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Research strategy and dedicated experimental setup
Research strategy
We chose an approach combining experimental field-like phenotyping and model-assisted phenotyping. N-use efficiency is usually measured as a crop balance at harvest: specifically, the ratio of seed yield to soil N available. The effects of low-N availability are quantified at the canopy scale at maturity, but a better understanding of the underlying NUE processes at the plant scale, as well as their modulation by N deficiency, is needed. Furthermore, considering the high plasticity of oilseed rape development in response to the climatic conditions, and compensatory capacities of the organs between the vegetative and the reproductive phase, the plant-scale processes deserve to be studied under field conditions or, at least, under field-like reconstructed canopies throughout the crop growth cycle. We therefore choose an adapted culture device called PERISCOPE, (Bissuel-Belaygue et al., 2015) ensuring field-like conditions in terms of shoot interactions between plants and root-explored soil volume, and procuring access to the individual root system of each plant from sowing to harvest. In our experiments, the plant response to N availability was investigated by evaluating NUE-related functional traits over the crop cycle (i.e. total shoot and root dry matter and C and N content) and integrative traits such as seed yield and NUE, assessed at the end of the crop cycle. Several intermediate destructive measurements allowed us to accurately characterize NUE dynamics, assessing how the individual developmental processes led to the final seed yield and NUE at harvest.
For the model-assisted phenotyping approach, we proposed a conceptual framework describing winter oilseed rape C and N functioning, including all shoot and root compartments. It was designed from the model called ARNICA developed for Arabidopsis thaliana, (Richard-Molard et al., 2009) (Chapter II. Figure II.11). The proposed conceptual framework was then used for the trait analysis, as it offers an explicit and dynamic description of whole-plant growth, allowing us to identify and hierarchize the main processes supporting the observed genotypic variation of biomass elaboration in response to N conditions, and therefore of NUE if considering the ratio of produced plant biomass to N-available. All the model state variables were chosen to be measurable on the plants grown on the PERISCOPE device. Modeling was only used here as a framework for analyzing and prioritizing traits likely to be phenotyped.
However, measuring those traits required destructive measurements throughout the crop cycle, including time-consuming measurements of root traits. Hence, depending on the experiment, we acquired those variables on a limited number of genotypes across different N supplies. We investigated a total of seven genotypes of winter oilseed rape (Brassica napus L.) in three different experiments, combining a maximum of three contrasting N supplies. The genotypes were chosen to represent winter oilseed rape diversity as they were released between 1980 and 2004 and represent ancient ‘++’ (high glucosinolates and high erucic acid contents) or modern ‘00’ types (low glucosinolates and low erucic acid). The genotypes were chosen from a large panel, previously phenotyped in the field, for their contrasting seed yield responses to N input levels (Bouchet et al., 2016).
Figure I.3. Experimental device used for phenotyping N-use-efficiency-related traits during the whole crop cycle. Winter oilseed rape plants were grown under field-like conditions in a reconstructed canopy system (PERISCOPE device) allowing individual root and shoot measurements. Plants were grown in individual tubes 1 m high and 0.16 m in diameter, grouped into containers of 1 m3, placed outside and therefore submitted to field climate (A). Each tube was filled with substrate and regularly supplied with nutrient solution. In containers, the space between tubes was filled with soil to ensure the thermal insulation of root parts (B). Two rows of border plants were sown around the tubes to mimic bioclimatic field conditions. Six seeds were sown in each tube (B). After thinning, a single plant was kept per column, leading to a homogeneous canopy of 35 plants/m2 (C, aerial view of one container), which were grown for the whole crop cycle until harvest (D, vegetative growth; E, flowering; F, seed filling and ripening). Photos correspond to the experiment conducted in Grignon in the 2017–2018 cropping season (GR18).
Experimental setup
Three experiments were conducted at two different sites and over two climatic years, in Le Rheu from 2014–2015 and in Grignon from 2014–2015 and 2017–2018, hereafter referred to as LR15, GR15, and GR18, respectively. The LR15 and GR15 experiments were conducted before I began my Ph.D., whereas I personally designed and conducted the GR18 experiment. Experiments were conducted using the PERISCOPE device (Bissuel-Belaygue et al., 2015). An overview of the experiments and a description of their specific characteristics and modalities similarities are presented in Figure I.2. More detailed descriptions of the growing conditions and trait measurements are given in Chapter III and Chapter IV. The experiments performed are complementary in terms of genotypes tested and sampling stages (Figure I.2), and used a common methodology for data measurements. We were careful to compare genotypes with contrasting seed yield in the field but similar growth-cycle durations and dates of flowering (no more than 8 days between the two extreme genotypes) to minimize confounding effects between phenology and NUE processes. The genotype AVISO was tested as a control in all experiments.
Plants were grown in tubes (Figure I.3) and grouped into containers of 1 m3 to obtain a reconstructed canopy with a density of 35 plants m-², commonly used in Europe (Terres Inovia, 2019). In containers, the space between tubes was filled with soil to ensure the thermal insulation of the root system. In addition, to avoid edge effects, two rows of plants were sown on each edge of the container. We sowed six seeds in each column: after thinning, only one medium-sized plant remained in each tube. We harvested plants at multiple growing stages throughout the crop cycle. Sampling dates depended on the experiment but altogether result in a comprehensive dataset on the growing dynamics (Figure I.2). To simulate constant low- and high-N conditions during the crop-growth cycle, a mineral solution was supplied every 200 growing degree days from emergence to seed maturity, resulting in 13 to 14 applications during the growth cycle. Moreover, we maintained the soil moisture above 85% of field capacity, thus avoiding other uncontrolled stress (i.e. water stress and nutrient loss through leaching).
At each sampling date, we divided harvested plants into fractions: taproots, fine roots, leaves (green, senescing, and fallen), main stem, branch stems, and pods (including immature seeds or, when dehiscent, seeds and pod walls). Shoot traits (i.e. stem diameter, plant height, leaf number, number of branches, and pod and seed number) and root traits (i.e. secondary root number and tap root depth and length) were measured manually (Figure I.4). Dry matter and C and N content of the different plant fractions were measured, and the green leaf area and pod area were assessed. Soil horizons were sampled for N-mineral characterization and soil moisture quantification. To characterize the crop N nutritional status, we used the nitrogen nutrition index (NNI), calculated using the equation developed by Lemaire and Gastal (1997) (Chapter II, Figure II.1). The NNI was measured on three key phenological stages, common to all experiments: at the beginning of rosette growth in autumn (BBCH 16-19), during stem elongation in spring (BBCH 30-32), and at the end of inflorescence emergence, immediately before flowering (BBCH 59). The characteristics of the N conditions generated, including mean values of N supplies, mineral N initially present in the substrate, and NNI, are presented in Chapter IV (Table IV.2).
Using the PERISCOPE device, all individual plant fractions were collected, including fine roots and fallen leaves of each plant, until harvest. Five key growing stages are presented: BBCH 16–19, autumnal growth; BBCH 32, beginning of stem elongation after overwintering; BBCH 59, end of the vegetative growth immediately before flowering; and BBCH 68, end of flowering and beginning of pod development. Photos correspond to the cv. AVISO growing under low-N conditions in the experiment conducted in Grignon in the 2017–2018 cropping season (GR18). Scale colored bands correspond to 10 cm.
The methodological specificities and main objectives of each experiment are described below.
LR15 experiment (6 genotypes x 2 N supplies): This experiment was intended to characterize the genotypic variation in six winter oilseed rape growth and NUE at four key stages in response to two contrasting N conditions. The experiment was conducted at the INRA research station in Le Rheu (LR), located in Brittany, France (latitude 48°06’29.0″N; longitude 1°47’37.3″W), during the 2014–2015 cropping season. Six contrasting genotypes winter oilseed rape genotypes (cv. AMBER, ASTRID, AVISO, EXPRESS, MOHICAN, and MONTEGO) for seed yield, biomass accumulation, and amount of N absorbed in field conditions, were cultivated under two N supplies (N-limiting: equivalent to 25 kg N ha-1; non-N-limiting: equivalent to 165 kg N ha-1). Genotypes were experimented using a split-plot design allowing five repetitions (genotype x N supply). Each tube was filled with a soil-sand mixed substrate and was regularly supplied with nitrogen-free Hoagland nutrient solution to prevent water and other mineral stresses, except N. Nitrogen solution was supplied 13 times over the cycle, at intervals of approximately 200 growing degree days. Plants were harvested on four sampling dates during the crop cycle, including three intermediate samplings at the growing stages—BBCH 18 (autumnal growth), BBCH 31 (beginning of stem elongation), and BBCH 68 (end of flowering and beginning of pod development)—and a final sampling at seed maturity (Figure I.2). These are key development stages determining changes in whole-plant C-N balance, as reported in the literature.
GR15 experiment (1 genotype x 3 N supplies): This experiment was intended to characterize in detail the dynamic growth of a single genotype (cv. AVISO) in response to three contrasting N conditions (equivalent to 29, 58, and 175 kg N ha-1). The experiment was conducted at the INRA research station in Thiverval-Grignon (GR), France (latitude 48°50’21.7″N; longitude 1°56’48.4″E) during the 2014–2015 cropping season. Nitrogen solution was supplied 14 times over the cycle. Each column was filled using a mixture of attapulgite and clay pebbles and regularly supplied with Hoagland solution. Plants were experimented using a complete block design allowing six repetitions. Plants were harvested on seven sampling dates during the crop cycle, including five samplings during the vegetative growth (BBCH 19, 20, 21, 30, and 59), one sampling at the end of flowering (BBCH 71), and one sampling at seed maturity.
GR18 experiment (5 genotypes x 1 N supply + 1 genotype x 2 N supplies): This experiment was intended to accurately characterize shoot and root growth throughout the vegetative phase of five oilseed rape genotypes (cv. AMBER, AVISO, EXPRESS, MOHICAN, and OLESKI) growing under low N supply. The experiment was conducted at the INRA research station in Thiverval-Grignon during the 2017–2018 cropping season. Plants were grown under a single N limiting supply (equivalent to 29 kg N ha-1), except for AVISO, which was additionally cultivated under a non-limiting N supply (equivalent to 200 kg N ha-1). Nitrogen solution was supplied 14 times over the cycle. Each column was filled using the same substrate than in GR15 and regularly supplied with Hoagland solution. Plants were experimented using an incomplete block design allowing seven to eight repetitions per genotype. Plants were harvested on four sampling dates during vegetative growth (BBCH 16, 19, 32, and 59), and one additional sampling was performed at harvest to quantify seed yield. In addition to the other measured plant traits described above for other experiments, the leaf area of the fallen leaves was measured.
Table of contents :
GENERAL INTRODUCTION
I. Societal issues
I.1. Importance and utilization of winter oilseed rape
I.2. Nitrogen fertilization and environmental impacts of oilseed rape
II. Scientific issues
II.1. Understanding of the carbon and nitrogen functioning and its genotypic variability
II.2. Phenotyping oilseed rape response to N-availability
CHAPTER I. PRESENTATION OF THE Ph.D. THESIS
1.1. From research issues to a Ph.D. objective
1.2. Research strategy and dedicated experimental setup
1.2.1. Research strategy
1.2.2. Experimental setup
1.2.2.1. LR15 experiment
1.2.2.2. GR15 experiment
1.2.2.3. GR18 experiment
CHAPTER II. BIBLIOGRAPHIC REVIEW
2.1.Several definitions exist for N use efficiencies and related parameters
2.2.Processes related to nitrogen use efficiency in oilseed rape
2.2.1. The oilseed rape plant: development, growth and N-metabolism
2.2.1.1. Vegetative growth
2.2.1.2. Reproductive phase
2.2.2. Interaction between C-N metabolism and between shoot-root compartments
2.3.N-availability has major role on oilseed rape functioning
2.3.1. Oilseed rape N-requirements
2.3.2. Impact of N-limitation in NUE-related processes
2.3.3. Impact of N-limitation on plant traits at canopy level
2.3.4. Impact of N-limitation on plant traits at the plant level
2.4.Genetic diversity of NUE-related processes and associated traits
2.4.1. Genetic variability of NUE-related processess
2.4.2. Genetic variability of NUE-associated traits
2.4.3. Genotype x Nitrogen interaction
2.5. Phenotyping for N-use efficiency related traits in oilseed rape
2.5.1. Phenotyping for NUpE-related traits requires adapted devices
2.5.2. A model-assisted phenotyping approach
CHAPTER III. Nitrogen Uptake Efficiency, mediated by fine root growth, early determines variations in Nitrogen Use Efficiency of rapseed
1. Introduction
2. Materials and methods
2.1. Plant Material
2.2. Experimental design
2.3. Climate conditions
2.4. Management of hydric and mineral conditions
2.5. Sampling and measurements
2.6. Variables calculated
2.7. Component-contribution analysis
2.8. Statistical analysis
3. Results
3.1. Relating NUE_Seed to NUE_DM at seed maturity and at earlier stages
3.2. Dynamic contribution of NUpE and NUtE to NUE_DM
3.3. Genotypic variation in NUE_DM and its components
3.4. Deciphering genotypic variation in NUpE-related processes
4. Discussion
CHAPTER IV. Which efficiencies explain oilseed rape genotypic variations in biomass accumulation and partitioning under low N-availability?..
1. Introduction
2. Material and methods
2.1. Site description and plant material
2.2. Experimental design
2.3. Nitrogen management
2.4. Sampling and measurements
2.5. State variables calculation, cumulative variable estimation, and global indicators.
2.6. Statistical analysis
3. Results
3.1. The winter oilseed rape conceptual modeling framework
3.2. Quantitative analysis of the CN dynamics of fallen leaves, main stem and fine root system
3.3. Estimation fo the model parameters during rosette growth period
3.4. Validity to the conceptual framework during the whole vegetative growth
3.5. Could we extend the conceptual modeling framework up to the seed filling period?
3.6. How did the model parameters vary according to N-condition?
3.7. How did the model parameters vary with genotype under the low-N condition ?
4. Discussion
CHAPTER V. GENERAL DISCUSSION AND PROSPECTS
5.1. Whole-plant dry matter NUE: a key variable for deciphering the dynamics of genotype response to nitrogen availability
5.2. N uptake efficiency: the neglected side of N use-efficiency worth addressing in depth
5.3. Lighting the dark side of winter oilseed rape: why fine roots matter in N-use efficiency
5.4. Think globally, act locally
5.5. Toward growing oilseed rape for sustainable agriculture production
