Evolution of lignin distribution at tissue level all along the internode development 

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In vivo or in vitro estimation of digestibility.

Fodder is normally characterized by its dietary value, which is expressed by the potential for milk production in dairy cows or weight gain in bull calves. However, this type of measurement on animals with high potential and on their performance zootechniques is not feasible routinely due to the heaviness of experiments. They can only be used to estimate the dietary value of some genotypes and validate the evaluation criteria that can be used routinely. In vivo, the most widely used tool used as a reference for estimating the energy value of corn is the standard sheep in a cage. In routine, it is mainly used in vitro in vitro digestibility estimation techniques where enzymatic cocktails (amylases, pepsins and a mixture of cellulases and hemicellulases) mimic digestion by ruminants (Aufrère and Michalet-Doreau, 1983, Lila et al., 1986, De Boever et al., 1988, Ronsin, 1990). The so-called Aufrère method is the one that refers in the world of maize silage selection to estimate the in vitro digestibility of the dry matter. Work developed over the past 60 years has also highlighted the strong relationship between the energy value of forage and the degradability of the walls (Aufrère et al., 2007). These in vitro estimates account for about 60% of the genetic variability observed in vivo (Argillier et al., 1998).
Degradability is the main target for improving energy value at the moment, and Near InfraRed Spectroscopy (NIRS) prediction equations are being developed to predict this character in broadband. The degradability of plants is very much limited by the degradability of the walls surrounding each cell. The walls consist of polysaccharides (highly degradable: cellulose and hemicelluloses) and phenolic compounds (non-degradable and limiting the degradability of polysaccharides in the parietal assembly: lignin and ferulic and p-coumaric acids). These walls are therefore at the center of targets for improving degradability.

Criteria for registration in the French official catalog and impact on varietal selection

In the 1950s, the goal of the official French catalog was to facilitate responses to post-war challenges to ensure food security. Improving productivity has therefore been the major objective of this era. Before the 1980s, the corn market was a grain market divided into six precocity groups corresponding to the agro-climatic zones. The « fodder » market appeared with the creation of varieties with high dry matter production of the whole plant and in 1985 the creation of a specific section of the official catalog amplified this segmentation. In fifty years forage maize has seen an incredible rise. Since 1986, entries in the official French catalog are made in « corn grain » or « corn fodder » (Surault et al., 2005). The varieties listed in « fodder » are evaluated for their traditional agronomic values (yield, precocity, and stem behavior) but also, since 1998, for their qualitative value via the consideration of their energy value (UFL). The goal was clearly to promote the UFL / kg MS energy concentration of the whole plant in the breeding objectives. Indeed, from 1985 to 2000, genetic progress was remarkable in productivity. Nevertheless, the value of food has made little progress because of the priority given to improving grain yield and agronomic qualities. Improving the energy value remains an important point to consider in breeding programs. For illustration, an improvement of 0.05 point of UFL allows the production of 1.8 kg of supplementary milk per day and per cow consuming a ration of 16 kg of MS (Barrière and Emile, 2000) or 600 kg per cow and per year, representative corn hybrids from the last 50 years of breeding, Baldy et al. (2017) showed that, up to 2012, the UFL value fell before stabilizing and starting to recover in the last 5 years (Figure 4.2). It was therefore necessary to wait 15 years for selection efforts on silage quality to be visible on the silage market. Strong differences exist between marketed hybrids and a further increase in UFL values is always possible by targeting the right traits to improve this quality without penalizing agronomic qualities.

Impacts of the modifications of the lignin biosynthesis pathway

Modification of monolignol metabolism has a strong impact on plant growth. It has been shown in arabidopsis mutants for c3’h, hct and cse (genes encoding for key enzymes in the monolignol biosynthesis pathway) which present a dwarf phenotype (Bonawitz and Chapple, 2010; Vanholme et al., 2012). These phenotypes were mainly explain by a great reduction in the lignin content. However, in 2015 Anderson et al., showed in Arabidopsis disrupted for genes encoding cinnamyl alcohol dehydrogenases that the dwarf phenotype was not associated to a reduction of the lignin content. Indeed, in these mutants, the lignin content did not differ from the wild type but the amounts of hydroxycinnamaldehyde monomer incorporated did differ. This demonstrates that the composition and structure of lignin is also very important for the mechanical properties of the cell wall, not only the lignin content.
More recently, mutants for cinnamoyl CoA reductase which leads to cell wall deficient in lignin content was shown to be associated to a decrease of the proportion of oriented cellulose fibrils in Arabidopsis (Liu et al., 2016). The modification of the orientation and the order of cellulose fibrils was observed in all tissues of the stem and it suggests that the impacts on the lignin biosynthesis pathway have direct consequences on the structure of other cell walls components.

Methods to investigate the tissue specificities

I chose to present actual methods of staining and imaging to quantify and investigate further the distribution of the lignification in tissues through the second part of the accepted chapter in Chimie Verte et IAA (Ed. Stéphanie Baumberger – Lavoisier) : « L i age ie, u outil puissant pour étudier les variabilités de répartition et de composition des tissus de la biomasse lignocellulosique – Pe spe ti es da s l a lio atio de la d g ada ilit des pa ois hez le aïs » by Valérie Méchin, Matthieu Reymond, David Legland, Mathieu Fanuel, Fadi El Hage, Aurélie Baldy, Yves Griveau, Marie-Pierre Jacquemot, Sylvie Coursol, Marie-Françoise Devaux, Hélène Rogniaux, and Fabienne Guillon. As far as phenolic constituents are concerned, all studies show that lignin content is the first factor that is negatively correlated with the degradability of the walls (Jung and Deetz 1993, Méchin et al., 2000 and Méchin et al., 2005). Lignins limit the degradability of the walls both by their bonds with the polysaccharides within the wall but also by their hydrophobic properties, absorbing and preventing the progression of cellulolytic enzymes. However, a drastic reduction in lignin levels can not be considered since this decrease also leads to a significant loss of agronomic performance of plants such as height, resistance to lodging and pest attacks. For this reason, several groups have focused their research on other factors besides the lignin content to improve the degradability of plants. Our work has repeatedly emphasized the strong impact of lignin structure and the amount of p-coumaric acid on the limitation of wall degradability (Méchin et al., 2000, Mechin et al., 2005; et al., 2011, Mechin et al., 2014, El Hage et al., 2018). The ferulic acid content is also a factor that can limit the degradability of the walls. As regards polysaccharides, the presence of acetyl groups or side chains on hemicelluloses negatively affect the release of sugars. It is the same with the level of crystallinity of cellulose.
In this chapter, we have chosen not to detail the impact of biochemical factors on degradability variations. The literature is already very rich in this respect and we decided to focus on the impact of the distribution of lignified tissues within the stems on the degradability of the biomass. This distribution varies according to the genotype, the development stage of the plant and the hydric conditions of culture, and it is an important lever to modulate the degradability of the walls. It is therefore crucial to develop powerful analytical tools to characterize biomass and update the parameters to be selected to improve degradability or to allow significant resilience to plants in the current context of global warming.

Imaging techniques

According to the representative scale of the structures of interest, various image acquisition devices can be used to study their morphology, their spatial organization or their chemical composition (Rousseau et al., 2015). Historically, microscopy was the technique used to study plant anatomy at the cellular or tissue level. Macroscopic image acquisition systems have also been developed. They allow to quickly observe surfaces of the order of the cm2 with a minimum sample preparation, which allows to observe large series. Confocal microscopy has emerged as a means of volumetric investigation of samples with a gain in axial resolution and minimal preparation.
For imaging at the cell wall or organelle level within cells, electron microscopy has often been the method of choice, reaching nanoscale resolutions. The 3D structure can also be explored, either by combining scanning electron microscopy with the serial section of the sample (Bhawana et al., 2014), or by adapting the tomography algorithms to transmission electron microscopy. Magnetic resonance imaging (MRI) and X-ray tomography are popular methods for the non-destructive study of the 3D architecture of biological samples, without requiring staining, sectioning or inclusion. The high resolution obtained by tomodensitometry (below the micron) is often the best method for the study of plant organs (Stuppy et al., 2003, Cloetens et al., 2006, Dhondt et al., 2010). We present briefly some techniques commonly used in our laboratories.

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Darkfield microscopy

Darkfield microscopy is a simple method that makes non-colored and transparent objects visible. It is used to visualize small objects such as bacteria or in the case of plant tissues, cell structures. In this mode, the central light rays along the optical axis of the microscope, which usually pass through and around the sample are blocked. Only oblique rays from large angles strike the sample. They are diffracted, reflected and / or refracted by optical discontinuities, such as the cell membrane, nucleus and internal organelles, allowing these rays to reach the objective. A macrovision device based on this principle was developed at INRA in Nantes to observe plant tissues (Devaux et al., 2009) (Figure 18.1). The speed of sample preparation and acquisition makes it possible to image large series on which image analysis processes can then be applied to extract morphological parameters such as cell size, beam size and density. at the scale of the observed section and compare different genotypes on morphological criteria (Legland et al., 2012; Legland et al., 2014).

Brightfield microscopy

Brightfield microscopy is very often the first technique used for histological studies. The sample is illuminated by transmission. The structures are made visible by their local differences in light transmission factors. The biological structures are naturally little contrasted. Contrast is obtained with the use of dyes that selectively interact with chemical functions of the molecules (Spence, 2001). In Table 3.1, some staining techniques used on lignocellulosic substrates are indicated.

Table of contents :

General introduction
I. The maize plant
a) Back to origins
b) Anatomy of the maize plant
c) Maize crop production and valorization
1. The digestibility of corn silage, degradability of the walls seen from the side of the cow
i. In vivo or in vitro estimation of digestibility.
ii. Criteria for registration in the French official catalog and impact on varietal selection
2. Bioethanol production from maize crop residues, degradability seen from the EZ process side
i. Biomass and 2nd generation biorefineries
ii. Pretreatment of lignocellulosic biomass for bioethanol production
1- Chemical pretreatments
2- Physicochemical pretreatments
II. Cell wall in grasses
a) Structure
b) Polysaccharidic compounds
1. Cellulose
2. Hemicelluloses
3. Pectins
c) Phenolic compounds
1. Lignins
i. Composition of the lignins
ii. Impacts of the modifications of the lignin biosynthesis pathway
2. p-hydroxycinnamic acids
i. Ferulic acids
ii. p-coumaric acids
III. Distribution of the lignification within the maize stem tissues
a) Tissue anatomy in maize
b) Methods to investigate the tissue specificities
1. Imaging techniques
i. Darkfield microscopy
ii. Brightfield microscopy
2. Conclusion
IV. Cell wall establishment during maize stem development.
a) Genetic variation for internode elongation, cross section surface and cell size within the three genotypes throughout internode development
b) The main differences for internode elongation and cell wall phenolic composition were positioned at young stages, before silking
c) Different developmental patterns were observed for lignin content, composition, structure and distribution
1. Evolution of lignin content all along the internode development
2. Evolution of lignin structure and composition all along development
3. Evolution of lignin distribution at tissue level all along the internode development
d) Throughout internode growth different developmental patterns were observed for phydroxycinnamic acids accumulation
1. Evolution of esterified p-coumaric acid content all along internode development
e) Esterified ferulic acids were deposed all along plant development but used to anchor lignification only at early stages
f) Combination of the biochemical and histological findings to propose a model of spatiotemporal cell wall development
V. Relationships between cell wall degradability and 1- biochemical cell wall components and 2- the distribution of lignification.
a) Impact of the cell wall biochemistry on the cell wall degradability
b) Impact of the distribution of the lignification on cell wall degradability
VI. Impact of water stress on cell wall composition and on lignification distribution
VII. Genetic determinism of the cell wall traits under different watering conditions.
a) QTLs of cell wall components
b) QTLs of tissue distribution
c) QTLs under different watering conditions
VIII. Objectives of my PhD
Chapter 1 Introduction
Material and Methods
Histological analyses.
(Accepted Article) Histological quantification of maize stem sections from FASGA-stained images.
Dedicated NIRS predictive equations establishment and NIRS predictions of cell wall related traits in maize internodes.
I. (Accepted article) Impact of the water deficit on the biochemical composition of the cell wall, the lignification distribution with the tissues and the degradability of the cell wall of maize stems
II. F2bm3 does not respond histologically or biochemically to water deficit.
a) Cell wall composition of F2bm3 internode and response to water deficit
1. F2bm3 and the NIRS predictions
2. Estimations of the F2bm3 cell wall components values
3. Biochemical relationships and response to water deficit.
b) Histological profiles of F2bm3 and response to water deficit
Conclusion
Chapter 2 Introduction
Material and Methods.
Plant materials and field experiments
NIRS predictive equations construction and NIRS predictions
I. Results
a) 2 years of field experiments declined in 4 irrigation conditions due to different environmental context.
b) Phenotypic variations
1. The parental inbred lines and their characteristics under different irrigation condition
2. Variations of biochemical and histological traits among the recombinant inbred lines showed transgression and allowed to obtain expected correlations between traits
c) Genetic determinism of histological traits under different irrigation conditions.
d) QTLs involved in the variation of biochemical traits and co-localizations with QTLs involved in the variation of histological traits
e) Genetic determinism for biochemical cell wall traits of the whole plant without ears matches with the genetic determinism for biochemical cell wall traits of the internode in maize and with
II. (Submitted article) Water deficit responsive QTLs for cell wall degradability and composition traits in maize at silage stage
Conclusion
Chapter 3 Introduction
I. Variation of cell wall biochemistry of the internode carrying the main ear reflects the variations of cell wall biochemistry of the whole plant without ear.
Material and Methods
Results and discussion
II. The histology of the internode carrying the main ear reflects the mean all the internodes of the stem.
Material and Methods
a) Histological pattern of the internode carrying the main ear reflects the average histological pattern of all the internodes of a maize stem.
b) Histological variations of the internode carrying the main ear partially reflect variation of the biochemistry of the whole plant without ears.
Conclusion
Chapter 4 What bricks do we bring to the cell wall degradability understanding?
a) Development and probation of high-throughput tools
b) The understanding of cell wall degradability
c) Impact of water deficit on the biomass quality
d) From the internode to the whole plant
II. All these results may be discussed further
a) Going further in the improvement of the high-throughput tools
b) Choice of the plant material and and monitoring of the water deficit conditions to address the PhD questions
c) Relationships between cell wall degradability and lignification at different scales
1. Targeting the general cell wall composition within the plant
2. Targeting tissues specificities within the plant
d) Impact of the water deficit on the lignification at different scales
III. Perspectives
References

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