Hi. I’m Luis Herrera-Estrella. I’m Director of the National Laboratory of Genomics for Biodiversity in Irapuato, Mexico, and I’ll be talking to you about plant nutrition and sustainable agriculture. I will be dividing my presentation into three sections, one talking about plant nutrition in general, the second about phosphorus, an essential plant nutrient, and in the third part about the use of genetic engineering to design an environmentally friendly phosphorus fertilization system. So, I will start talking about some very important issues that we need to consider in food production. New projections by the United Nations indicate that the world’s population might reach over 9 billion people by 2050. These will be mainly in developing countries, and not so much in industrialized countries that have a more steady, stabilized growth. These will not only be an increase in population, but there will be also a switch in the percentage of people living in urban regions and rural zones. So, before, in the 1950s, most of the people lived in rural zones and they were doing activities related to food production, but in 2050 about only one-third to one-fourth of the people will be living in rural areas and will be involved in food production. So, less people producing food, more people consuming high levels of calories in the cities. So, to achieve the goal of satisfying the food needs of people, we need to double food production in the same cultivated area. We cannot increase cultivated area because we will have to deforest and we will have to destroy jungle and forest to be able to incorporate more area to cultivation. So, we need to increase production but maintain the same cultivated area. So, we need to increase about 69% of the calorie intake by 2050, and to increase this 69% we need to double food production, because the global meat demand is increasing and we need to produce feed for animals to be grown. So, we will increase the level of beef, pork, and poultry that people are consuming, and because these animals are not very efficient, particularly beef, in the conversion of energy from feed into meat, we will need to increase more the production of agricultural product. To reach the project needs in food production, we need to increase plant productivity. So, this graph shows in the green line the level of increasing productivity that we currently have using the technology we have in hand. So, even though things have improved in the past and we have better crops and we have better ways of irrigation and better ways of fertilization, we still have a gap in productivity that is required to reach the amount of food we will need by 2050. And to close this gap we will need to make use of all the technology that we can develop to increase crop yield per area unit. So, we need to increase food production, but we need to decrease the environmental impact of agriculture. So, agriculture is one of the activities, human activities, that causes more environmental problems. For instance, 24% of the greenhouse gas emissions are produced by agriculture, 37% of the Earth’s landmass is just for agricultural purposes – either for cropping or grassland for cattle or other animals – and over 70%, between 70 and 80%, of the fresh water used in the world is done for agriculture. So, we need to reduce greenhouse emissions, we need to optimize the use of Earth’s landmass for cropping and grassing, and also we need to decrease and make more efficient the use of water because this is going to be one of the most limiting factors for the future of humanity. So, how can we make agriculture more sustainable? We need to increase plant productivity, we need to use all technology available to increase the capacity of plants to produce grains and fruits and biomass, so we need to use traditional breeding, we need to use genetic engineering, genetically modified organisms (GMOs), and new breeding technologies such as genome editing that are promising major changes to incorporate new traits into crops. At the same time, we need to increase yield but reduce agrochemical use. We need to optimize fertilization schemes, so we need to reduce the application of chemical fertilizer and increase the use of organic and biofertilizers – biofertilizers are bacteria that help plants to be more efficient in fertilizer use. We need to reduce also the use of pesticides and herbicides. We need more food, but we also need healthier food. We need food with less chemicals. So, we need to use biocontrol agents that prevent diseases and pests in crops… biological and genetic weed control systems as well, to reduce herbicide use. To optimize fertilization schemes, we need to understand plant nutrition. That’s why it is important to learn what the plant needs and how we can optimize it so that we can make a more sustainable agriculture. But, also, we need to understand how plants interact with soil microbes, because microbes play a very important role in the capacity of the plant to take up… to assimilate nutrients from the soil, and also how fast it grows using these nutrients. And we also need to understand the molecular, physiological, and developmental mechanisms that plants use to optimize nutrient uptake and use. And, just some basic concepts about nutrition… there are two types of organisms in our planet. Autotrophs, that produce their own food… these are essentially photosynthetic organisms… plants, algae, and photosynthetic bacteria. There are also a few microbes that can obtain their food using chemical energy. And heterotrophs, that get their food from other organisms. So these are animals, including humans, fungi, insects, and so on. So, all these organisms feed on autotrophs, so we need to make sure that these get enough nutrients to grow, so that we can feed heterotrophs. And the ecosystem allows that every organism is connected to its environment by a continuous exchange of energy and nutrients, and environmental cues that make them interact with the other organisms to maintain an ecological equilibrium. So, why is plant nutrition important? Because food production requires an adequate crop plant nutrition. If we feed well our plants, if they get enough nutrients, we will have optimal yields. This in turn impacts food production. We need more production and we need healthier food. And to make a sustainable agriculture, we need to consider not only the environmental part, but also the social and economical part of a sustainable agriculture. People doing agricultural activities need to make a living in a way that is satisfactory and has the same standards as people living in urban areas. It has to be also economically feasible, because we can make an environmentally friendly agriculture that is economically not feasible because the productivity is not sufficient to supply enough food. So, we need to make a sustainable agriculture in social, economical, and environmental terms. So, some important aspects of plant nutrition… the plant organic mass is produced from atmospheric CO2 and water from the soil, so most of the carbohydrates that form part of a plant come from these two compounds. But also plants require mineral nutrients from the soil, so they take up through the roots minerals and they are essential for plant growth. There are about 50 inorganic elements found in plants, but they are not all essential. An element is essential only if it required to complete the life cycle of a plant. Where do plant nutrients come from? So, water and essential minerals are taken up from the soil by the root system. So, essentially all water and minerals are taken up by the root from the soil. Carbon dioxide diffuses into the leaves from the atmosphere through pores in the leaves that are called stomata. So, CO2 enters the leaves and, through the process of photosynthesis, is used to produce carbohydrates. A large portion of the water that’s taken up by plants is evaporated through leaves to facilitate the transport of minerals from the root to the upper parts of the plant. So, not all the water needed by plants is maintained in the plant; a large percentage is evaporated through a streaming process to help nutrients go up to the upper parts of the plant. About 95% of the dry weight of the plant is organic matter. Only 5% is inorganic. Most of the material in a plant is carbohydrate, such as cellulose. We can see this very large tree, in which most of the matter is cellulose composing the cell walls of the plant. Therefore, carbon, hydrogen, and oxygen are the most abundant elements in the dry weight of a plant, because they form part of carbohydrates. However, some organic molecules contain nitrogen, sulfur, and [phosphorus]. Therefore, these elements are also relatively abundant in plants. So, how do we define an essential nutrient? So, an essential nutrient is required for the plant to complete its life cycle, as I said before, but it has to be shown that the specific effects of a deficiency can only be corrected by supplying that same element, and the element must be directly involved in plant physiology, independently of possible effects on the microbiological or chemical soil conditions. To date, 17 elements have been identified as essential nutrients for plant growth. Nine of these are macronutrients that are required in relatively large amounts: carbon, oxygen, hydrogen, nitrogen, potassium, calcium, magnesium, phosphorus, and sulfur. And eight are considered micronutrients because they are required in very small amounts. These are chlorine, iron, zinc, manganese, boron, copper, nickel, and molybdenum. There are other elements such as sodium, silicon, and selenium that are required for maximum biomass production in some plant species and therefore can be considered as functional nutrients, but since they are not needed by all plant species, they cannot be considered essential nutrients. So, how is CO2 and carbon used as a nutrient in plants? So, this is through the process of photosynthesis. Plants use the energy of sunlight and water to produce sugars. So, CO2 enters the plant leaf through the stomata, it reaches the chloroplast, which is an organelle in the cell that takes care of photosynthesis. So, sunlight and water are used to produce high energy compounds that, through the cycle called the Calvin cycle, are used to produce sugars, and these sugars are later on converted into amino acids, lipids, and all the organic molecules that are present in a plant. So, among the essential nutrients, nitrogen is one of the most important. It’s the most abundant element in the Earth’s atmosphere. It is the 4th most abundant element in a plant, after carbon, hydrogen, and oxygen. And often it is the most limiting nutrient for plant growth. Nitrogen is essential for plants because it is a component of amino acids, nucleic acids, chlorophyll, and many other small molecules that participate in the biochemical processes of a plant. Phosphorus is also a very important nutrient. It is the 5th most abundant element in a plant and it is the 1st or 2nd most commonly limiting nutrient for plant growth in agricultural land. It plays important roles in energy and information transfer, as it is a component of DNA, RNA, phospholipids, and also as part of high energy compounds such as ATP. The small micronutrients have either structural activities or they can act as catalytics in enzyme reactions. So, boron acts as a structural component of cell walls by linking cellulose fibers in the cell wall. Some other nutrients remain in ionic form and help enzymatic activities and maintain osmotic pressure. So, for instance, potassium is a co-factor for over 40 enzymes, and is the principal cation to maintain cell turgor and control of membrane potential. Calcium contributes to cell wall and membrane structure and also acts in signaling processes for different biochemical reactions in the plant cell. Magnesium is a co-factor for enzymes required for phosphate transfer, and it is also a structural component of chlorophyll. Here we show the molecule of chlorophyll, in which a molecule of magnesium coordinates a porphyrin-like ring structure. There are also micronutrients that act as redox agents. These are involved in the transfer of electrons in photosynthesis and also in the respiratory chain to produce high energy compounds in the physiology of the plant and other organisms. Now, plant nutrition involves two major components. One is nutrient intake, in which the plant takes up nutrients from the soil, and the other one is how efficient is the plant in utilizing these nutrients to produce biomass, seeds, and fruits. So, nutrient uptake efficiency has several components. One of them is root architecture. Root architecture influences what is the capacity of the plant to explore the soil to acquire nutrients. Also the presence of transporters in the roots… we need nitrogen transporters, we need phosphate transporters, sulfur transporters… the interaction with microbes that help the plant to be more efficient in nutrient uptake, and also how much exudates… organic molecules the plant exudates to interact with microbes. Nutrient utilization, on the other hand, also involves different processes. So, for instance, transport from the roots into the upper parts of the plant, how efficiently the plant can transport these molecules… how it regulates the homeostasis of different nutrients… and also what is the efficiency of assimilating and mobilizing these nutrients from older parts of the plant to younger parts, and finally for seed production and fruit production. How efficient… how much biomass can the plant produce with the same amount of nutrients? Now, how do nutrients get into the plant? So, nutrients are present in the form of ions in the soil, and they enter the plant through the roots, through the epidermal cells of the roots, and a large portion of the epidermal cells of the roots form these protrusions that are called root hairs. These root hairs comprise about 70-80% of the root surface and it is here in these root hairs in which nutrients enter the plant. So, we have transporters for ammonium and nitrate, we have transporters for phosphate, we have transporters for phosphite, and all other nutrients, and these nutrients are transported from root hairs into the rest of the root, until they reach the xylem, and then through the xylem they are sent to the rest of the plant. So, how do the ions get into the plant? They get into the plant through transporters. These transporters can be facilitators that just help the ion enter the cell if the concentration is higher outside than inside, but in most of the cases the concentration of the nutrient is lower outside the cell than inside, so we need transporters that require a potential – either a potential in the membrane, or that needs energy in the form of ATP – to transport nitrate, magnesium, sodium, calcium, zinc, and phosphate. So, how much nutrient do we need for plant growth? So, if we have very little nutrient, the plant almost doesn’t grow. It achieves a little bit of growth from whatever is stored in the seed. If we increase the amount of nutrient, we will see that there is an increase in growth and yield, until it reaches a maximum, in which if we keep adding nutrient it will not help the plant to produce more, but eventually we will reach a zone in which if we add too much nutrient it will be toxic, it will become toxic. So, this is particularly important for certain nutrients such as phosphate, that if we supply to the plant in excessive amounts can become toxic for plant growth. So now, what happens if we don’t have sufficient nutrients in the soil? The plant suffers and you can see the symptoms on the plant. So, nutrient deficiency symptoms appear when one or more nutrients are present in limiting concentrations, and deficiency can occur not because the nutrient is not in sufficient amounts in the soil, but because the soil chemistry doesn’t allow the presence of the nutrient in a form that is available for the plant uptake. Here we see, for instance, the symptoms of deficiency of manganese on leaves. So, the most important mineral deficiencies in plants are those caused by nitrogen, phosphorus, and potassium deficiencies, and that’s why these three compounds are the most common elements used in fertilizers. So, a healthy plant looks green and shiny, but if you have phosphorus deficiency you see the accumulation of a purple staining in the edges of the leaves, which is due to the accumulation of anthocyanins, which directly point toward a phosphorus deficiency. If you have potassium deficiency, you see the dying of the edges of the leaf, and if you have nitrogen deficiency you can see this yellowing of the central part near the central vein of the leaf. So, the nutrition of the plant is not only determined by the availability of nutrients from the soil. Bacteria and soil microbes play a very important role in plant nutrition. So, there are bacteria that are endophytic bacteria – they are inside the plant, they live inside the plant – and these bacteria are associated intimately with the plant, and it has been proposed that these plants can be even… these bacteria can even be inherited, through the seed, to the next generation. The plant also produces root exudates that attract beneficial microbes that help the plant to have better nutrition. For instance, the plant associates with mycorrhizae and nitrogen-fixing bacteria that help them provide the plant with nitrogen and phosphorus. The plant also associates with plant growth promoting bacteria – bacteria that produce substances that make the plant healthier and promote their growth and productivity. There are also bacteria that help the plant to get nutrients from the soil, because they solubilize compounds which are not available for the plant to take. And of course it can also associate with pathogens, which are detrimental for plant nutrition and plant growth. So, the metabolism of bacteria, of soil bacteria, is very important because one of the things that can happen is these bacteria can make nitrogen available for plants. One very important issue to consider is that plants cannot use nitrogen in the form of N2, the elemental nitrogen that is present in the atmosphere. So, although nitrogen is very abundant in our planet, plants cannot directly use it from the atmosphere. It first has to be converted into ammonium or nitrate, and this is being done by nitrogen-fixing bacteria that restock nitrogenous minerals in the soil by converting atmospheric nitrogen into ammonia via a process called nitrogen fixation. These bacteria can be free living in the soil or they can be associated with the plant in symbiosis. The metabolism of soil bacteria makes nitrogen available in the plant and helps to have the nitrogen cycle. So, atmospheric nitrogen is fixed by nitrogen fixing bacteria that convert it into ammonia. Soil bacteria can also produce ammonia from organic materials present in the soil. This ammonium is then converted into nitrate by nitrifying bacteria. This nitrate is taken up by the root, through the root hairs, and then converted into ammonia, which is then used to produce amino acids and other compounds inside the plant. But this nitrogen can also be converted into elemental nitrogen by denitrifying bacteria. This elemental nitrogen goes to the atmosphere and completes the cycle… starting again for nitrogen fixation. So, those… the presence of soil microbes and the role of soil microbes in plant nutrition is quite important, because they can supply nitrogen to the plant. This interaction between plants and bacteria can be even in a symbiotic interaction, in which bacteria help the plant by fixing nitrogen. This is particularly important for legumes, such as peas and beans, that form symbiotic relationships with nitrogen fixing bacteria. The bacteria supplies the plant with fixed nitrogen for assimilation into organic compounds such as amino acids, and the plant in turn provides the bacteria with carbohydrates and other organic compounds to sustain bacterial growth. Legumes form nodules in which their bacteria lives inside, they fix nitrogen, and exchange organic compounds with the plant. So, this is a picture. I guess we can see these round protrusions in the root – they are called nodules – and in these nodules is where nitrogen fixation takes place. The nitrogen fixing bacteria lives inside these nodules and helps the plant to get nitrogen in the form of organic compounds. This topic has been presented in detail by professor Sharon Long in this series of iBiology seminars, so I recommend you to see the presentation of professor Sharon Long to understand the details of the molecular biology of nitrogen fixation. So, there are other types of organisms that help plants to improve nutrition. These are mycorrhizae. They are symbiotic associations of roots with fungi that enhance plant nutrition. The fungi increases the root surface area to increase water uptake and nutrient uptake. So, it’s like the root gets enhanced to cover a much larger area because the fungi grows much faster than the root. The fungi also is a great growth factor that stimulates root growth and branching, and also produces antibiotics that protect that plant against infections by bacteria and other fungi. In turn, the association provides the fungi with a beneficial environment and a steady supply of sugars to grow. Mycorrhizae takes two major forms: ectomycorrhizae and endomycorrhizae. In the ectomycorrhizae, the fungi covers the root and allows the plant to have a larger area of nutrient absorption, and some in fact grows into the spaces between root cells, but they don’t enter the root cells. In the case of the endomycorrhizae, these endomycorrhizae actually enter the cell and they produce this ramified structures that are called arbuscules, and that’s where the exchange of nutrients and carbon takes place between the fungal cells and the plant cells, helping the plant to absorb more water and nutrients. So, these extensions serve as a way of being more efficient in exploring the soil to have better access to water and nutrients. Mycorrhizae and nodules have an evolutionary relationship. Mycorrhizae evolved 400 million years ago, during the adaptation of aquatic plants to land. They probably played a very crucial role to help aquatic plants adapt to the low level of nutrient availability in the soil. We should remember that these primitive aquatic plants had very primitive root systems, and they required an association with mycorrhizae to be able to extract nutrients from the soil. Root nodules in legumes originated between 65 and 150 million years ago, during the early evolution of angiosperms, and they have been proposed to have a common evolutionary organism, because the genes activated during the initial stages of root nodule formation and mycorrhizae interaction are the same. And mutations in these genes affect not one, but both processes, suggesting that they use the same system that was established for mycorrhizae, then was used by the interaction with nitrogen-fixing bacteria. The root microbiome is much more than rhizobium and mycorrhizae. If we consider that the plant has between 37,000 and 40,000 genes, we know should consider that it interacts with fungi, with nematodes, with arthropods, with bacteria, with algae… and we should take into account that all these organisms play an important role in helping the plant achieve an optimal nutrition and also to sustain better growth. So, this should be considered as the whole genome of a plant, in which we should take into account not only the genes present in the plant genome, but also all the genes that are present in the microbes that are associated in what we call the root of the plant microbiome, because they will provide biochemical and physiological functions that are needed for optimal plant growth. So, the root microbiome contributes to plant nutrition, health, and development. So, there are microbes that help the plant to grow and develop better. They activate the immune system, so that they are better prepared to withstand infection by pathogens. They also help the plant to better tolerate drought. They assist the plant to enhance nutrient uptake. And they can also directly protect the plant against pathogens because they can produce antibiotics and enzymes that actually act against these pathogens. So, as in the case of humans, the microbiome associated with plants plays an essential role for the physiology and survival of plants, and we have to think about this as a major tool for agriculture because it has been proposed that if we have the right microbiome we could enhance plant productivity and achieve a more sustainable agriculture, because it will help us to reduce the need of water and fertilizers, herbicides, and pesticides required for optimal agricultural production, if we associate the right microbiome with a plant. Here is just an example – these are coffee plants in a nursery, grown with a biofertilizer and chemical fertilizer alone. They have the same amount of chemical fertilizer applied in both cases, but if the plant is inoculated with a bacteria and a fungi, they sustain much better growth and produce more biomass than when you add only the chemical fertilizer. So, this shows the importance of the presence of the right microbiome associated with the plant to achieve better growth and also to have healthier plants. So, nutrient availability in the soil is an important issue that we should consider to produce more sustainable agriculture. Nutrients are naturally present in all soils, and bacteria and fungi assist in nutrient uptake. So, we have care of understanding what is the role of the microbiome and how we can use this to enhance plant productivity. Often, nutrients have low availability in the soil and limit plant growth and productivity, so we need to make sure that the soil conditions are sufficient to have an efficient supply of nutrients to the plant. We should remember that harvesting plant biomass or seeds removes nutrients from the soil that need to be replaced. So, what do we have to do? We need to apply fertilizers to supplement soils with nutrients to achieve high yields. This is to replace what we have removed. Each time we harvest, w e remove nutrients from the soil… we have to replace. And to achieve a sustainable agriculture, we need to fully understand plant nutrition to design a more sustainable agriculture. What is the best form to apply fertilizers? What is the chemical form of the fertilizer that we need to apply? How do we need to apply the fertilizer? And how is the association of plants with microbes necessary to achieve a better nutrient uptake and utilization? So, that’s all of what I wanted to tell you about this introductory talk about plant nutrition, and I am just showing a picture of my students that have worked with me and discussed many of these issues for the past few years in my laboratory. Thank you very much.