisions in lateral root primordium (LRP) development is not stereotypical. (A) Number of cells in LRP median slices as a function of the developmental stage. Overlaps between stages are highlighted in red. (B) Developmental paths of two distinct LRP (red or blue dots). Adapted from (Lucas et al., 2013).
Although the shape of the lateral root primordium in this study was considered as highly regular (Lucas et al., 2013), significant deformations have been found quite often in Arabidopsis plants (19% of 756 primordia analysed) such as a lack of symmetry in relation to the primordium axis or a flattened surface of the dome (Szymanowska-Pulka, 2013).
Lastly, developing lateral root primordia must pass through several parental tissues to emerge. However, not all primordia reach this step (Dubrovsky et al., 2006; Lucas et al., 2008). The emergence of the lateral root primordium requires a coordinated separation of outward, adjacent cell layers to minimize the damage of parental tissues (Péret et al., 2009).
Remarkably, abnormal shapes reported in the study of Szymanowska-Pulka (2013) were usually observed in primordia that had not emerged outside the parent root surface, suggesting that both the shape and emergence of the lateral root primordium is affected by the overlying tissues of the parent root (Lucas et al., 2013; Szymanowska-Pulka, 2013). The coordination between the progression and emergence of the lateral root primordium was confirmed in lax3 Arabidopsis mutant, where the inhibition of lateral root primordium emergence was accompanied by an increased proportion of stage I primordia (Swarup et al., 2008). This mutant failed to express several cell-wall remodeling enzymes necessary for the loosening and separation of overlying tissues during lateral root primordium emergence.
Lateral root primordium morphogenesis therefore appears to be orchestrated by mechanical signals between the developing organ and the parental tissues.
The idea has already been put forward that auxin could play a determinant role in lateral root primordium morphogenesis, supported by the observation of aberrant root morphologies in a number of auxin-related Arabidopsis mutants (Szymanowska-Pulka, 2013). Combined with recent experimental evidence of auxin distribution being sensitive to mechanical stresses (Nakayama et al., 2012), these findings evoke a mechano-induced, auxin-regulated primordium development.

Table of contents :

CHAPTER I. INTRODUCTION 
1 WHICH ROLE FOR ROOTS IN GLOBAL FOOD PRODUCTION?
1.1 Challenges for global agriculture
1.2 Roots for a second Green Revolution
2 ROOT SYSTEMS AND THEIR PHENOTYPIC VARIATION
2.1 Components of phenotypic variation: Phenotypic plasticity and developmental instability
2.2 Root developmental instability as a foraging strategy to optimize efficiency of resource uptake
2.3 Origins of root developmental instability
2.3.1 Initiation of lateral root primordia
2.3.2 Development of lateral root primordia
2.3.3 Elongation of lateral roots
2.4 Signaling clues involved in lateral root development
2.5 Modelling root growth variations
2.5.1 Various modelling approaches of the root growth variations
2.5.2 Towards a spatio-temporal analysis of root growth and root system architecture
3 OBJECTIVES OF THIS THESIS
AUTHORS CONTRIBUTIONS
ACKNOWLEDGEMENTS
CHAPTER REFERENCES
CHAPTER II. LATERAL ROOT GROWTH PATTERN IN MAIZE 
1 SPATIO-TEMPORAL ANALYSIS OF EARLY ROOT SYSTEM DEVELOPMENT IN TWO CEREALS, PEARL MILLET AND MAIZE, REVEALS THREE TYPES OF LATERAL ROOTS AND A STATIONARY RANDOM BRANCHING PATTERN ALONG THE PRIMARY ROOT
AUTHORS CONTRIBUTIONS
ABSTRACT
1.1 INTRODUCTION
1.2 RESULTS
1.2.1 Model-based clustering of lateral root growth rate profiles reveals three growth patterns for pearl millet and maize lateral roots
1.2.2 Comparison of apical diameter profiles and growth rate profiles for the three classes of lateral roots identified in maize
1.2.3 Linking root growth profile with root anatomy
1.2.4 Analyzing the primary root branching pattern
1.3 DISCUSSION
1.3.1 An original methodology to classify lateral roots
1.3.2 Origin and roles for the three lateral root types
1.3.3 Positioning of the three lateral root classes is random along the primary root
1.3.4 Extending the longitudinal modeling framework for studying the whole growth profile of type A lateral roots
1.3.5 A new look at lateral roots in future high-throughput phenotyping analyses?
1.4 MATERIALS AND METHODS
1.4.1 Experimental
1.4.2 Imaging and image processing
1.4.3 Image analysis
1.4.4 Correction of growth rate profiles
1.4.5 Model description
1.4.6 Root anatomy
2 ANALYZING THE MODULATION OF THE LATERAL ROOT GROWTH PATTERN IN DIFFERENT CONTEXTS
2.1 METHODS
2.1.1 Description of rhizotron experiments and associated post-harvesting analyses
2.1.2 Aeroponic experiment in collaboration with UCL
2.2 RESULTS
2.2.1 Model-based analysis of the influence of treatments on lateral root growth rate profiles and apical diameters
2.2.2 Comparison between treatments
CHAPTER REFERENCES
APPENDIX AND SUPPLEMENTARY MATERIAL
SUPPLEMENTARY REFERENCES
CHAPTER III. IDENTIFYING DEVELOPMENTAL ZONES IN MAIZE LATERAL ROOT CELL LENGTH PROFILES  AUTHORS 
ABSTRACT
1 INTRODUCTION
2 MATERIAL AND METHODS
2.1 Plant material, growth conditions and lateral root apex harvest
2.2 Image analysis and acquisition of lateral root cell length profiles and morphological properties
2.3 Multiple change-point models for identifying development zones in lateral root cell length profiles
2.3.1 Definition of heteroscedastic piecewise Gaussian linear models and Gaussian change in the variance models
2.3.2 Illustration of the application of multiple change-point models on selected maize lateral root apices
3 RESULTS
3.1 Selection of the number of developmental zones
3.2 Discontinuity of the selected piecewise linear functions
3.3 The limits between developmental zones is explained both by a change in slope and in residual standard deviation
3.4 Consistency of the EZ-MZ limit with the first root hair position
3.5 A strong modulation of the developmental pattern was observed among lateral roots
3.6 Choice of the variables summarizing lateral root development for the metaanalysis
3.7 Exploration of the diversity of lateral roots using principal components analysis
4 DISCUSSION
4.1 Successive developmental zones in the root apex are well characterized by piecewise linear functions
4.2 Interpretations of the changes in residual standard deviation at the limit between developmental zones
4.3 Comparison between segmented regression models and multiple change-points models
5 CONCLUSION
ACKNOWLEDGEMENTS
AUTHOR CONTRIBUTIONS
CHAPTER REFERENCES
APPENDIX AND SUPPLEMENTARY MATERIAL
SUPPLEMENTARY REFERENCES
CHAPTER IV. EXPLORING THE INTRINSIC ORIGIN OF GROWTH VARIATIONS IN MAIZE LATERAL ROOTS 
1 METHODS
1.1 Observation of lateral root primordia
1.2 Root anatomy
1.2.1 Plant material
1.2.2 Root cross-sectioning
1.2.3 Image acquisition and processing
1.2.4 Root measurements
1.3 Epidermal cell length profiles
1.4 Expert labelling of lateral roots: the A-B-C classification
1.5 Sugar content
1.5.1 Root sampling
1.5.2 Sugar content quantification
1.6 Gene expression
1.6.1 Total RNA extraction
1.6.2 Quantitative real-time PCR (qRT-PCR) analysis
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
2.1.2 Longitudinal window for the initiation of lateral root primordia
2.1.3 Relationship between inter-lateral organ distance and lateral root primordium development
2.1.4 Lateral root emergence
2.1.5 Summary
2.2 Anatomical lateral root structure
2.2.1 Root labeling and growth profiles associated to anatomical
2.2.2 Root anatomical features are tightly correlated, but much less to root elongation
2.2.3 Longitudinal variations in lateral root anatomy
2.2.4 Relationship between longitudinal variations in root anatomy and root elongation
2.2.5 Summary
2.3 Epidermal cell length pattern in the growing zone of lateral roots
2.3.1 Root labeling
2.3.2 Analysis of longitudinal cell length profiles reveals a large range of lengths of the growing zone
2.3.3 Summary
2.4 Glucose concentration and its distribution in lateral root apices
2.4.1 Glucose concentration
2.4.2 Longitudinal gradient in glucose concentration
2.4.3 Summary
2.5 Gene expression in lateral root apices
2.5.1 Gene description
2.5.2 Gene expression pattern associated to lateral root classes
2.5.3 Summary
CHAPTER REFERENCES
CHAPTER V. GENERAL DISCUSSION AND PERSPECTIVES 
1 LATERAL ROOT GROWTH VARIATIONS: STRUCTURED IN TIME, RANDOM IN SPACE 204
2 EXPLORING THE ORIGIN OF LATERAL ROOT TYPES
2.1 Lateral root fate is only partially determined before root emergence
2.2 Lateral root growth variations are related to the cellular pattern in root apices
2.3 Carbohydrate supply emerges as an important factor for the determination of root growth variations
3 TOWARDS ROOT SYSTEM BREEDING THROUGH MULTI-SCALE PHENOTYPING
CHAPTER REFERENCES 

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