Effets de l'environnement lumineux et de l'âge foliaire sur la croissance, la capacité photosynthétique et la production protéique chez Nicotiana benthamiana

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(1)Effets de l’environnement lumineux et de l’âge foliaire sur la croissance, la capacité photosynthétique et la production protéique chez Nicotiana benthamiana Mémoire. Steffi-Anne Béchard Dubé. Maîtrise en biologie végétale Maître ès sciences (M.Sc.). Québec, Canada © Steffi-Anne Béchard Dubé, 2015. . .

(2) Résumé Cette étude visait à caractériser la croissance, la capacité photosynthétique, la concentration en azote et protéines totales solubles, la production de protéines recombinantes (HA) ainsi que la quantité de lumière interceptée à différents stades de développement de plants de Nicotiana benthamiana afin d’optimiser la production de vaccins. L’évolution des réponses physiologiques étudiées fut similaire chez toutes les feuilles primaires, suggérant que le processus de sénescence s’initie et progresse de façon semblable indépendamment de leur ordre d’initiation. Toutefois, la superposition des patrons temporels de sénescence et de croissance foliaire a mené à un rendement HA maximal se situant invariablement dans la partie médiane du plant lorsqu’exprimé sur une base foliaire. À l’échelle du plant entier, nos résultats suggèrent qu’il est possible d’augmenter la production de vaccins en récoltant les plants à un stade de développement plus tardif, ou en augmentant la densité de culture et en récoltant ces plants plus tôt.. . iii .

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(4) Abstract Nicotiana benthamiana is a wild relative of tobacco increasingly used as a plant protein expression platform to produce recombinant vaccine antigens against the influenza virus. Investigation on the physiological determinants of this production is essential to optimize and regulate vaccines production following a new flu outbreak. We examined the photosynthetic photon flux density, growth, light-saturated photosynthesis, total soluble protein, nitrogen content and recombinant protein production at different phenological stages. The similar evolution of the studied physiological responses suggested that the senescence process is initiated and progresses in a similar way in all primary leaves, regardless of the order of initiation. In contrast, the superposition of the time pattern of senescence with that of leaf growth shows that maximal HA yield expressed on a leaf basis is invariably located in the middle part of the plant. At the whole plant scale, our results suggest that it is possible to increase the production of antigens by harvesting plants at a later developmental stage, or by increasing plant density and harvesting these plants earlier.. . v .

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(6) Table of content Résumé ................................................................................................................................. iii Abstract .................................................................................................................................. v List of Tables ........................................................................................................................ ix List of Figures ....................................................................................................................... xi List of Appendices .............................................................................................................. xiii List of Abbreviations ........................................................................................................... xv Remerciements.................................................................................................................... xix Introduction............................................................................................................................ 1 1. Literature Review............................................................................................................ 3 1.1 Influenza vaccines ........................................................................................................ 3 1.1.1 Methods for vaccine production ............................................................................ 3 1.1.2 Nicotiana benthamiana ......................................................................................... 3 1.1.3 Transient expression .............................................................................................. 4 1.1.4 Virus-like particles ................................................................................................ 5 1.2 Physiological determinants of growth and development ............................................. 6 1.2.1 Irradiance ............................................................................................................... 6 1.2.2 Nitrogen content and photosynthetic capacity ...................................................... 7 1.2.3 Total soluble protein content and senescence ....................................................... 8 2. Hypotheses ...................................................................................................................... 9 3. Objectives ....................................................................................................................... 9 4. Material and Methods ................................................................................................... 11 4.1 Plant production ......................................................................................................... 11 4.2 Growth cycle .............................................................................................................. 11 4.3 Experimental design ................................................................................................... 12 4.4 Leaf sampling ............................................................................................................. 12 4.5 Leaf growth ................................................................................................................ 13 4.6 Canopy profiles of light intensity............................................................................... 14 4.7 Light-saturated photosynthesis rate ........................................................................... 14 4.8 Total nitrogen and soluble protein content ................................................................ 15 4.9 Agroinfiltration .......................................................................................................... 15 4.10 HA hemagglutination assay ..................................................................................... 16 . vii .

(7) 5. Results ........................................................................................................................... 19 5.1 Time course of leaf-level PPFD ................................................................................. 19 5.2 Leaf growth and physiology....................................................................................... 22 5.2.1 Photosynthetic capacity ....................................................................................... 22 5.2.2 Leaf growth ......................................................................................................... 23 5.2.3 Nitrogen content .................................................................................................. 23 5.2.4 Total soluble protein content ............................................................................... 23 5.2.5 Whole leaf scaling ............................................................................................... 24 5.3 Incubation phase......................................................................................................... 24 5.3.1 Agroinfiltration .................................................................................................... 24 5.3.2 Growth during the incubation phase.................................................................... 24 5.3.3 Leaf biomass at harvest ....................................................................................... 26 5.3.4 Whole plant total soluble protein content after incubation ................................. 26 5.3.5 Plant profile of HA activity ................................................................................. 28 5.3.6 HA yield per leaf ................................................................................................. 28 5.3.7 Relation between total soluble proteins and HA activity .................................... 31 6. Discussion ..................................................................................................................... 33 6.1 Growth of the Nb canopy and mutual shading among leaves .................................... 33 6.2 Canopy light vs. N allocation profiles........................................................................ 34 6.3 Photosynthetic nitrogen use efficiency ...................................................................... 36 6.4 Light environment and leaf senescence ..................................................................... 38 6.5 Post-incubation soluble protein content vs. VLP yield .............................................. 39 6.6 Crop density vs. HA activity yield per unit greenhouse surface ................................ 40 7. Conclusion générale ...................................................................................................... 41 8. Bibliographie ................................................................................................................. 43 9. Annexes ......................................................................................................................... 49. viii .

(8) List of Tables Table 4.1. Electrical conductivity (EC) of the nutrient solution during the growing phases. ...................................................................................................................... 12 Table 5.1. Fresh leaf biomass of plants agroinfiltrated at 15, 30 and 45 g fresh weight following 7 days of incubation in growth chambers ................................................ 26 Table 6.1. Light extinction coefficient (k) estimated with the formula of Monsi & Saeki (1953) using the leaf area index (LAI) and the photosynthetic photon flux density (PPFD) at the bottom of the plant canopy relative to that measured above the canopy (PPFDabove) for plants agroinfiltrated at 15, 30 and 45 g fresh weight, corresponding respectively to 29, 32 and 35 days after seeding (DAS). ................. 34 Table 6.2. Potential yields of VLPs expressed per group of plants agroinfiltrated at 15, 30 and 45 g and the crop density required to equal the HA yield obtained with 30 g and 45 g plants at a plant density of 33 plants per m2. ............................................. 40. . ix .

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(10) List of Figures Figure 1.1. Diagram illustrating the infection process of a plant cell with Agrobacterium and the transient integration of recombinant DNA information in the host cell nucleus (adapted from Tzfira & Citovsky, 2005). ......................................... 4 Figure 1.2. External and internal distinctions between the real influenza virus (left) and Virus-Like Particles (right) (adapted from Medicago, 2014)....................................... 5 Figure 1.3. N. benthamiana plant canopy at 30 grams of fresh biomass (~32 days after seeding), corresponding approximately to a canopy LAI of 2.7. ................................. 6 Figure 4.1. Illustration of the order of primary leaf sampling adopted in this study. Leaf A corresponds to the 5th primary leaf originating from the apical meristem, leaf B to the 7th, and so on ......................................................................................................... 13 Figure 4.2. Illustration of the positioning of photodiodes over target primary N. benthamiana leaves .................................................................................................... 14 Figure 4.3. Illustration of the N. benthamiana plant canopy at the three different times of agroinfiltration, that is when the plants reached 15, 30 and 45 g fresh weight of foliar biomass, respectively. ....................................................................................... 16 Figure 4.4. Example of HA hemagglutination assays used in this study. Shown here is the agglutination result for the pooled 9th primary leaves (i.e. leaves C, labelled “Sample 1” on the plate of N. benthamiana plants from the second (July) trial agroinfiltrated at 45 g fresh weight. Original crude leaf extract dilutions were in this case 1/12, 1/16 and 1/20. Control columns (C- and C+) are used to confirm that different plates can be compared on the same basis. .................................................. 17 Figure 5.1. Comparison of the photosynthetic photon flux density (PPFD) incident above the Nb cultivation greenhouse (A, B, C) and inside the greenhouse at crop height (D, E, F) during selected days of two (June and September 2014) of the three “summer” trial months selected for this study and one contrasting “winter” month (February 2014). ......................................................................................................... 20 Figure 5.2. Time course of the photosynthetic photon flux density (PPFD) measured at the level of primary leaves A, B, C, D to E relatively to the PPFD intercepted above the plant for three trials: A) June, B) July and C) September 2014 ........................... 21 Figure 5.3. Time course of A) light-saturated photosynthetic rate (Asat), B) projected leaf area, C) nitrogen content, and D) total soluble protein content of N. benthamiana primary leaves A, B, C, D, E to F .............................................................................. 22 Figure 5.4. Light-saturated photosynthetic rate (Asat) as a function of total soluble protein content for primary leaves A, B, C, D, E to F. ............................................... 23 Figure 5.5. Whole leaf A) photosynthetic capacity, B) nitrogen content, and C) total soluble protein content expressed as a function of the projected leaf area for leaves A, B, C, D, E to F ....................................................................................................... 25 Figure 5.6. Fresh leaf biomass profile of plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g fresh weight and incubated 7 days inside a growth chamber. ....................... 27. . xi .

(11) Figure 5.7. Whole plant total soluble protein content following a week of incubation for plants agroinfiltrated at 15, 30 and 45 g fresh weight [trials of June, July, September, and mean of three trials]. ............................................................................................ 28 Figure 5.8. Hemagglutinin (HA) activity profile of Nb plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g fresh weight............................................................................... 29 Figure 5.9. Whole leaf hemagglutinin (HA) yield profile of Nb plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g fresh weight ................................................................ 30 Figure 5.10. Whole plant hemagglutinin (HA) yield of Nb plants agroinfiltrated at 15, 30, and 45 g fresh weight............................................................................................ 31 Figure 5.11 Relationship between the whole plant total soluble protein content expressed in A) per gram of leaf and in B) for the entire plant and HA activity yield expressed in A) per gram of leaf and in B) for the entire plant of Nb plants agroinfiltrated at 15, 30 and 45 g fresh weight [trials of June, July, September, and mean of three trials] .................................................................................................... 32 Figure 6.1. Vertical profiles of leaf PPFD (relative to incident light above the canopy; orange bars) and leaf nitrogen content (relative to maximum leaf N content; green bars) as a function of leaf position for leaves A–F (corresponding to leaf position 5 to 15, respectively) of Nb plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g ......... 35 Figure 6.2. Changes in photosynthetic nitrogen use efficiency (PNUE) of primary leaves A, B, C, D, E to F during the growing phase................................................... 37 Figure 9.1. Comparison of the cumulative photosynthetic photon flux density (PPFD) incident above the cultivation greenhouse (A) and inside the greenhouse at crop height (B) during two (June and September 2014) of the three “summer” trial months selected for this study and one contrasting “winter” month (February 2014). ........... 52. xii .

(12) List of Appendices Appendix 1: Greenhouse experimental design ............................................................... 49 Appendix 2: Greenhouse photosynthesis measurement system ..................................... 50 Appendix 3: Infiltration system ...................................................................................... 51 Appendix 4: Cumulative PPFD above greenhouse and inside the greenhouse at plant height .......................................................................................................................... 52. . xiii .

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(14) List of Abbreviations Asat. Light-Saturated Photosynthetic Rate. BSA. Bovine Serum Albumin. DAS. Days After Seeding. EC. Electrical Conductivity. FW. Fresh Weight. HA. Hemagglutinin. HPS. High Pressure Sodium. k. Light Extinction Coefficient. LAI. Leaf Area Index. LED. Light-Emitting Diode. ME. Malic Enzyme. N. Nitrogen. NA. Neuraminidase. Nb. Nicotiana benthamiana. PDC. Pyruvate Dehydrogenase Complex. PNUE Photosynthetic Nitrogen Use Efficiency PPFD. Photosynthetic Photon Flux Density. PPVs. Particules Pseudo-Virales. VLPs. Virus-Like Particles. VPD. Vapour Pressure Deficit. . xv .

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(16) Pour ceux et celles qui désirent entreprendre une maîtrise : Préparez-vous à une expérience qui va changer votre vie… Steffi-Anne Béchard-Dubé . . xvii .

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(18) Remerciements Il y a de ces gens exceptionnels dans la vie qui nous changent. Par l’énergie positive qu’ils dégagent, la détermination qu’ils transpirent, par leur ténacité, débrouillardise, ingéniosité et sens de l’observation, ils vous transmettent un véritable intérêt pour la recherche, et vous ne pouvez que le partager. J’ai eu l’énorme privilège d’évoluer au cours des deux dernières années sous la direction de deux chercheurs passionnés par leur travail. Je remercie sincèrement Steeve Pepin et Gilbert Éthier de m’avoir accompagnée tout au long de mon projet de maîtrise. La route vers la rédaction finale de ce mémoire n’a pas été la plus courte, mais combien enrichissante. Je sors de cette expérience complètement grandie; j’ai acquis un énorme bagage de connaissances scientifiques et je suis extrêmement reconnaissante pour toutes ces qualités personnelles que vous m’avez transmises. Il y a aussi ces personnes très spéciales à mes yeux qui ont embelli ma vie au cours des deux dernières années et qui n’ont jamais cessé de croire en mes forces. Vous avez été des éléments essentiels à ma réussite. Merci à Jean-Samuel qui m’a appuyée tout au long de mon projet; à mes parents, Michel et Gaétane, qui étaient toujours là pour m’encourager à persévérer; à trois amies merveilleuses qui ont su m’épauler : Jennifer, Johane et Marianne. De plus, il y a toute l’équipe de l’Université Laval qui travaillait sur le même projet que moi. Je tiens à vous remercier de l’aide que vous m’avez apportée et des différentes discussions partagées qui m’ont permis d’évoluer à l’intérieur de ce projet : André Gosselin, Dominique Michaud, Linda Gaudreau, Ann-Catherine Laliberté, Marie-Claire Goulet, Jennifer Corriveau Boulay, Marielle Gagné, Sara Venegas Yuste et Lingling Shang. Finalement, j’aimerais remercier le personnel de Medicago Inc. pour leur précieuse collaboration, entre autres pour la préparation des inocula et pour les rencontres qui ont été bénéfiques à la progression de mon projet.. . xix .

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(20) Introduction Selon l’Organisation mondiale de la Santé (OMS), l’influenza représente une très grande menace dans le domaine de la santé. À chaque année, cette infection des voies respiratoires est responsable de 250 000 à 500 000 décès à l’échelle de la planète (OMS, 2007). Ce sont les virus de l’influenza de types A, B et C qui causent l’infection. Le virus de type A possède deux groupes de protéines à sa surface, les hémagglutinines (HA) et les neuraminidases (NA), qui peuvent muter avec le temps et produire de nouvelles souches du virus. Jusqu’à maintenant, HA a été trouvé sous forme de 14 sous-types, tels que H1, H2 et H3, etc., alors que du côté de NA, 9 sous-types (N1, N2, etc.) ont été recensés jusqu’ici. Deux lignées du virus de type B ont été découvertes, soit B/Yamagata or B/Victoria. Enfin, le virus de type C est rare et beaucoup moins dangereux (PHAC, 2011). Puisque les protéines à la surface du virus de l’influenza peuvent changer, la production de vaccins spécifiques aux nouvelles souches du virus s’avère essentielle. Bien qu’il existe différentes méthodes pour produire des vaccins, le développement récent de plates-formes permettant la production de protéines d’intérêt dans les plantes semble être une solution prometteuse en matière de rendement, de coûts, de rapidité et de sécurité de production par rapport à la méthode traditionnelle (Redkiewicz et al., 2014). Une des espèces végétales les plus utilisées pour la production de protéines recombinantes est le Nicotiana benthamiana (Nb). Jusqu’à maintenant, les recherches sur l’utilisation du Nb comme système transitoire d’expression de protéines ont pour la plupart porté sur les aspects moléculaires. Par exemple, Robert et al. (2013) ont démontré qu’il existe une corrélation entre l’activité protéolytique et l’âge foliaire, et que l’utilisation d’inhibiteurs de protéases peut mener à une augmentation du rendement en protéines recombinantes chez Nb. Afin d’approfondir les connaissances sur les aspects physiologiques de la production de protéines recombinantes dans les feuilles du Nb, un partenariat entre l’Université Laval, la compagnie Medicago Inc. et le Conseil de recherches en sciences naturelles et en génie a été établi. Medicago Inc. est une compagnie biopharmaceutique au stade clinique de recherche qui possède une expertise dans le développement de vaccins et de protéines thérapeutiques contre différentes maladies et infections. Elle s’intéresse particulièrement à l’expression . 1 .

(21) transitoire de gènes dans les plantes qui codent pour l’accumulation de particules pseudosvirales (PPVs) (D’Aoust et al., 2008). La compagnie possède trois unités de recherche dans la ville de Québec qui s’étendent sur une surface de 53 000 pieds carrés (i.e. ~4924 m2). De plus, une filiale de Medicago Inc., Medicago USA, est localisée en Caroline du Nord avec des unités de recherche qui s’étendent sur près de 100 000 pieds carrés (i.e. ~9290 m2). Par le passé, Medicago a réalisé différentes expérimentations pour augmenter l’expression des PPVs dans la plante. Une des limitations rencontrées chez Nb est que la partie inférieure du plant (laquelle peut atteindre jusqu’à ~50 % de la biomasse totale) ne produit qu’une faible part du rendement en PPVs. C’est donc dans le but de clarifier les déterminants physiologiques associés à cette baisse de rendement que l’évolution dans le temps de caractères physiologiques des feuilles du Nb, ainsi que leur rendement en PPVs à différents stades phénologiques, ont été étudiés dans le cadre de ce projet de maîtrise.. 2 .

(22) 1.. Literature Review. 1.1 Influenza vaccines 1.1.1 Methods for vaccine production . The influenza virus was first isolated in 1933 and, through time, numerous techniques to produce vaccines have been developed in order to counter the disease. Antigenic drift, productivity, tolerability, immunogenicity, and time-scale are some of the major hurdles faced in vaccine production that justify the continuous development of new technologies. Vaccines against influenza can be produced using different technologies, such as cultivation of a live attenuated strain of the virus in chicken’s egg embryo, the production of subunit vaccines and the creation of vaccines via genetic engineering (Crovari et al., 2011). Clinical trials are continuously conducted in order to target the best suitable technology to fight against an influenza outbreak. Plant-made proteins are reported to be a very successful and promising alternative to meet the vaccine production requirements (Redkiewicz et al., 2014). Unlike the traditional extraction of secondary plant compounds of medical interest in plants like mint, sage, and poppy, nowadays plants can be modified in a permanent or in a temporary way to produce specific protein antigens used as vaccines (Hofbauer & Stoger, 2013). Tobacco, lettuce, and tomato are good examples of widely used model organisms in plant genetic engineering (Bombarely et al., 2012; Chen & Lai, 2012). 1.1.2 Nicotiana benthamiana . Nicotiana benthamiana (Nb) is a Solanaceous species indigenous from Australia (Goodin et al., 2008). There are 76 species of Nicotiana found in South America and Australia, including Nicotiana tabacum, widely adopted by the tobacco industry (Knapp et al., 2004). As the large growing body of scientific literature about Nb shows, Nb has become the most widely used plant in virology and the prevalent model in plant-pathogen interactions (Goodin et al., 2008; Liu et al., 2012). Three major technical advances explain why Nb has become an extremely important subject in molecular biology. First, it possesses the ability to express genes after the insertion of a vector plasmid in its genome (Goodin et al., 2008); in other words, it has a huge susceptibility to different genes (Yang et al., 2004). Secondly, genes of Nb were historically used to describe a commonly known process, the down-. . 3 .

(23) regulation of genes using the virus-induced gene silencing technology (Ruiz et al., 1998). Thirdly, Nb was a plant model for the development of the agroinfiltration technique. 1.1.3 Transient expression . Agrobacterium transient gene expression is a system developed in 1996 in Belgium, which consists in inoculating a specific strain of the bacterium Agrobacterium tumefaciens with a plasmid construction containing the recombinant DNA of interest. The A. tumefaciens culture is grown to a specific optical density and used for infection of a plant host (Kapila et al., 1997). Following this step, the bacteria penetrate the intracellular spaces of the leaf, and a mobile part of the tumour-inducing plasmid (T-DNA) is transferred to the nucleus of host cells (Fig.1.1). In the nucleus, the new DNA is integrated in the plant genome by a recombination mechanism, ultimately leading to the temporary expression of transgene to produce the foreign protein of interest (Bundock et al., 1999). In this technique, plants are considered as transient expression vectors because the genetic viral information is not integrated permanently into the plant genome. Rather, the DNA fragment stays active only a couple of days in the nucleus of the plant cells (Vézina et al., 2011). Many benefits are found by using plants for transient expression, including cost efficiency, high yield, ease of genetic manipulation, rapidity and feasibility of production, safety, and no need for a «cold chain» (Sala et al., 2003). . Plant cell. Nucleus T-DNA. Figure 1.1. Diagram illustrating the infection process of a plant cell with Agrobacterium and the transient integration of recombinant DNA information in the host cell nucleus (adapted from Tzfira & Citovsky, 2005). . 4 .

(24) 1.1.4 Virus-‐like particles . Following the agroinfiltration, plants transfected to produce influenza virus proteins express these proteins (or antigens) and assemble them into virus-like particles (VLPs) that subsequently merge with the plasma membrane of the plant cells. In the case of the influenza virus, VLPs consist of a protein shell covered with a lipid membrane containing little strands of the most abundant viral glycoprotein of the real virus, the recombinant hemagglutinin, HA (Vézina et al., 2011; Quan et al., 2007). In contrast to the real influenza virus, the interior of the envelop of VLPs is empty because it does not incorporate genetic information, making it non-infectious and unable to replicate (Fig. 1.2) (D’Aoust et al., 2010). In the laboratory, the expressed VLPs are purified and used to produce vaccines. Because their structural characteristics resemble that of the real influenza virus, VLPs are capable of stimulating an immune response against the virus (Vézina et al., 2011). In addition, vaccines produced using the VLPs technology overcome strain-specific protection and provide cross-protective immunity against other strains of the influenza virus (Quan et al., 2007). Influenza virus. Virus-Like Particles. NA proteins HA proteins Genetic material. Figure 1.2. External and internal distinctions between the real influenza virus (left) and Virus-Like Particles (right) (adapted from Medicago, 2014).. . 5 .

(25) 1.2 Physiological determinants of growth and development 1.2.1 Irradiance . Light is a key input in the photosynthetic cycle, and different light intensities and wavelengths can impart morphological and physiological modifications to the leaf and the plant, to potentially trigger or accelerate the process of senescence (Walters, 2004). All these changes have considerable impacts on the production and accumulation of proteins, and thus on the synthesis of VLPs. Because plant density in Medicago’s greenhouses is relatively high in order to produce a profitable amount of vaccines per unit surface, leaves positioned in the lower portion of the plant canopy receive a light considerably reduced in intensity and drastically altered in spectrum compared to the light incident on leaves located at the top. In the case of randomly distributed, uniform leaf canopies, Monsi and Saeki (1953) have demonstrated that the vertical light intensity profile of the plant canopy can be approximated by the BeerLambert Law. For a uniform N. benthamiana canopy with a Leaf Area Index (LAI; m2 of leaf surface area per m2 of ground area) of approximately 2.7 (equivalent to 33 plants of 30 g fresh weight per m2 – see Fig. 1.3), Monsi & Saeki’s model predicts that the average light intensity at the level of the bottom leaves would be only ~25% of that incident above the canopy.. Figure 1.3. N. benthamiana plant canopy at 30 grams of fresh biomass (~32 days after seeding), corresponding approximately to a canopy LAI of 2.7. . 6 .

(26) With regards to the spectral light quality, upper canopy leaves absorb preferably blue and red wavelengths (Schmitt & Wulff, 1993), leaving an attenuated, green-enriched visible light spectrum and a predominance of far-red wavelengths in the lower canopy (Kasperbauer, 1971). Consequently, the photomorphogenetic light environment (i.e. the red to far-red ratio) in the lower canopy is drastically different than that of the upper canopy (Schmitt & Wulff, 1993). In the particular case of N. tabacum, Colgan et al. (2010) showed that different light regimes (i.e. different light intensities as well as day lengths) did not influence recombinant protein expression in comparison to temperature. Rather, supplementary light affected total soluble protein yield and plant biomass production. 1.2.2 Nitrogen content and photosynthetic capacity . There is a strong correlation between the nitrogen (N) content of a leaf and its photosynthetic capacity because proteins of the Calvin cycle and of the thylakoids make up a large portion of the total leaf nitrogen content (Evans, 1989). Depending on the growth irradiance profile, nitrogen is allocated into the different strata of the plant canopy to maximise the overall canopy photosynthesis (Moreau et al., 2012; Martins et al., 2014). Within leaves, nitrogen is also differentially allocated depending on the growth irradiance. For example, if leaves become acclimated to low light levels, more nitrogen is driven into light-harvesting complexes rich in antenna pigments (e.g. chlorophyll b) at the expense of the nitrogen investment into components making up the electron transport capacity (e.g. light reaction centers rich in chlorophyll a, cytochrome b6f complexes, etc.) (Evans & Poorter, 2001; Walters et al., 2003). Such dynamic regulation due to light environment is commonly named photosynthetic acclimation (Walters, 2004). To assess photosynthetic acclimation, indicators such as the chlorophyll a/b ratio or the light-saturated rate of photosynthesis are commonly used (Walters, 2004). In some cases, the light environment can lead to the initiation of the senescence process, characterized by a decline of the photosynthetic rate following the reception of senescence signals (Biswal et al., 2012). Once senescence is initiated, older leaves that once were a sink of nutrients become a source of nutrients for younger organs such as developing leaf and/or flower primordia (Fischer, 2007).. . 7 .

(27) 1.2.3 Total soluble protein content and senescence . Up to 70 % of the total leaf proteins are located within the chloroplast. Therefore, a direct relationship has been established between the photosynthetic rate and the total soluble protein content (Zhou & Gan, 2010). This strong relation can in large part be explained by the preponderance of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplast stroma, the most important (more than 50%) protein of the Calvin Cycle (Malkin & Niyogi, 2000). It is well known that (i) biotic factors like insects and bacteria and (ii) abiotic factors such as drought and heat stress induce plant and leaf senescence (Carp & Gepstein, 2007). When the senescence process is initiated, Rubisco and other proteins are degraded and the resulting amino acids and organic acids are reallocated to younger parts of the plant (Marschner, 1995). In fact, when senescence is triggered, genes are regulated to activate enzymes, such as proteases, that contribute to the degradation of photosynthetic proteins like Rubisco (Schaller, 2004). For instance, Inada et al. (1998) have demonstrated that plant cells normally loose approximately 66% of their total soluble protein content during senescence. Even in an optimal growth environment, senescence can be triggered by endogenous factors (Zentgraf et al., 2004). Zentgraf (2007) established that leaf age is the principal factor provoking leaf senescence. In addition to being regulated by leaf age, senescence associated genes are also controlled by the developmental stage of the entire plant (Zentgraf et al., 2004).. 8 .

(28) 2.. Hypotheses. A. The limiting amount of light received by older leaves located at the bottom of the N. benthamiana canopy causes the initiation and/or accelerated evolution of senescence, leading to low yield of VLPs. B. In N. benthamiana, as in many other species, the phenological stage of the plant has an influence on the evolution of senescence in newly initiated leaves.. 3.. Objectives. In order to assess changes in the light environment and the parallel ontogeny of photosynthetic capacity and protein accumulation in Nb leaves, three objectives were established: 1. To characterize the light environment (photosynthetic photon flux density (PPFD) intercepted) at different canopy depths throughout the growth phase of Nb plants in greenhouse. 2. To assess changes in growth, photosynthetic capacity, and total nitrogen and soluble protein content in primary leaves of greenhouse-grown Nb plants as they mature during the pre-infiltration period. 3. To compare HA yields obtained following agroinfiltration at three different ontogenic phases of Nb development, that is at 15, 30 and 45 g of fresh biomass. As previously discussed, light has numerous impacts on the development and physiology of plants. Therefore, evaluating the time course of changes in PPFD at the leaf level could help explaining the evolution of photosynthetic and protein productivity in aging leaves of Nb. Examination of the evolution of different physiological indicators of leaf productivity will help understand the process of leaf senescence and its impact on protein accumulation and vaccine antigen production. Measurements of light profiles and physiological determinants of photosynthetic productivity will also help understand if light acclimation is operating at the time scale of Nb pre-agroinfiltration cultivation, or if it is rather the leaf senescence process that is initiated before significant leaf shading occurs. Either way, it is. . 9 .

(29) important to identify the physiological determinants of the loss of VLP productivity in the lower Nb canopy. The current agroinfiltration protocol followed by Medicago Inc. uses Nb plants that have reached 30 g fresh weigh (FW), a target biomass above which the onset of flowering is expected to accelerate the senescence of a large portion of the accumulated leaf tissue. To verify this hypothesis, and to specifically follow the progress of leaf senescence beyond the 30 g ontogenic stage, we extended the biomass production period to 45 g to see if, from a whole plant perspective, the addition of new leaf tissues could offset the loss of HA expression in older biomass. Another goal was to assess the HA yield per unit biomass of younger plants agroinfiltrated at 15 g. By comparing the HA yield per unit biomass of plants agroinfiltrated at three different ontogenic stages, we wished to verify if the age of the plant had a significant impact on the overall VLP production capacity of the leaves. Put differently, we wished to answer the question: “Would three Nb plants agroinfiltrated at 15 g FW achieve an overall greater HA yield than a single Nb plant agroinfiltrated at 45 g FW?” This question has important practical implication with regards to cultural practices (e.g. use of shorter production times at greater plant densities).. 10 .

(30) 4.. Material and Methods. 4.1 Plant production Experiments were conducted in one greenhouse (A2) at the Pavillon des services, Laval University (lat. 46°46’33’’ N, long. 71°16’49’’ W) from June to October 2014. Measurements were repeated three times, corresponding to seeding dates June 9th, July 4th and August 13th.. 4.2 Growth cycle Because this project is in partnership with Medicago Inc., the company’s standard growth cycle was adopted in order to produce comparable plants. Seeds produced by Medicago Inc. were mixed with an agar medium, and 500 µL of the solution was dispensed into each cell of seedling trays. Seedling trays were placed in a growth chamber for two weeks under 250 µmol m–2 s–1 PPFD (16 h photoperiod) provided by fluorescent tubes (F72T8 TL841 4100 K, Philips, Amsterdam, The Netherlands) and an air temperature of 28°C. Water irrigation was applied manually three times during this period. On day 7 after seeding, trays were verified and if there was more than one plant per cell, the extra plants were removed in order to leave only one plant per cell. At the end of the two-week period, each plantlet was transplanted in a 0.4-L pot (diameter = 10 cm) and transferred in greenhouse for a growth period varying between 15 and 21 days, depending on the target agroinfiltration fresh weight. Climate conditions inside the greenhouse were controlled to maintain a temperature regime of 29°C/27°C (day/night) and a relative humidity of 60%. Incident PPFD over the greenhouse was measured with a quantum sensor (LI-190, LI-COR Inc., Lincoln, NE, USA). When light intensity at the top of the greenhouse decreased below 800 µmol m–2 s–1, high pressure sodium (HPS) lamps placed above the plants were turned on to supply 160 µmol m–2 s–1 PPFD at crop height. This supplemental light regime was applied throughout a continuous 24 h photoperiod. Plants were fertigated with a complete nutrient solution (N:P:K:Ca:Mg of 14:1:14:7:3) of electrical conductivity varying from 0 to 3.9 mS cm–1, depending on the maturity stage of plants (Table 4.1). Fertigation was applied by a drip irrigation system manually set on two or three times per day, based on the growth stage.. . 11 .

(31) The growing substrate was a potting and seeding soil mix (Agromix (0.4-0-0.02), Fafard Inc., Saint-Bonaventure, Qc, Canada).. Table 4.1. Electrical conductivity (EC) of the nutrient solution during the growing phases. Values are the average of the three trials. Days after seeding EC (mS cm–1). 1 à 16. 16. 18. 24. 28. 33. 0 (H2O). 1.0. 1.6. 2.6. 3.6. 3.9. Upon reaching the target pre-agroinfiltration weight, plants were agroinfiltrated (see Section 4.10), then placed inside a growth chamber for a 7-day incubation period. Air temperature inside the growth chamber was 20°C and the PPFD at the level of the plants was 200 µmol m–2 s–1 provided by fluorescents (F72T8 TL841 4100 K, Philips). Additional fluorescent tubes were installed between the rows of plants to supply supplementary lighting to the lower canopy. The growth chamber photoperiod was 16 hours per day.. 4.3 Experimental design A total of four plots were located on two rectangular tables in the greenhouse (2 plots per table). Each plot consisted of nine pot trays (3 x 3) with 5 plants per tray disposed in an alternate pattern. A total of 180 plants (45 plants x 4 plots) were used for each crop replicate (Appendix 1). Plant population was 33 plants per square meter.. 4.4 Leaf sampling Leaves of Nb were arranged in an alternate (spiral) pyllotactic pattern. We denoted leaves originating directly from the main stem as “primary leaves”, and leaves growing on stems emerging from axillary buds of primary leaves as “secondary leaves”. All physiological measurements described in the next sections were performed on primary leaves 5, 7, 9, 11, 13 and 15, hereafter referred to as leaves A to F, counting from the base of the plant (Fig. 4.1).. 12 .

(32) D B. EF C A. Figure 4.1. Illustration of the order of primary leaf sampling adopted in this study. Leaf A corresponds to the 5th primary leaf originating from the apical meristem, leaf B to the 7th, and so on. A total of six primary leaves were sampled on each N. benthamiana plant. . 4.5 Leaf growth Non-destructive and destructive leaf growth measurements were carried out. In the first case, the length of all primary leaves of six plant randomly distributed in the greenhouse was measured using a ruler, starting as soon as the leaf primordium could be clearly distinguished on the shoot apical meristem. In a previous experiment, leaf length measurements were related to the projected leaf surface area measured with a scanning planimeter (LI-3100C, LI-COR Inc.) according to the following relationship: 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑐𝑐𝑐𝑐 = 0.38598 ∗ 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙ℎ 𝑐𝑐𝑐𝑐. .. ; 𝑅𝑅 = 0.97. Eq. 4.1. In the second case, destructive measurements were performed on three occasions during the pre-agroinfiltration growth cycle, that is when the total fresh biomass reached 15, 30 and 45 g (corresponding, on average for the three crop replicates, to 29, 32, and 35 days after seeding). At each sampling date, the leaf area and fresh weight of each primary and secondary leaf of four plants were recorded. Additionally, the fresh weight of each leaf of four more plants per agroinfiltration cycle was recorded at the end of the 7-day incubation period.. . 13 .

(33) 4.6 Canopy profiles of light intensity Twenty-four GaAsP photodiodes (G2711-01, Hamamatsu Photonics, SZK, Japan) connected to a data logger (CR 23X, Campbell Scientific, Logan, UT, USA) were used to measure incident PPFD at six levels in the plant canopy. The photodiodes were deployed successively over target primary leaves (leaves A to E, plus one photodiode placed at the top of the plant) of four plants (one plant in each of the four greenhouse plots). The leaf photodiodes were initially covered with shading material, which was then removed upon positioning the photodiode above the target leaf as soon as it was visible on the shoot apical meristem. As the leaves unfolded, photodiodes were repositioned daily over the middle portion of each target leaf (see Fig. 4.2). The data logger sampling frequency was 5 seconds, and the stored PPFD values represented 5 min averages.. Figure 4.2. Illustration of the positioning of photodiodes over target primary N. benthamiana leaves. Shown here are the photodiodes placed over the middle portion of leaves A and B at the early stage of growth. . 4.7 Light-‐saturated photosynthesis rate Light-saturated photosynthetic rates (Asat) of target primary leaves were measured repeatedly throughout the growth cycle using two cross-calibrated portable gas exchange systems (LI-6400XT, LI-COR Inc.). In each of the four greenhouse plots, one plant was. 14 .

(34) randomly chosen and the Asat measurements were carried out on that same plant over the complete growth cycle. Measurements were repeated every two or three days, from the moment the leaf length reached five centimeters and until approximately 40 days after seeding (DAS). Each LI-6400XT unit was individually connected to a dew point generator (LI-610, LI-(+"E: J<KKF8;<NGF@EKK<DG<I8KLI<F=

(35) n in order to obtain a leaf-toair vapour pressure deficit (VPDL) approximately equal to 1 kPa at an operating leaf temperature equal to 28°C (see Appendix 2). Plant illumination was provided by a red-blue (10% blue) light-emitting diode (LED) panel (TI SmartLamp, LED Innovation Design, Laval, Quebec, Canada) whose spectrum matched that of the 6400-02B LED light source of the 6 cm2 LI-6400XT leaf chamber. Air flow through the leaf chamber was set at 400 µmol s–1, and the chamber CO2 mole fraction controlled at 400 µmol mol–1 (the latter being comparable to the ambient greenhouse CO2 mole fraction). Knowing from previous full light responses curve measurements on leaves of Nb that, under similar growth conditions, net photosynthesis reached a plateau above a PPFD of 800 µmol m–2 s–1, we determined Asat from measurements under the following order of PPFD levels: 1600, 1400, 1200 and 1600 µmol m–2 s–1.. 4.8 Total nitrogen and soluble protein content On the same days as the Asat measurements days were taken, equivalent target leaves from other plants (3 plants per plot) were destructively sampled for total nitrogen and soluble protein content determination. Leaves were immediately immersed in liquid nitrogen (around –195°C) and then stored in a freezer at –80°C until analysis. Soluble proteins were extracted in 50 mM Tris HCl, pH 8.0, containing 500 mM NaCl, 1 mM PMSF and 0.04% (w/v) sodium metabisulfite. Soluble protein content in the extract was quantified following the method of Bradford (1976) using bovine serum albumin (BSA) as a standard. Absorbance (595 nm) measurements were done with a microplate reader (FLUOstar OMEGA, BMG LABTECH, Ortenberg, Germany). Total nitrogen content in leaves was measured using the micro-Kjeldahl digestion assay (Kjeldahl, 1883).. 4.9 Agroinfiltration Agroinfiltration was performed on eight plants at three moments during the growth cycle, that is when the total foliar biomass reached approximately 15, 30 and 45 g FW (Fig. 4.3). The infiltration solution contained bacteria that are naturally present in the soil, . 15 .

(36) Agrobacterium tumefaciens. To perform transient expression of recombinant proteins, a recombinant strain is used, which contains the genes coding for VLPs (H1 antigens) expression in planta. The bacterial strain containing the plasmid construction required to express HA was provided by Medicago Inc. Plants were individually immersed upside down in a 10-L polycarbonate tank containing the Agrobacterium solution and connected to a vacuum pump. The tank was put under vacuum for a period of one minute, after which sudden release of the vacuum forced the penetration of the solution inside the extracellular spaces of the leaf (Appendix 3).. Figure 4.3. Illustration of the N. benthamiana plant canopy at the three different times of agroinfiltration, that is when the plants reached 15, 30 and 45 g fresh weight of foliar biomass, respectively.. 4.10 HA hemagglutination assay We use the hemagglutination assay developed by Hierholzer & Killington (1996) to determine the activity of the H1 antigen in our leaf samples, following the protocol used by Medicago Inc. This method used to determine the activity level of Influenza virus in a sample is based on the fact that the virus sticks to blood cells (the agglutination). When the activity level of Influenza virus particles in solution is high enough to fix all blood cells, an amorphous network between the cells and viruses is formed and the resulting solution in the plate well appears pink and uniform (see Fig. 4.4). At the opposite, if the activity of the virus is not high enough to fix all blood cells, the remaining cells sediment by gravity and form a distinct red point at the bottom of the well.. 16 .

(37) One hundred microliters of three different dilutions (adjusted according to leaf age) of crude leaf extract (see Section 4.8) were pipetted on the first line of a 96-well microtiter plate, then underwent a series of two-fold dilutions in phosphate buffered saline (PBS) solution until the 12th row of wells (Fig. 4.4). One hundred microliters of a 0.25 % turkey blood cells suspension in PBS were added to each sample (turkey blood in Alsever solution, Bio Link Inc.) and the plate was left to incubate at room temperature for two hours. Following incubation, the titer (i.e. the greatest dilution before the appearance of a clear large isolated point in the center of the well) of each of the three leaf sample dilution series was determined by visual inspection (see Fig. 4.4) and the one with the greatest overall dilution factor was selected as the final titer (i.e. as the number of HA activity units in 100 µL) for the leaf sample. For example, titer values of the three dilution series of “Sample 1” (Figure 4.4) were respectively 1/6144, 1/4096 and 1/5120, yielding 1/6144 as the final titer of the leaf sample (i.e. 6144 HA units per 100 µL of sample).. Figure 4.4. Example of HA hemagglutination assays used in this study. Shown here is the agglutination result for the pooled 9th primary leaves (i.e. leaves C, labelled “Sample 1” on the plate of N. benthamiana plants from the second (July) trial agroinfiltrated at 45 g fresh weight. Original crude leaf extract dilutions were in this case 1/12, 1/16 and 1/20. Control columns (C- and C+) are used to confirm that different plates can be compared on the same basis.. . 17 .

(38)

(39) 5.. Results. 5.1 Time course of leaf-‐level PPFD Comparative values of the incident PPFD above the greenhouse vs. inside the greenhouse at the height of the Nb crop are presented in Figure 5.1 for two (June and September 2014) of the three trials, plus during the month of February 2014 (to allow comparison with typical “winter” light conditions). Overall, the greenhouse light transmission factors, i.e. the ratio between the slopes of the curves for cumulative incident PPFD at crop height and cumulative incident PPFD above greenhouse, were 0.888, 0.438 and 0.765 in February, June and September, respectively. While above greenhouse cumulative PPFD varied considerably among the three trial months, from 338 mol m–2 in February to 1005 mol m–2 in June and 650 mol m–2 in September, corresponding values of cumulative PPFD were only 310, 412 and 491 mol m–2 at plant height inside the greenhouse (see Appendix 4). Indeed, a direct comparison of the right-hand vs. left-hand sides of Figure 5.1 highlights the two-faceted disturbance of the natural light environment that took place inside the greenhouse during Nb cultivation: 1) the implementing of a 24 h photoperiod with a minimum PPFD of 160 µmol m–2 s–1 at crop height, and 2) the limiting of the maximum PPFD at crop height to 600 µmol m–2 s–1, at most times, through the deployment of radiation screens. These two cultural practices have previously been identified as optimal for maximising biomass while preventing photoinhibitory damage in Nb crops (unpublished results). Figure 5.2 shows the time course of the incident PPFD on the five target primary leaves measured during each of the three summer trials. To facilitate comparison among the three crop replicates, leaf-level PPFD values are expressed relative to the above canopy PPFD. For all leaves, the normalised PPFD remained closed to one from the day of their initiation to approximately 8–10 days, showing that during this initial period no significant shading from the leaves initiated above took place. After this period, the leaf-level PPFD dropped rapidly, reaching near zero values after approximately 7 days, thereby suggesting rapid. . 19 .

(40) foliar expansion and closing of the canopy above the measuring point during that period.. PPFD (µmol m–2 s–1). The rate of PPFD decrease was similar for all leaves.. 2500. A). D). February 2014. February 2014. 2000 1500 1000 500 0 27. 29. 31. 33. 35. 37. 39. 41. 43. 45. 47. 27. 29. 31. 33. PPFD (µmol m–2 s–1). Day of year 2500. B). 37. 39. 41. 43. 45. 47. Day of year E). June 2014. June 2014. 2000 1500 1000 500 0 149. 151. 153. 155. 157. 159. 161. 163. 165. 167. 149. 151. 2500. C). 153. 155. 157. 159. 161. 163. 165. 167. Day of year. Day of year PPFD (µmol m–2 s–1). 35. September 2014. F). September 2014. 2000 1500 1000 500 0 239 241 243 245 247 249 251 253 255 257 259 261 263. Day of year. 239 241 243 245 247 249 251 253 255 257 259 261 263. Day of year. Figure 5.1. Comparison of the photosynthetic photon flux density (PPFD) incident above the Nb cultivation greenhouse (A, B, C) and inside the greenhouse at crop height (D, E, F) during selected days of two (June and September 2014) of the three “summer” trial months selected for this study and one contrasting “winter” month (February 2014). . 20 . .

(41) Leaf PPFD relative to above canopy Leaf PPFD relative to above canopy Leaf PPFD relative to above canopy. A). June 2014. 1.0 0.8 0.6 0.4. A B C D E. 0.2 0.0. 15. 20. 25. 30. 35. Days after seeding. B). 40. July 2014. 1.0 0.8 0.6 0.4 0.2 0.0. 15. 20. 25. 30. 35. Days after seeding. C). 40. 45. September 2014. 1.0 0.8 0.6 0.4 0.2 0.0. 15. 20. 25. 30. 35. Days after seeding. 40. 45. Figure 5.2. Time course of the photosynthetic photon flux density (PPFD) measured at the level of primary leaves A ( ), B ( ), C ( ), D ( ) to E ( ) relatively to the PPFD intercepted above the plant for three trials: A) June, B) July and C) September 2014. Values indicated are the means of four plants ± SD. . . 21 .

(42) 5.2 Leaf growth and physiology 5.2.1 Photosynthetic capacity . The light-saturated photosynthetic rate (Asat) of primary leaves was maximal soon after leaf initiation, reaching a similar value (~18 µmol m–2 s–1) for all leaves (Fig. 5.3a). Following a period of approximately 8–10 days, Asat started to decrease monotonically with leaf age at a rate comparable for all leaves. Interestingly, the onset of this eventual decline in Asat coincided with the time when shading from younger leaves expanding above progressed rapidly (see Fig. 5.2). Examination of the time course of Asat in the oldest sampled leaves (leaves A, B, and C) showed that Asat invariably fell below a value of 5 µmol m–2 s–1 some 11 days after the onset of the decline.. 200. A). Projected leaf area (cm–2). Asat (µmol m–2 s–1). 25 20 15 10. A B C D E F. 5 0 15. 20. 25. 30. 35. 150. 100. 50. 0. 40. B). 15. 20. 100. C). 80 60 40 20 0 15. 20. 25. 30. Days after seeding. 25. 30. 35. 40. 35. 40. Days after seeding. 35. 40. Total soluble protein content (g m–2). Nitrogen content (mmol m–2). Days after seeding 5. D). 4 3 2 1 0. 15. 20. 25. 30. Days after seeding. Figure 5.3. Time course of A) light-saturated photosynthetic rate (Asat), B) projected leaf area, C) nitrogen content, and D) total soluble protein content of N. benthamiana primary leaves A ( ), B ( ), C ( ), D ( ), E ( ) to F ( ). Values indicated are the means for the three trials ± SD.. 22 .

(43) 5.2.2 Leaf growth . Figure 5.3b indicates that the primary leaves we sampled were initiated approximately every two days, thereby inferring a leaf primordium initiation rate at the apical meristem equal to one leaf per day. The maximum projected leaf area, as well as the time required to reach it, depended on the order of leaf initiation. For instance, 40 days after seeding, the leaf area of primary leaves A to D was 50, 110, 170 and 180 cm2, respectively. 5.2.3 Nitrogen content . In comparison to Asat, the leaf nitrogen content of primary Nb leaves started to decline sooner after leaf initiation and followed a similar rate of decrease in all leaves (Fig. 5.3c). Unlike the decrease in Asat, which appeared monotonic throughout the leaf ageing process, the rate of leaf nitrogen decline increased in concert in all leaves after DAS 34. 5.2.4 Total soluble protein content . In the case of leaves A to C, maximal values of total soluble protein content were reached very soon after leaf initiation, whereas the leaf protein content of leaves D to F peaked a few days later than in the older leaves (Fig. 5.3d). In addition, the maximal value of total soluble protein content tended to decrease proportionally to the order of leaf initiation. The relationship between Asat and soluble proteins content was well described by a MichaelisMenten function (R2 = 0.93) with a half-saturation protein content of 1.12 g m–2 and a maximal Asat of 23.2 µmol m–2 s–1 (Fig. 5.4). . . . . . . Asat (µmol m–2 s–1). 20. 15. 10. 5. 0 0.0. . . . A B C D E F. 0.5. 1.0. 1.5. 2.0. 2.5. 3.0. Total soluble protein content (g. 3.5. m–2). 4.0. . Figure 5.4. Light-saturated photosynthetic rate (Asat) as a function of total soluble protein content for primary leaves A ( ), B ( ), C ( ), D ( ), E ( ) to F ( ). Data are means for the three trials ± SD. . . 23 .

(44) 5.2.5 Whole leaf scaling . Because the aforementioned time series of Asat and leaf nitrogen and soluble protein content were expressed on a per unit area basis, and concomitantly leaf expansion proceeded in sigmoidal fashion, it is of interest to re-scale the physiological attributes of the leaves on a per whole leaf basis to gain a more holistic view of the evolution of leaf photosynthetic capacity and nitrogen/soluble protein accumulation. Figure 5.5 shows that in all the primary leaves studied whole leaf photosynthetic capacity, nitrogen content, and soluble protein accumulation followed a “bell shape” curve with respect to projected leaf area. In the case of whole leaf soluble protein content (Fig. 5.5c), the peak of the curve was reached at around 50–60% of the maximal leaf size, after which the rate of net protein degradation appeared to overtake the rate of leaf area production, thereby leading to a decrease in overall leaf content. Comparatively, for whole leaf photosynthetic capacity and nitrogen content, the peak of the curve was reached at a later stage during leaf expansion (i.e. ~70– 80% and ~75–85% of the maximal leaf size, respectively – see Fig 5.5a,b). This lag in decrease of photosynthetic potential may be indicative of an initial overinvestment of nitrogen in the soluble protein fraction of the photosynthetic machinery.. 5.3 Incubation phase 5.3.1 Agroinfiltration . To ensure that we agroinfiltrated plants near the target 15, 30 and 45 g, we followed the increase in biomass every day by selecting random plants from the four greenhouse plots. On average for the three trials (June, July and September 2014), the total leaf fresh biomass prior to agroinfiltration was equal to 17, 29 and 45 g, corresponding to day 29, 32 and 35 after seeding (Table 5.1). 5.3.2 Growth during the incubation phase . After being agroinfiltrated, plants were placed in an incubation growth chamber for 7 days (see Section 4.2). During this period, although visibly stressed from the agroinfiltration treatment, the plants continued to grow. We assessed the growth of the three groups of agroinfiltrated plants (15, 30 and 45 g) following their 7-day incubation period. The increases in fresh leaf biomass were 59, 48 and 27% for the 15, 30 and 45 g plants,. 24 .

(45) respectively (Table 5.1). The larger increase in plants of 17 g was probably caused by more. Total soluble protein content (mg leaf –1). Nitrogen content (mg leaf –1). Photosynthetic capacity (µmol s–1 leaf –1). space and better access to light during the incubation phase compared to bigger plants.. 0.25. A B C D E F. 0.20 0.15. A). 0.10 0.05 0.00. 0. 50. 100. 150. Projected leaf area. 200. (cm2). 14. B). 12 10 8 6 4 2 0. 0. 50. 100. 150. Projected leaf area. 200. (cm2). 25. C). F 20. E 15. D. 10. C. 5 0. B. A 0. 50. 100. 150. 200. Projected leaf area (cm2). Figure 5.5. Whole leaf A) photosynthetic capacity, B) nitrogen content, and C) total soluble protein content expressed as a function of the projected leaf area for leaves A ( ), B ( ), C ( ), D ( ), E ( ) to F ( ). Data are means of three trials. . . 25 .

(46) Table 5.1. Fresh leaf biomass of plants agroinfiltrated at 15, 30 and 45 g fresh weight following 7 days of incubation in growth chamber. Values are means for the three trials. Target fresh weight (g) 15. 30. 45. Pre-incubation fresh weight (g). 17. 29. 45. Post-incubation fresh weight (g). 27. 43. 57. Biomass increase (%). 59. 48. 27. 5.3.3 Leaf biomass at harvest . At Medicago Inc., the VLP yield is quantified for the entire Nb plant. In order to gain a better understanding of the VLP expression profile among the different leaves, we measured the soluble protein content and VLP activity of primary leaves 5 to 17 separately, and similarly for their corresponding secondary leaves. For plants agroinfiltrated at 15 and 30 g, 72% and 28% of the fresh biomass was allocated to the primary and secondary leaves, respectively (Fig. 5.6). In comparison, plants agroinfiltrated at 45 g allocated less of their total leaf biomass to the primary leaves (63% against 37% for the secondary leaves). The vertical profile of the primary leaf biomass followed a “bell shape” curve with its peak skewed towards the lower half of the plant (Fig. 5.6). For the secondary leaves, the biomass increased more or less gradually from top to bottom. 5.3.4 Whole plant total soluble protein content after incubation . Figure 5.7 shows the overall soluble protein accumulation in the entire Nb plant following the 7-day incubation period. For plants agroinfiltrated at 15 g, the protein content was around 210 mg of soluble protein per plant, and increased only slightly (~258 mg protein per plant on average for the three trials; P= 0.24) for plants agroinfiltrated at 30 g. In comparison, there was a significant increase (P= 0.019) of whole plant protein yield in plants agroinfiltrated at 45 g (~425 mg of protein per plant on average). It should be noted that such high plant protein yields were achieved in part due to a careful manual agroinfiltration followed by incubation at relatively low plant density inside the growth chamber. Comparatively at Medicago Inc., the plant protein yield is usually inferior because some leaves are broken during the large-scale mechanical agroinfiltration and. 26 .

(47) manipulation of plants. Broken and/or incompletely agroinfiltrated leaves are removed from the plant prior to incubation and thus cannot contribute to the plant total protein content. Additionally, the significantly higher plant density used inside Medicago’s incubation chambers results in more limiting light conditions.. A. B). C. Figure 5.6. Fresh leaf biomass profile of plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g fresh weight and incubated 7 days inside a growth chamber. The Y-axis represents the numbering of the different leaves starting from the bottom of the plant. The red and blue horizontal bars are for the primary and secondary leaves, respectively. Values are means of three trials ± SE.. . 27 .

(48) Total soluble protein content (mg plant –1). 500. June July September Mean. 400 300 200 100 0. 0. 10. 20. 30. 40. 50. Incubation weight (g) Figure 5.7. Whole plant total soluble protein content following a week of incubation for plants agroinfiltrated at 15, 30 and 45 g fresh weight [trials of June ( ), July ( ), September ( ), and mean of three trials ( )]. 5.3.5 Plant profile of HA activity . The profile of hemagglutinin (HA) activity (expressed in HA activity units per gram of fresh leaf tissue) among the various leaves of the Nb plant is given in Figure 5.8. For plants agroinfiltrated at 15 g, the peak primary leaf HA activity was found around the mid-canopy level and decreased gradually towards the upper and lower parts of the plant, whereas for the secondary leaves, HA activity was highest for the bottom leaves and gradually decreased towards the upper leaves. As plants grew bigger before the agroinfiltration, the peak HA activity of both primary and secondary leaves was shifted upward on the plant, leading in the case of secondary leaves to a complete reversal of the HA activity profile relative to plants agroinfiltrated at 15 g. 5.3.6 HA yield per leaf . The overall HA yield (obtained by multiplying the number of HA activity units per g leaf FW by the total fresh biomass of the leaf) of the different leaves of the Nb plant is given in Figure 5.9. For plants agroinfiltrated at 15 g, 72% of the total plant HA yield was found in the primary leaves against 28% in the secondary leaves. As plants grew bigger before the agroinfiltration, the distribution of the plant’s HA yield between primary and secondary leaves gradually became more even, with 59% and 51% of the total plant HA yield being found in primary leaves for plants agroinfiltrated at 30 and 45 g, respectively. 28 .

(49) In the case of the primary leaves, the HA yield profile followed a normal distribution with a peak slightly skewed towards the bottom leaves despite its moving upward with plant age. For the secondary leaves, most of the HA yield of the younger plants (agroinfiltrated at 15 and 30 g) was found in the leaf lower strata, but for plants agroinfiltrated at 45 g, the HA yield was evenly distributed between the upper and lower plant regions (Fig. 5.9).. A). B). C). Figure 5.8. Hemagglutinin (HA) activity profile of Nb plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g fresh weight. The Y-axis represents the numbering of the different leaves starting from the bottom of the plant. The red and blue horizontal bars are for the primary and secondary leaves, respectively. Values are means of three trials ± SE.. . 29 .

(50) A). B). C). Figure 5.9. Whole leaf hemagglutinin (HA) yield profile of Nb plants agroinfiltrated at A) 15 g, B) 30 g, and C) 45 g fresh weight. The Y-axis represents the numbering of the different leaves starting from the bottom of the plant. The red and blue horizontal bars are for the primary and secondary leaves, respectively. Values are means of three trials ± SE.. In terms of total per plant yield, plants that were agroinfiltrated at approximately 45 g produced 30 million HA units, which essentially doubled and tripled the yields of plants agroinfiltrated at 30 and 15 g, respectively. The HA productivity ratio of primary and secondary leaves changed appreciably among the three target agroinfiltration weights (Fig. 5.10).. 30 .

(51) HA yield. (activity units per plant x 106). 35 30 25. Primary leaves Secondary leaves Total. 20 15 10 5 0. 15. 30. 45. Agroinfiltration fresh weight (g) Figure 5.10. Whole plant hemagglutinin (HA) yield of Nb plants agroinfiltrated at 15, 30, and 45 g fresh weight. The red columns are for primary leaves, blue columns for secondary leaves and green columns for the total plant (primary + secondary leaves). Values are means of three trials ± SE. 5.3.7 Relation between total soluble proteins and HA activity . We found a remarkably close linear relationship between whole plant HA activity units and total soluble protein content following incubation (R2 = 0.976) (Fig. 5.11b). When expressed on a weight basis (in gram of leaf), the relationship between HA activity and the protein content was less linear (R2 = 0.57) (Fig. 5.11a). We suppose that the decrease in the coefficient of determination is due to the very different types of leaves and protein contents in into a single plant. On the other hand, at the entire plant scale, the relationship was very consistent among the three greenhouse trials, which leads us to suggest that, at the whole plant level, the total soluble protein content can be used as a robust indicator of HA expression under experimental conditions similar to ours.. . 31 .

(52) Figure 5.11. Relationship between the whole plant total soluble protein content expressed in A) per gram of leaf and in B) for the entire plant and HA activity yield expressed in A) per gram of leaf and in B) for the entire plant of Nb plants agroinfiltrated at 15, 30 and 45 g fresh weight [trials of June ( ), July ( ), September ( ), and mean of three trials ( )].. 32 .

(53) 6. Discussion 6.1 Growth of the Nb canopy and mutual shading among leaves The amount of light required in a single day (i.e. the daily PPFD integral) to maximize plant CO2 uptake and produce consistent biomass accumulation in greenhouse crops may be achieved through a combination of daylight, supplementary lighting and deployment of radiation screens (Albright et al., 2000). In this study, Nicotiana benthamiana plants were grown under a 24 h photoperiod because previous works had demonstrated that increasing the photoperiod from 16 to 24 h significantly enhanced plant biomass (Gosselin et al., unpublished data). Unlike photosynthesis, leaf respiration releases an important amount of fixed CO2 into the air (Atkin et al., 2000). However, because the pyruvate dehydrogenase complex (PDC) and NAD+-malic enzyme (ME) are inactivated in the presence of light, foliar respiration is partially inhibited under natural or artificial light conditions, thereby reducing the respiratory losses of CO2 (Padmasree & Raghavendra, 1998). Hence, the 24 h continuous light regime used in this study resulted in the production of a sustained and adequate supply of photosynthetic assimilates to fuel plant growth. N. benthamiana is a fast growing species, which exhibited rapid leaf expansion and canopy closing inside the greenhouse. Measurements of light intensity on target leaves A–F showed that normalized PPFD values (i.e. relative to PPFD measured at the top of the plant) remained closed to one up to approximately 8–10 days after leaf initiation. Following this growth period, substantial leaf shading occurred and the values of incident leaf PPFD declined rapidly, reaching near zero values after ~7 days. The superposition of the overall pattern of leaf expansion and time course of shading indicated that the rate of PPFD decrease was similar among all leaves and was caused by mutual shading. We used the formula of Monsi & Saeki (1953) to estimate the light extinction coefficient (k) for the three trials using the PPFD incident on leaf A (fifth leaf from the bottom of the plant) relative to that measured above the Nb canopy (PPFDabove). Mean k values were 0.61, 1.02 and 1.15 for plants agroinfiltrated at 15, 30 and 45 g fresh weight, respectively (Table 6.1). As reported by Monsi & Saeki (1953), k values in a plant canopy composed predominantly of horizontal leaves, such as in N. benthamiana crop, typically ranged between 0.7 and 1.0. These results thus suggest that the attenuation of light inside the canopy increased with. . 33 .