In a previous study, we noticed a significant reduction in leaf nitrogen concentration in the phase that precedes the maturation of sugarcane in field conditions. Although we know that there is a strong relationship between nitrogen and photosynthesis in higher plants, the processes underlying the assimilation of CO2 affected by nitrogen are poorly understood in C4 plants, such as sugarcane. Low photosynthesis associated with low leaf nitrogen content could be a consequence of the inefficiency of the CO2 concentration mechanism in the bundle-sheath cells, where the carboxylation by the enzyme Rubisco occurs. In C4 metabolism, not only the enzymes PEPC and Rubisco have a key role in assimilation of CO2, but also the resistances that determine the CO2 concentration at the carboxylation sites. The importance of stomatal conductance for photosynthesis has been extensively studied, while mesophyll conductance is a current research topic on C4 photosynthesis. However, the role of bundle-sheath conductance has been underestimated to such an extent that we do not know the importance of this variable in photosynthesis regulation of C4 plants such as sugarcane. Considering the current literature, this proposal has as hypothesis that low leaf nitrogen content reduces sugarcane photosynthesis due to decreases in activities of both carboxylation enzymes PEPC and Rubisco, without affecting the mechanism of CO2 concentration in bundle-sheath cells. The proposal also aims to reveal the relative importance of stomatal, mesophyll and bundle-sheath conductances for regulation of sugarcane photosynthesis under varying nitrogen availability. The major photosynthetic variables linked to C4 metabolism in mesophyll cells and C3 metabolism in bundle-sheath cells will be estimated in vivo using a biochemical model of C4 photosynthesis based on combined measurements of leaf gas exchange and chlorophyll fluorescence. These evaluations will be carried out in sugarcane plants grown under five nitrogen doses, with leaf nitrogen content and accumulation of biomass being also evaluated.

Funding: FAPESP (2017/12302-2); Period: Feb to Dec 2018; R$ 10,230.00 + US$ 48,368.44

People: RV Ribeiro (PI); PC Struik; Y Yin; EC Machado; VR Tofanelo

The climatic variations will cause drought in cultivated areas, reducing the crop yield and affecting the food security of millions of people around the World. Research devoted to understand the crop physiology under water deficit is useful for plant breeding and also to guide researches on transgenic plants. At the beginning of water deficit, plant roots produce signals for stomatal closing, avoiding excessive water loss. Plant signaling is a promising research theme when considering the water use efficiency. This research has as aim to evaluate the role of chemical and hydraulic signals on citrus tolerance to drought. Two experiments will be carried out: firstly, seedlings of 'Valencia' sweet orange, 'Rangpur' lime or 'Swingle' citrumelo plants will grow in pots with the root system divided two fractions, where one fraction will be subjected to water deficit and the other will be irrigated; secondly, 'Valencia' sweet orange plants grafted on 'Rangpur' lime and also on 'Swingle' citrumelo rootstocks (plants with two root systems) will be subjected to water deficit, maintaining one root system well-hydrated. Evaluations of plant water relations, gas exchange, plant hydraulic conductance, growth, abscisic acid concentration in xylem and leaves, xylem pH, sap flow, aquaporin activity and resistance to cavitation will be done, as well as the anatomical study of leaves and roots for a better understanding of plant strategies under water deficit.

Funding: FAPESP (2011/22383-3); Period: Jun 2012 to Nov 2014; R$ 74,190.17 + US$ 66,183.15

Coordination: RV Ribeiro

The water transport in plants is not a completely passive process and the presence of ions in the sap can increase the xylem conductance. Hypothetically, hydrogels (pectins) on intervessel pit membrane may shrink if ions are present in the sap, which results in reduced resistance to water flow. However, this explanation is challenged by the lack of pectins in mature intervessel pit membranes. Alternatively, we hypothesize that the ionic effect is related to radial flow, i.e., water transport from dead xylem conduits to surrounding living cells that are able to store water. Along this pathway, pectins occur in vessel-parenchyma pit membranes, which may explain the increment of hydraulic conductance in the presence of ions. In our main FAPESP post-doctoral project, we develop a method to study radial flow that can be validated and improved using pre-characterized species in relation to the ionic effect, as those ones studied by Dr. Steven Jansen's group. Thus, our goal is to test if the ionic concentration of the xylem liquid determines radial flow in various Acer species, and whether this may explain the ion-mediated effect. Radial flow in plant stems is fundamental for the understanding of plant water transport by the functional link between xylem and phloem and drought resistance mechanisms, which also determines the plant productivity under varying water conditions.

Funding: FAPESP (2019/08596-6); Period: Sep to Dec 2019; R$ 28,514.30 + US$ 13,295.84

People: L Pereira (PI); S Jansen; RV Ribeiro (co-PI)

Sugarcane is largely grown in rainfed areas, where water deficit is a frequent environmental challenge. In previous experiments and independent of water availability, we found higher growth of plants originating from mother plants (plants in which propagation material was obtained) subjected to drought, an evidence of transgenerational memory. If this mechanism of drought resistance is proved, plant performance in field conditions could be improved by choosing propagation material or even by new techniques of producing pre-sprouted plants under nursery. Thus, the objectives of this study are (i) to test the hypothesis that plants obtained from mother plants cultivated under water deficit are more resistant to drought and have higher growth than those obtained from mother plants grown without water restriction and (ii) to evaluate the persistence of memory and the resilience to drought of daughter plants, considering the leaf gas exchange. Sugarcane plants (mother plants) will be submitted to three cycles of water deficit by water withholding (MDH) or grown with daily irrigation (MI) until the maturation phase. Then, plants (daughter ones) will be obtained from MDH and MI plants and submitted to one or two cycles of water deficit at the tillering phenological stage. As references, plants obtained from MDH and MI plants will remain well irrigated. In addition to leaf gas exchange measurements, photochemical activity, chlorophyll content, leaf water potential, relative water content, hydrogen peroxide, lipid peroxidation and the antioxidant metabolism, biomass production and leaf area will be evaluated.

Funding: FAPESP (2018/21194-1); Period: Mar to Jun 2019; R$ 8,588.05

People: KA Vitti; RV Ribeiro (PI)

Water and nitrogen are some of the most limiting factors in world agricultural production, being both factors strongly associated with plant photosynthesis. Water deficit reduces CO2 assimilation as a consequence of decreases in stomatal conductance and metabolic impairment. Changes in nitrogen metabolism under water deficit are poorly understood in C4 plants and any information is still fragmented. It is known that the radical nitric oxide (NO) is an important signaling molecule in several biological systems, protecting cells by reducing free radicals formed under oxidative stress. In such scenario, the influence of nitrogen nutrition on physiological responses of sugarcane to water deficit will be evaluated, considering the photosynthetic activity, antioxidant metabolism and plant growth. The hypothesis to be tested is that plants that receive nitrate as source of nitrogen will have higher activity of nitrate reductase and produce more NO under water deficit, reducing the oxidative damage caused by ROS and favoring the photosynthetic metabolism under low water availability. Sugarcane plants will be supplied with different sources of N (ammonium or nitrate) and subjected to water deficit by adding PEG-8000 to the nutrient solution (osmotic potential of -0.75 MPa).

Funding: FAPESP (2017/11279-7); Period: Aug 2017 to Jul 2019; R$ 47,256.00

People: MD Pissolato; RV Ribeiro (PI)

Agriculture is the human activity with the world's highest consumption of water, the main environmental resource limiting crop yield. Citrus plants are subject to seasonal variation in water availability in many areas of cultivation and an effective strategy to mitigate the impacts of water scarcity in the citrus industry is the use of rootstocks tolerant to drought. Studies suggest that a significant amount of water can be transported from a wet portion to a dry portion of the root system, with the dry fraction of the root system becoming an additional water sink. The osmotic adjustment is a process by which the cell water potential can be reduced by the active accumulation of osmotic substances, being an important physiological adaptation to drought. As plants are able to uptake water of dry surrounding environment, the osmotic adjustment favors the cell turgor, gas exchange and growth in dry conditions. This study aims to test the hypothesis that drought tolerance in citrus species is associated with the ability of redistribute water within root system, revealing which morpho-physiological characteristics are associated with such hydraulic redistribution. To test this hypothesis will carry out an experiment with Valencia orange trees grafted on three different rootstocks, using a split-root system. One part of the root system will be subjected to water withholding while the other part will be well-watered. Measurements of sap flow in both rootstocks and in scion, leaf turgor, leaf water potential and leaf gas exchange will be taken under growth chamber conditions. Osmotic adjustment and plant biometry will also be evaluated. We expected that the water redistribution varies with the rootstock used and that the osmotic adjustment is an important physiological strategy associated with root water redistribution.

Funding: FAPESP (2016/02199-7); Period: Mai 2016 to Feb 2018; R$ 47,256.00

People: MT Miranda; RV Ribeiro (PI)

Water shortage is likely to be one of the major global environmental stresses of the 21st century. There are recent evidence that support that nitric oxide (NO) play an important role in the defense mechanisms of plants under environmental stress, including drought. However, the relation between drought tolerance of plants and NO metabolism is still unclear. As NO is a molecule related to environmental stresses, we hypothesize that NO accumulate in sugarcane plants under water deficit, with such response being associated with the degree of drought-tolerance of different genotypes. The aim of the present study is to investigate the NO metabolism by analyzing the synthesis and degradation of NO in two sugarcane genotypes with differential tolerance to water deficit. Water deficit will be induced in plants growing in nutrient solution by adding PEG-8000. This treatment will reduce the osmotic potential of nutrient solution to -0.75 MPa. The activities of nitric oxide synthase (NOS), nitrate reductase (NR), Snitrosoglutathione reductase (GSNOR) will be analyze and the production the NO will be detect by fluorescence microscopy technique.

Funding: FAPESP (2015/21546-7); Period: Mai to Jun 2016; R$ 930.00 + US$ 6,336.09

People: NM Silveira; JT Hancock; RV Ribeiro (PI)

An effective strategy to mitigate the impacts of water scarcity on citrus production is the use of rootstocks tolerant to drought. The Rangpur lime rootstock is able to change the sensitivity of plant shoot to drought, maintaining the availability of carbohydrates and root growth under unfavourable conditions. These responses indicate the ability to coordinate the source-sink relationship, adjusting the balance between synthesis and consumption of carbohydrate through changes in photosynthesis, respiration and carbohydrates partitioning. In such context, this proposal will test the hypothesis that carbohydrate metabolism is more active in drought-tolerant rootstock, which would lead to further root growth and increase the sink strength. As consequence, there would be an increase in import of photoassimilates in roots and a stimulation of photoassimilates exportation from leaves. The three rootstocks most used in Brazilian citrus industry, Swingle citrumelo, Sunki tangerine and Rangpur lime will be used in two experiments to test this hypothesis. Inside a growth chamber, plants of each rootstock will be divided into two lots and one of the lots will be subjected to water withholding for 18 days following substrate rehydration. Gas exchange, leaf water potential, biometry, contents of carbohydrates in leaves, stem and roots, activity of key enzymes of sucrose metabolism and the determination of ATP will be evaluated. In the second experiment, the plants will receive a gas mixture containing 13C before the water deficit, and measurements of gas exchange and leaf water potential and analysis of 13C partitioning and phloem sap will be taken.

Funding: FAPESP (2015/14817-4); Period: Nov 2015 to Jul 2019; R$ 185,293.02

People: SF Silva; EC Machado; RV Ribeiro (PI)

Ozone (O3) is the most toxic air pollutant with detrimental effects on plant health. Tropospheric O3 is produced by complex photochemical reactions involving biogenic volatile organic compounds (BVOCs). O3 is seasonally distributed in temperate climates, however, it occurs during the entire year with toxic concentration being registered along the whole year in tropical and subtropical climates. Most of the important agricultural crops responds to O3 stress. However, the detrimental effect of O3 is unknown in sugarcane, one of the most important crop species in Brazil, especially in the state of Sao Paulo. Inside the leaves, O3 phytotoxicity is due to the generation of reactive oxygen species (ROS). When ROS formation exceeds the antioxidant capacity of cells, an oxidative stress occurs leading to the specific structural oxidative marks and visible injuries. Based on the hypothesis that O3 levels are toxic to sugarcane plantations, the present study aims to: (i) assess the morphological, anatomical, biochemical and physiological responses of sugarcane varieties to O3. A fumigation experiment will be carried out in a free-air facility, where plants will be subject to O3 treatments and (ii) understand the BVOCs production by sugarcane varieties. During the experiment, biomass production, leaf gas exchange, photochemistry, antioxidant metabolism and structural oxidative stress marks will be evaluated. Stomatal O3 uptake will be calculated and BVOCs emission will be verified. For the first time, we will be able to understand how sugarcane reacts to O3 stress and if O3 sensitivity is genotype-dependent.

Funding: FAPESP (2014/23839-9); Period: May 2015 to Aug 2017; R$ 75,965.12 + US$ 38,430.25

People: BB Moura; E Paoletti; RV Ribeiro (PI)

Ozone (O3) is the most toxic air pollutant with detrimental effects on plant health. Tropospheric O3 is produced by complex photochemical reactions involving biogenic volatile organic compounds (BVOCs). O3 is seasonally distributed in temperate climates, however, it occurs during the entire year with toxic concentration being registered along the whole year in tropical and subtropical climates. Most of the important agricultural crops responds to O3 stress. However, the detrimental effect of O3 is unknown in sugarcane, one of the most important crop species in Brazil, especially in the state of Sao Paulo. Inside the leaves, O3 phytotoxicity is due to the generation of reactive oxygen species (ROS). When ROS formation exceeds the antioxidant capacity of cells, an oxidative stress occurs leading to the specific structural oxidative marks and visible injuries. Based on the hypothesis that O3 levels are toxic to sugarcane plantations, the present study aims to: (i) assess the morphological, anatomical, biochemical and physiological responses of sugarcane varieties to O3. A fumigation experiment will be carried out in a free-air facility, where plants will be subject to O3 treatments and (ii) understand the BVOCs production by sugarcane varieties. During the experiment, biomass production, leaf gas exchange, photochemistry, antioxidant metabolism and structural oxidative stress marks will be evaluated. Stomatal O3 uptake will be calculated and BVOCs emission will be verified. For the first time, we will be able to understand how sugarcane reacts to O3 stress and if O3 sensitivity is genotype-dependent.

Funding: FAPESP (2014/13524-0); Period: Oct 2010 to Aug 2017; R$ 175,701.24

People: BB Moura; RV Ribeiro (PI)

Drought is a major limiting factor in crop yield, plants generally show a reduction in the rates of photosynthesis and growth, often associated with changes in plant metabolism. Nitric oxide (NO) has emerged as an important signaling molecule in various biological systems. It is postulated that NO plays a protective role by acting mainly on the interruption of free radicals formed under oxidative stress. Given the above, our objective is to evaluate the influence of NO in physiological responses of sugar cane (Saccharum spp) to drought stress. Additionally, we intend to determine the main sources of NO in genotypes with differential sensitivity to drought. Genotypes will be subjected to drought by adding PEG 8000 to the nutritive solution (osmotic potential of -0.75 MPa). In addition to evaluating the differential production of NO by plants limiting conditions, we also analyze the role of NO delivered through nutrient solutions of sodium nitroprusside (SNP). Evaluations will be divided into three experiments; the first will deal with the activity of enzymes synthesis, beyond the detection of NO in plants. The second will address the physiological analyzes such as gas exchange, photochemical activity, photosynthetic pigments, enzymes of photosynthetic metabolism and growth analysis in order to verify if NO is capable of attenuating growth inhibition and impairment of the electron transport chain in photosynthesis under stress conditions. The last experiment answer is NO potentiate antioxidant mechanisms, therefore, we will perform analyzes of reactive oxygen species and antioxidant enzyme system.

Funding: FAPESP (2012/19167-0); Period: Apr 2013 to Nov 2016; R$ 147,169.67

People: NM Silveira; EC Machado; RV Ribeiro (PI)

The earth's climate change will cause drought in many regions, reducing plant productivity and threatening the food security of millions of people. Researches to understand the physiology of plants under water stress will be useful to selection of drought tolerant materials and to guide transgenic plants researches. At the beginning of soil water deficit, plant roots produce signals that induce stomatal closure, which prevent excessive water losses. So, the study of signals is one of the most promising areas in the water-use efficiency in plants. The proposed research aims to evaluate the participation of hydraulic and chemical signals in the drought tolerance of two citrus rootstocks. To this end, two experiments will carry out: first, Valencia sweet orange, Rangpur lime and Swingle citrumelo seedlings will be grown with the root system split between two containers, with approximately half the system in each container. It will allow that half of the root system is expose to dry soil while the remaining half is irrigate. In the second experiment Valencia sweet orange seedlings will be inarching on two rootstocks (Rangpur lime and Swingle citrumelo), so it keeping the water deficit in one rootstock at a time. We will evaluate the water relations, gas exchange, hydraulic conductivity, growth, xylem ABA concentration, xylem pH, sap flow, and aquaporins expression.

Funding: FAPESP (2011/16263-5); Period: Nov 2011 to Oct 2014; R$ 228,743.28

People: E Espinoza-Nunez; EC Machado; MT Miranda; RV Ribeiro (PI)