Knowledge about the length of xylem vessels is essential to understand water transport in plants because these multicellular units show a 100-fold variation, from less than a centimeter to many meters. However, the available methods to estimate vessel length (VL) distribution are excessively time consuming and do not allow large and in-depth surveys. Here, we describe a semi-automated method to measure hydraulically-weighted VL (VLH) using an automated Pneumatron device. Gas conductivity of a xylem tissue with a certain length is estimated with the Pneumatron in a straightforward and precise way, theoretically similar to the air-injection method. Besides giving results similar to the silicone-injection method, the pneumatic-based method enables faster and easier measurements of more than 50 samples per day, which is a significant advantage. Herein, a detailed description of the methodology is presented as well as the software and an R-script for data analysis. The method described represents an important contribution to studies on plant hydraulic architecture and can improve our understanding about the role of VLH in plant performance under varying water availability.

Luciano Pereira, Marcela Trevenzoli Miranda, Gabriel Silva Pires, Vinícius Sacramento Pacheco, Xinyi Guan, Lucian Kaack, Zohreh Karimi, Eduardo C. Machado, Steven Jansen, Melvin T. Tyree& Rafael Vasconcelos Ribeiro

It is time to recognize the existence of plant memory, a phenomenon that allows plants to store and recall information from previous events and then change their responses to future stressful conditions. Although many recent publications have addressed this topic and concluded about plant memory using either classical or advanced approaches, several scientists are still armored against this important concept inherent to life and found in plants. Herein, our aims are to clarify the concept of memory and its use in plants and to overview the mechanisms underlying plant memory. How do plants store information? Where is such information stored? How long can information be stored? How does memory affect the organization of a given system with such ability? These are some questions that deserve attention from the scientific community rather than inadequate and incoherent ones challenging the existence of plant memory. Let us move on to the next page and deal with plants as living beings.

Yutcelia C. F. Galviz, Rafael Vasconcelos Ribeiro& Gustavo M. Souza

Nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. Nitrate reductase (NR) not only mediates the reduction of NO3− to NO2− but also reduces NO2− to NO, a relevant pathway for NO production in higher plants. Herein, we hypothesized that sugarcane plants supplied with more NO3− as a source of N would produce more NO under water deficit. Such NO would reduce oxidative damage and favor photosynthetic metabolism and growth under water limiting conditions. Sugarcane plants were grown in nutrient solution and received the same amount of nitrogen, with varying nitrate:ammonium ratios (100:0 and 70:30). Plants were then grown under well-watered or water deficit conditions. Under water deficit, plants exhibited higher root [NO3−] and [NO2−] when supplied with 100% NO3−. Accordingly, the same plants also showed higher root NR activity and root NO production. We also found higher photosynthetic rates and stomatal conductance in plants supplied with more NO3−, which was associated with increased root growth. ROS accumulation was reduced due to increases in the activity of catalase in leaves and superoxide dismutase and ascorbate peroxidase in roots of plants supplied with 100% NO3− and facing water deficit. Such positive responses to water deficit were offset when a NO scavenger was supplied to the plants, thus confirming that increases in leaf gas exchange and plant growth were induced by NO. Concluding, NO3− supply is an interesting strategy for alleviating the negative effects of water deficit on sugarcane plants, increasing drought tolerance through enhanced NO production. Our data also provide insights on how plant nutrition could improve crop tolerance against abiotic stresses, such as drought.

Maria Dolores Pissolato, Neidiquele Maria Silveira, Paula Joyce Carrenho Prataviera, Eduardo Caruso Machado, Amedea Barozzi Seabra, Milena Trevisan Pelegrino, Ladaslav Sodek & Rafael Vasconcelos Ribeiro

Understanding hydraulic failure of angiosperm xylem and its underlying mechanisms are relevant to reveal how plants respond to climate change, especially during severe drought events. Various methods to quantify embolism resistance have been developed over the last decades, including hydraulic (e.g., bench dehydration and centrifuge methods), and non-hydraulic approaches (e.g., the optical method and microCT). However, there is a need to develop a low-cost and relatively fast method that can be applied easily by plant scientists to hundreds of samples. Pneumatic measurements offer a highly promising approach to fulfil these requirements. The Pneumatron measures the kinetics of pressure change under partial vacuum while extracting gas from a cut open xylem tissue. The method is based on Fick's law for diffusion, Henry's law for partitioning of gas concentration between liquid and gas phases at equilibrium, and the ideal gas law. Our pneumatic measurements demonstrate that the molar air discharge shows a striking similarity to embolism resistance of xylem based on traditional methods. A major advantage of the Pneumatron is that potential artifacts associated with hydraulic measurements are avoided, while high accuracy is obtained with the automated design of the device. It can be applied to various organs (stem, roots, leaves), and will be useful to study the mechanisms behind air-seeding.

Steven Jansen, Xinyi Guan, Lucian Kaack, C. Trabi, Marcela Trevenzoli Miranda, Rafael Vasconcelos Ribeiro& Luciano Pereira

Downy mildew caused by Plasmopara viticola is one of the most destructive diseases of Vitis vinifera worldwide. Grapevine breeding programs have introgressed P. viticola-resistant traits into cultivated V. vinifera genotypes and launched interspecific hybrids with resistance against downy mildew. In general, pathogen infection affects primary metabolism, reduces plant growth and development and modifies the secondary metabolism toward defense responses, which are costly in terms of carbon production and utilization. The objective of this work was to evaluate the photosynthesis impairment by inducible defenses at the leaf level in V. vinifera cultivars resistant to P. viticola. Photosynthetic limitations imposed by P. viticola in susceptible and resistant grapevine cultivars were evaluated. Histochemical localization of hydrogen peroxide and superoxide and the activity of ascorbate peroxidase were assessed. Measurements of leaf gas exchange, chlorophyll fluorescence and the response of leaf CO2 assimilation to increasing air CO2 concentrations were taken, and photosynthetic limitations determined in cultivars Solaris (resistant) and Riesling (susceptible). The net photosynthetic rates were reduced (−25%) in inoculated Solaris plants even before the appearance of cell death-like hypersensitive reactions (“HR”). One day after “HR” visualization, the net photosynthetic rate of Solaris was reduced by 57% compared with healthy plants. A similar pattern was noticed in resistant Cabernet Blanc and Phoenix plants. While the susceptible cultivars did not show any variation in leaf gas exchange before the appearance of visual symptoms, drastic reductions in net photosynthetic rate and stomatal conductance were found in diseased plants 12 days after inoculation. Decreases in the maximum Rubisco carboxylation rate and photochemical impairment were noticed in Riesling after inoculation with P. viticola, which were not found in Solaris. Damage to the photochemical reactions of photosynthesis was likely associated with the oxidative burst found in resistant cultivars within the first 24 h after inoculation. Both chlorophyll degradation and stomatal closure were also noticed in the incompatible interaction. Taken together, our data clearly revealed that the defense response against P. viticola causes a photosynthetic cost to grapevines, which is not reversible even 12 days after the pathogen infection.

Antonio F. Nogueira Júnior, Merle Tränkner, Rafael Vasconcelos Ribeiro, Andreas von Tiedemann & Lilian Amorim

New practices to improve the germination and the initial establishment of seedlings are needed to improve crop development in rainfed areas, where drought events may happen just after sowing. Here, we tested the hypothesis that pre-treatment of common bean seeds with S-nitrosoglutathione (GSNO)—a nitric oxide donor (NO)—or calcium (Ca2+) improves the germination and initial seedling growth under water deficit and both, together, will act synergistically. Firstly, common bean seeds were soaked in GSNO solutions at 50, 100, 500, 1000 and 2000 µM and calcium chloride (CaCl2) at 1, 5 and 10 mM. Then, seeds were subjected to well-watered and water deficit conditions. In another assay, seeds were treated as described above and then the seedlings were transferred to nutrient solution and subjected to water deficit imposed by polyethylene glycol (– 0.3 MPa). In general, our data indicated that pre-treatment of common bean seeds with GSNO or Ca2+ improved germination and root growth under water deficit in a dose-dependent manner. Further tests with a NO scavenger (cPTIO), a Ca2+ chelator (EGTA) and reduced glutathione confirmed the roles of both GSNO and Ca2+ in improving seed germination. However, such improvement in early development of seeds pre-treated with GSNO or Ca2+ was short and no differences among treatments were found at the end of the experimental period. Additionally, seed pre-treatment combining GSNO and Ca2+ aggravated the effects of water deficit on germination and initial development of common bean seedlings, indicating that GSNO nulled any positive effect of Ca2+ and vice versa.

Neidiquele Maria Silveira, Rafael Vasconcelos Ribeiro, Paula Joyce Carrenho Prataviera, Maria Dolores Pissolato, Joana C. Pieretti, Amedea Barozzi Seabra& Eduardo Caruso Machado

Drought tolerance is defined by several morpho-physiological mechanisms that together improve plant development under water-limiting conditions. Previously, we found root hydraulic redistribution is one of those mechanisms for water stress avoidance. Herein, we aimed to verify the physiological mechanisms associated with root hydraulic redistribution and its consequences for leaf gas exchange and plant growth. Valencia sweet orange scions were grafted onto either Rangpur lime or Swingle citrumelo rootstock. Each plant had two root systems of the same rootstock in distinct pots, which allowed partial irrigation. Our results revealed that citrus species redistribute water under drought and this varies when comparing rootstocks, with Rangpur lime showing higher ability to redistribute water than Swingle citrumelo. For the first time, root hydraulic redistribution in Rangpur lime was associated with osmotic adjustment in well-watered roots of plants facing water deficit. Rangpur lime also presented an effective stomatal regulation of water loss and decreases in leaf transpiration likely allowed water transport to roots under water deficit. As conclusion, we found that root hydraulic redistribution, osmotic adjustment and stomatal control of leaf gas exchange are important physiological mechanisms associated with drought tolerance induced by Rangpur lime rootstock.

Marcela Trevenzoli Miranda, Simone Ferreira da Silva, Neidiquele Maria Silveira, Luciano Pereira, Eduardo Caruso Machado& Rafael Vasconcelos Ribeiro

The aim of this study was to evaluate the sugarcane responses to varying ammonium:nitrate (NH4+:NO3−) ratio and to reveal how much NH4+ plants can tolerate before showing impairment of photosynthesis and growth. Sugarcane plants were grown in nutrient solution with the following NH4+:NO3− ratios (%): 20:80; 30:70; 40:60; 60:40; 70:30; and 80:20. The lowest photosynthetic rates, stomatal conductance, instantaneous carboxylation efficiency and leaf chlorophyll a content were found in plants supplied with higher than 60% NH4+. The leaf content of chlorophyll b proved to be more sensitive than chlorophyll a and decreases were found from 40% NH4+. We did not observe significant differences in leaf NO3− concentration under varying NH4+:NO3− ratio. However, plants that received 80% NH4+ showed the highest leaf NH4+ concentration and lowest leaf [NO3−]:[NH4+] ratio. The estimated leaf nitrogen content was higher in plants supplied with 20% and 30% NH4+. Taken together, our data revealed that sugarcane plants are sensitive to NH4+, with photosynthesis and plant growth being impaired when NH4+ supply was higher than 30% in nutrient solution. Root biomass was significantly reduced under high NH4+ supply, which explains decreases in stomatal conductance. Besides stomatal limitation, photosynthesis was also limited by low carboxylation efficiency under high NH4+ supply. Apparently, leaf NH4+ concentrations higher than 1.0 µmol g−1 were enough to impair photosynthesis. The balance between [NO3−] and [NH4+] in leaves was more correlated to photosynthesis than either [NO3−] or [NH4+] alone.

Maria Dolores Pissolato, Neidiquele Maria Silveira, Eduardo C. Machado, Fernando C. Bachiega Zambrosi, Ladaslav Sodek & Rafael Vasconcelos Ribeiro

Xylem vulnerability to embolism represents an important trait to determine species distribution patterns and drought resistance. However, estimating embolism resistance frequently requires time‐consuming and ambiguous hydraulic lab measurements. Based on a recently developed pneumatic method, we present and test the “Pneumatron”, a device that generates high time‐resolution and fully automated vulnerability curves. Embolism resistance is estimated by applying a partial vacuum to extract air from an excised xylem sample, while monitoring the pressure change over time. Although the amount of gas extracted is strongly correlated with the percentage loss of xylem conductivity, validation of the Pneumatron was performed by comparison with the optical method for Eucalyptus camaldulensis leaves. The Pneumatron improved the precision of the pneumatic method considerably, facilitating the detection of small differences in the (percentage of air discharged [PAD] < 0.47%). Hence, the Pneumatron can directly measure the 50% PAD without any fitting of vulnerability curves. PAD and embolism frequency based on the optical method were strongly correlated (r2 = 0.93) for E. camaldulensis. By providing an open source platform, the Pneumatron represents an easy, low‐cost, and powerful tool for field measurements, which can significantly improve our understanding of plant–water relations and the mechanisms behind embolism.

Luciano Pereira, Paulo R. L. Bittencourt, Vinícius Sacramento Pacheco, Marcela Trevenzoli Miranda, Ya Zhang, Rafael S. Oliveira, Peter Groenendijk, Eduardo C. Machado, Melvin T. Tyree, Steven Jansen, Lucy Rowland & Rafael Vasconcelos Ribeiro

Grapevine rust Phakopsora euvitis is an important disease in tropical and subtropical grapevine-producing areas, and both American (Vitis labrusca) and European (Vitis vinifera) grapevine species are sensitive to rust. Grapevine rust can cause early plant defoliation, reducing photosynthesis and photoassimilate supply and storage. This study evaluated the photosynthetic damage induced by varying grapevine rust intensities in leaves of V. vinifera cv. Moscato Giallo and V. labrusca cv. Niagara Rosada. The disease effect in tissues adjacent to the lesion, called virtual lesion (β), was estimated for photosynthetic variables, considering measurements taken in both healthy and diseased tissues. The average lesion size was higher in V. vinifera (0.66 mm2) than in V. labrusca (0.24 mm2). A water-soaked halo around the lesion was observed only in V. vinifera. The β-values estimated for the photosynthetic rate were 4.99 for V. labrusca and 5.93 for V. vinifera, taking into account the water-soaked and sporulating areas. When considering only the sporulating area, the β-value increased to 11.49 in V. vinifera. Grapevine rust had similar negative effects on photosynthesis in both species with increasing disease severity. While stomatal conductance was slightly affected, the pathogen caused significant reductions in the effective quantum efficiency of photosystem II and the instantaneous carboxylation efficiency of V. vinifera and V. labrusca with increasing disease severity. The β-value of instantaneous carboxylation efficiency was higher in V. vinifera (9.26) than in V. labrusca (4.76). In conclusion, Phakopsora euvitis dramatically affects photosynthesis in both V. vinifera cv. Moscato Giallo and V. labrusca cv. Niagara Rosada, causing large virtual lesions and substantial damage to the photochemical and biochemical reactions associated with CO2 assimilation.

Barbara L. Navarro, Antonio F. Nogueira Júnior, Rafael Vasconcelos Ribeiro& M. B. Spósito

Downy mildew caused by Plasmopara viticola is the most destructive disease of grapevines. However, the mechanisms of damage caused by P. viticola to photosynthesis in Vitis labrusca remain unknown. Our aim was to quantify the effects of downy mildew on leaf gas exchange in V. labrusca, estimating virtual lesions (regions surrounding lesions where photosynthesis is null) and evaluating limitations imposed on photosynthesis. Grapevine plants cv. Niagara Rosada were inoculated with varying sporangia concentrations of P. viticola to obtain a range of disease severities. Leaf gas exchange and photochemical activity were measured in both diseased and healthy leaves and the eq. Y = (1 - x)β was used to correlate photosynthetic variables (Y) and disease severity (x), with β representing the ratio between virtual and visual lesions. Our results revealed a β-value of 2.79, indicating moderate virtual lesions. Photosynthetic rates decreased by about 83% in leaves with 50% of downy mildew severity and there were significant impairments in the maximum Rubisco carboxylation rate, maximum rate of electron transport driving regeneration of ribulose-1,5-bisphosphate, effective quantum efficiency of photosystem II and mesophyll conductance, even in plants showing less than 10% of downy mildew severity. In conclusion, our results not only reveal how P. viticola affects photosynthesis in V. labrusca, but also give insights into how crop modelling would be improved by taking into account virtual lesions and photosynthetic damage. Such key information is needed for simulation models that quantitatively integrate the interaction between disease and crop growth.

Antonio F. Nogueira Júnior, Rafael Vasconcelos Ribeiro, Fernanda Castro Correia Marcos& Lilian Amorim

Many assays focus on determining NO content within plant tissues to assess the actual concentration that impacts on cellular processes. Diaminofluorescein fluorescent dyes (DAFs) have been very widely used by plant scientists to reveal likely sites of NO production inside and outside cells. In general, DAFs dyes react with N2O3, a byproduct of NO oxidation, resulting in fluorescence. It is initially available in the form of diacetate (DAF-2DA), which allowed the ready absorption by the cells. The diacetate group is removed by cell esterases leaving the membrane impermeable to DAF-2 and available for N2O3 nitration to generate the highly fluorescent triazole (DAF-2T). Here, we describe two methods for detection of NO by fluorescence, one for NO extracellular detection by DAF-2 and the other one for NO intracellular detection, in this case using DAF-2DA.

Neidiquele Maria Silveira, Eduardo Caruso Machado& Rafael Vasconcelos Ribeiro

The entrapment of NO donors in nanomaterials has emerged as a strategy to protect these molecules from rapid degradation, allowing a more controlled release of NO and prolonging its effect. On the other hand, we have found beneficial effects of S-nitrosoglutathione (GSNO) – a NO donor – supplying to sugarcane plants under water deficit. Here, we hypothesized that GSNO encapsulated into nanoparticles would be more effective in attenuating the effects of water deficit on sugarcane plants as compared to the supplying of GSNO in its free form. The synthesis and characterization of chitosan nanoparticles containing GSNO were also reported. Sugarcane plants were grown in nutrient solution, and then subjected to the following treatments: control (well-hydrated); water deficit (WD); WD + GSNO sprayed in its free form (WDG) or encapsulated (WDG-NP). In general, both GSNO forms attenuated the effects of water deficit on sugarcane plants. However, the encapsulation of this donor into chitosan nanoparticles caused higher photosynthetic rates under water deficit, as compared to plants supplied with free GSNO. The root/shoot ratio was also increased when encapsulated GSNO was supplied, indicating that delayed release of NO improves drought tolerance of sugarcane plants. Our results provide experimental evidence that nanotechnology can be used for enhancing NO-induced benefits for plants under stressful conditions, alleviating the negative impact of water deficit on plant metabolism and increasing biomass allocation to root system.

Neidiquele Maria Silveira, Amedea Barozzi Seabra, Fernanda Castro Correia Marcos, Milena Trevisan Pelegrino, Eduardo Caruso Machado& Rafael Vasconcelos Ribeiro

A sugarcane gene encoding a dirigent-jacalin, ShDJ, was induced under drought stress. To elucidate its biological function, we integrated a ShDJ-overexpression construction into the rice Nipponbare genome via Agrobacterium-mediated transformation. Two transgenic lines with a single copy gene in T0 were selected and evaluated in both the T1 and T4 generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of OsNTS1/2 and OsMYB58/63 secondary cell wall biosynthesis genes. ShDJ-overexpression up-regulated the expression of the OsbZIP23, OsGRAS23, OsP5CS, and OsLea3 genes in rice stems under well-watered conditions. Taken together, our data suggest that ShDJ has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency.

Larissa Mara Andrade, Rafael Fávero Peixoto Junior, Rafael Vasconcelos Ribeiro, Paula Macedo Nóbile, Michael dos Santos Brito, Paulo Eduardo Ribeiro Marchiori, Samira Domingues Carlin, Alexandre Palma Boer Martins, Maria Helena de Souza Goldman, Juan Pablo Portilla Llerena, Caroline Fregonesi, Dilermando Perecin, João Felipe Carlos de Oliveira Nebó, Antonio Figueira, Thiago Romanos Benatti, Jorge da Silva, Paulo Mazzafera& Silvana Creste

This chapter summarizes how Nitric Oxide (NO•) modulates physiological and biochemical responses and improves plant acclimation to drought, flooding, heavy metal, salinity, and thermal stresses. It discusses the mechanisms of action of NO• donors in plants under those abiotic stress conditions. The chapter addresses the most commonly used NO• donors in experiments as well as the potential use of nanoparticles that release this radical. Plants are frequently exposed to environmental stresses in both natural and agricultural systems. Abiotic stresses dramatically impact plant growth and crop yield, with more than 50% of the major crop species being impaired by limiting conditions. Plants activate a complex network of multiple responses at molecular, cellular, and physiological levels. Such variety of responses occurs because plants have an effective signaling system, which processes and transfers information about environmental changes to other organs and tissues. These stress-linked signaling networks involve not only classical phytohormones, but also other signaling molecules, including nitric oxide.

Neidiquele Maria Silveira, Amedea Barozzi Seabra, Eduardo C. Machado, John T. Hancock& Rafael Vasconcelos Ribeiro

Drought stress can imprint marks in plants after a previous exposure, leading to plant acclimation and a permissive state that facilitates a more effective response to subsequent stress events. Such stress imprints would benefit plants obtained through vegetative propagation (propagules). Herein, our hypothesis was that the propagules obtained from plants previously exposed to water deficit would perform better under water deficit as compared to those obtained from plants that did not face stressful conditions. Sugarcane plants were grown under well-hydrated conditions or subjected to three cycles of water deficit by water withholding. Then, the propagules were subjected to water deficit. Leaf gas exchange was reduced under water deficit and the propagules from plants that experienced water deficit presented a faster recovery of CO2 assimilation and higher instantaneous carboxylation efficiency after rehydration as compared to the propagules from plants that never faced water deficit. The propagules from plants that faced water deficit also showed the highest leaf proline concentration under water deficit as well as higher leaf H2O2 concentration and leaf ascorbate peroxidase activity regardless of water regime. Under well-watered conditions, the propagules from plants that faced stressful conditions presented higher root H2O2 concentration and higher activity of catalase in roots as compared to the ones from plants that did not experience water shortage. Such physiological changes were associated with improvements in leaf area and shoot and root dry matter accumulation in propagules obtained from stressed plants. Our results suggest that root H2O2 concentration is a chemical signal associated with improved sugarcane performance under water deficit. Taken together, our findings bring a new perspective to the sugarcane production systems, in which plant acclimation can be explored for improving drought tolerance in rainfed areas.

Fernanda Castro Correia Marcos, Neidiquele Maria Silveira, Paulo Eduardo Ribeiro Marchiori, Eduardo C. Machado, Gustavo M. Souza, Marcos Guimarães de Andrade Landell & Rafael Vasconcelos Ribeiro

This study aimed to test the hypothesis that increases in leaf nitrogen concentration would reduce the sensitivity of sugarcane photosynthesis to low temperature. IACSP95-5000 plants were grown inside a growth chamber at 30/20oC (day/night) and we evaluated the effects of leaf nitrogen spraying (2.5% urea) on plants facing low temperature (22/12°C) for eight days. The leaf nitrogen supply increased leaf nitrogen concentration and plants exhibited higher leaf gas exchange as compared to nonsprayed ones. We also found higher activity of the carboxylation enzymes, Rubisco and phosphoenolpyruvate carboxylase, as well as a higher chlorophyll content in plants sprayed with nitrogen. Such enhancement of photosynthetic performance was associated with an increase in number of leaves and in total leaf area. Our results suggest that the effects of low temperature on photosynthesis of field-grown sugarcane plants could be alleviated by leaf nitrogen supply, with likely consequences for biomass production and crop yield.

Giovana Cerqueira, M.C. Santos, Paulo Eduardo Ribeiro Marchiori, Neidiquele Maria Silveira, Eduardo Caruso Machado, Rafael Vasconcelos Ribeiro

Drought is the most significant environmental stress for agricultural production worldwide, and tremendous efforts have been made to improve crop yield under the increasing water scarcity. Transcription factors are major players in the regulation of water stress-related genes in plants. Recently, different MYB transcription factors were characterized for their involvement in drought response. A sugarcane R2R3-MYB gene (ScMYBAS1) and its four alternative forms of transcript (ScMYAS1-2, ScMYBAS1-3, ScMYBAS1-4 and ScMYBAS1-5) were identified in this study. The subcellular localization, in Nicotiniana benthamiana, of the TFs fused in frame with GFP revealed that ScMYBAS1-2-GFP and ScMYBAS1-3-GFP were observed in the nucleus. The overexpression of ScMYBAS1-2 and ScMYBAS1-3 spliced transcripts in rice promoted change in plant growth under both well-watered and drought conditions. The ScMYBAS1-2 and ScMYBAS1-3 transgenic lines revealed a higher relative water content (RWC) compared to the wild type before maximum stress under drought conditions. The ScMYBAS1-2 transgenic lines showed a reduction in biomass (total dry weight). Conversely, ScMYBAS1-3 showed an increased biomass (total dry weight) relative to the wild-type. The overexpression of ScMYBAS1-3 in rice transgenic lines showed involvement with drought tolerance and biomass and, for this reason, was considered a good target for plant transformation, particularly for use in developing genotypes with drought tolerance and biomass accumulation.

Rafael Fávero Peixoto Junior, Larissa Mara de Andrade, Michael dos Santos Brito, Paula Macedo Nóbile, Alexandre Palma Boer Martins, Samira Domingues Carlin, Rafael Vasconcelos Ribeiro, Maria Helena de Souza Goldman, João Felipe Nebó Carlos de Oliveira, Antonio Vargas de Oliveira Figueira& Silvana Creste

Stress memory and an effective signaling among individuals in a given community are recognized to improve plant performance under recurrent stressful conditions. As living beings with memory and signaling abilities, plants can be considered as processing units and then be trained – or programmable from a computational viewpoint – and prepared for facing biotic and abiotic stresses. Here, we propose that sentinel plants could improve the resilience of agricultural and natural communities by reducing the impact of biotic or abiotic stressors on their neighbors. Modeling plants as programmable (or trainable) processing units compels us to think about a multidisciplinary perspective for integrating stress memory, signaling, and resilience of biological systems into executable programs, fostering the creation of applications and technologies that would benefit from the spatiotemporal dynamics related to plant-plant and plant-environment interactions.

Rafael Vasconcelos Ribeiro& Ricardo da Silva Torres

Rubber tree (Hevea brasiliensis) cultivation is the main source of natural rubber worldwide and has been extended to areas with suboptimal climates and lengthy drought periods; this transition affects growth and latex production. High-density genetic maps with reliable markers support precise mapping of quantitative trait loci (QTL), which can help reveal the complex genome of the species, provide tools to enhance molecular breeding, and shorten the breeding cycle. In this study, QTL mapping of the stem diameter, tree height, and number of whorls was performed for a full-sibling population derived from a GT1 and RRIM701 cross. A total of 225 simple sequence repeats (SSRs) and 186 single-nucleotide polymorphism (SNP) markers were used to construct a base map with 18 linkage groups and to anchor 671 SNPs from genotyping by sequencing (GBS) to produce a very dense linkage map with small intervals between loci. The final map was composed of 1,079 markers, spanned 3,779.7 cM with an average marker density of 3.5 cM, and showed collinearity between markers from previous studies. Significant variation in phenotypic characteristics was found over a 59-month evaluation period with a total of 38 QTLs being identified through a composite interval mapping method. Linkage group 4 showed the greatest number of QTLs (7), with phenotypic explained values varying from 7.67 to 14.07%. Additionally, we estimated segregation patterns, dominance, and additive effects for each QTL. A total of 53 significant effects for stem diameter were observed, and these effects were mostly related to additivity in the GT1 clone. Associating accurate genome assemblies and genetic maps represents a promising strategy for identifying the genetic basis of phenotypic traits in rubber trees. Then, further research can benefit from the QTLs identified herein, providing a better understanding of the key determinant genes associated with growth of Hevea brasiliensis under limiting water conditions.

André R. O. Conson, Cristiane H. Taniguti, Rodrigo R. Amadeu, Isabela A. A. Andreotti, Livia M. de Souza, Luciano H. B. dos Santos, João R. B. F. Rosa, Camila C. Mantello, Carla C. da Silva, Erivaldo José Scaloppi Junior, Rafael Vasconcelos Ribeiro, Vincent Le Guen, Antonio A. F. Garcia, Paulo de Souza Gonçalves & Anete P. de Souza

The objective of this work was to evaluate the effect of different gibberellin biosynthesis inhibitors on shoot growth above the panicle, as well as on fruit yield, size, and shape of nonirrigated 'Hass' avocado (Persea americana) trees. The experiment was carried out in a randomized complete block design, with five treatments, four replicates, and 40 trees. From 2013 to 2015, the following treatments were applied at full bloom, as foliar sprayings: water (control), 250 mg L-1 prohexadione-calcium, 2,500 mg L-1 trinexapac-ethyl, 1,750 mg L-1 paclobutrazol, and 350 mg L-1 uniconazole; the first two are acylcyclohexanediones and the last two, triazoles. Fruit yield, yield efficiency, and alternate bearing were not affected by the application of plant growth regulators. However, the gibberellin inhibitors increased fruit size and modified the fruit shape of nonirrigated 'Hass' avocado. Uniconazole reduces shoot growth above the panicle, and this effect may vary depending on environmental conditions, especially water supply.

Bruna do Amaral Brogio, Simone Rodrigues da Silva, Tatiana Cantuarias-Avilés, Sérgio Figueiredo Angolini, Edypol Guilherme Baptista & Rafael Vasconcelos Ribeiro

Hydraulic redistribution could contribute significantly to plant water balance under limiting conditions, reducing plant sensitivity to drought. We hypothesized that citrus species are able to redistribute water under drought, transporting water from a wet to a dry portion of root system. Using an unusual experimental design, ‘Rangpur’ lime and ‘Swingle’ citrumelo rootstocks were inarched into Valencia sweet orange trunk and subjected to water withholding. In a split root system, each plant was composed by two rootstocks, isolated from each other. During water deficit, only one rootstock faced low water availability while the other remained well watered. Sap flow, leaf water potential and soil moisture were monitored for 42 days. We found reserve sap flow in both rootstocks, with water being transported from the irrigated rootstock to the non-irrigated one. Nocturnal sap flow of well-watered rootstocks revealed that water redistribution continued at night, with ‘Rangpur’ lime showing higher hydraulic redistribution than ‘Swingle’ citrumelo. The ability of Citrus roots to redistribute water is genotype-dependent and may be an important physiological mechanism associated with drought tolerance in Citrus rootstocks.

Marcela Trevenzoli Miranda, Simone Ferreira da Silva, Bárbara Baêsso Moura, Adriana Hissae Hayashi, Eduardo Caruso Machado& Rafael Vasconcelos Ribeiro

Biomass accumulation, physiological and biochemical responses of sugarcane to ozone (O3) were evaluated in two genotypes (IACSP94-2094 and IACSP95-5000) in a realistic free-air O3 exposure system. Our aims were to investigate the mechanisms involved in O3 detoxification and verify whether such mechanisms are genotype-dependent. Plants were exposed to ambient air (Amb.) and ambient air plus 40% increase in O3 concentration (x1.4) for 90 days. Plants at x1.4 O3 showed reductions in leaf gas exchange and biomass production with consistent differences between the genotypes. Photosynthesis of IACSP95-5000 was more sensitive to elevated O3 as compared to IACSP94-2094. As a primary response, IACSP94-2094 showed reductions in stomatal conductance due to elevated O3, thus avoiding oxidative stress and maintaining CO2 assimilation for longer. As a consequence, IACSP94-2094 produced more biomass, and stalks had more soluble sugar concentration than IACSP95-5000. Leaf concentrations of O2−radical dot and H2O2 in plants exposed to x1.4 O3 were not enhanced, with such unexpected result suggesting that sugarcane plants have an effective antioxidant system to avoid O3-induced damage. In both genotypes, increases in ascorbate peroxidase activity was directly related to decreases in leaf H2O2 concentration. In IACSP94-2094, however, catalase pathway was also stimulated for detoxifying H2O2. We concluded that sugarcane sensitivity to O3 is genotype-dependent and then it is possible to reduce the deleterious effects of high O3 levels on sugarcane physiology and biomass production by growing tolerant genotypes in polluted areas.

Bárbara Baêsso Moura, Yasutomo Hoshika, Neidiquele Maria Silveira, Fernanda Castro Correia Marcos, Eduardo C. Machado, Elena Paoletti & Rafael Vasconcelos Ribeiro

The flexibility between C4 photosynthetic subtypes NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK), recently identified in some C4 species, confers high photosynthetic efficiency under varying light conditions. Theoretically, PEPCK decarboxylation uses less quanta per CO2 fixed than NADP-ME, suggesting an increase in PEPCK activity could be advantageous under shading, as CO2 leakiness increases under low light. Thus, we hypothesize that sugarcane plants have flexibility among the decarboxylation pathways, i.e., more than one decarboxylation route occurs independent of the environmental condition; furthermore, low light availability induces biochemical and anatomical adjustments resulting in increased PEPCK activity, which could contribute to maintaining or even increasing quantum efficiency of CO2 assimilation under limiting light. Two sugarcane varieties were evaluated and both presented activities of the three decarboxylases, either under full sunlight or shading. In vitro PEPCK activity increased in plants grown under low light, suggesting an upregulation of this decarboxylation pathway. Accordingly, changes in chloroplast arrangement of bundle sheath cells from centrifugal to evenly distributed were found. Our data suggest that such biochemical and anatomical adjustments found in sugarcane grown under shading were important to maintain the maximum quantum efficiency of CO2 assimilation. Finally, we propose a model highlighting the integration between the decarboxylation pathways under shading, considering carboxylation and decarboxylation pathways in sugarcane plants.

Cristina Rodrigues Gabriel Sales, Rafael Vasconcelos Ribeiro, Adriana Hissae Hayashi, Paulo Eduardo Ribeiro Marchiori, Karina I. Silva, Márcio O. Martins, Joaquim Albenisio Gomes Silveira, Neidiquele Maria Silveira& Eduardo C. Machado

Despite the importance of water transport efficiency for plant productivity, the current methods to measure the hydraulic conductance in stem segments are limited and can be tricky. These measurements may be unstable for several hours and there are no satisfactory procedures that allow choosing the moment to get reliable measures. Such instability may be generated by background flow, when there is a flow even without applying water pressure to induce the axial flow. Underlying mechanisms related to background flow are still unknown and based on available literature, we propose that the background flow is affected, or even explained, by the radial water flow and tissue capacitance. For this reason, both phenomena are fundamental for understanding plant water transport, the coupling between the phloem and xylem, and water relations under drought conditions. Besides addressing this issue, we suggest ways to study the radial water flow and capacitance in stem segments.

Luciano Pereira& Rafael Vasconcelos Ribeiro

Abstract Background and Aims Climate forecasts suggest that [CO2] in the atmosphere will continue to increase. Structural and ecophysiological responses to elevated air [CO2] (e[CO2]) in tree species are contradictory due to species-dependent responses and relatively short-term experiments. It was hypothesized that long-term exposure (4 year) to e[CO2] would change canopy structure and function of Coffea arabica trees. Methods Coffee plants were grown in a FACE (free air CO2 enrichment) facility under two air [CO2]: actual and elevated (actual + approx. 200 μL CO2 L–1). Plants were codified following the VPlants methodology to obtain coffee mock-ups. Plant canopies were separated into three 50 cm thick layers over a vertical profile to evaluate their structure and photosynthesis, using functional–structural plant modelling. Key Results Leaf area was strongly reduced on the bottom and upper canopy layers, and increased soil carbon concentration suggested changes in carbon partitioning of coffee trees under e[CO2]. Increased air [CO2] stimulated stomatal conductance and leaf photosynthesis at the middle and upper canopy layers, increasing water-use efficiency. Under e[CO2], plants showed reduced diameter of the second-order axes and higher investment in the youngest third to fifth-order axes. Conclusions The responses of Arabica coffee grown under long-term exposure to e[CO2] integrated structural and functional modifications, which balanced leaf area loss through improvements in leaf and whole-plant photosynthesis.

Miroslava Rakocevic, Rafael Vasconcelos Ribeiro, Paulo Eduardo Ribeiro Marchiori, Heloisa Ferreira Filizola & Eunice Reis Batista

Ozone (O3) is a toxic oxidative air pollutant, with significant detrimental effects on crops. Sugarcane (Saccharum spp.) is an important crop with no O3 risk assessment performed so far. This study aimed to assess O3 risk to sugarcane plants by using exposure-based indices (AOT40 and W126) based on O3 concentrations in the air, and the flux-based index (PODy, where y is a threshold of uptake) that considers leaf O3 uptake and the influence of environmental conditions on stomatal conductance (gsto). Two sugarcane genotypes (IACSP94-2094 and IACSP95-5000) were subjected to a 90-day Free-Air Controlled Experiment (FACE) exposure at three levels of O3 concentrations: ambient (Amb); Amb x1.2; and Amb x1.4. Total above-ground biomass (AGB), stalk biomass (SB) and leaf biomass (LB) were evaluated and the potential biomass production in a clean air was estimated by assuming a theoretical clean atmosphere at 10 ppb as 24 h O3 average. The Jarvis-type multiplicative algorithm was used to parametrize gsto including environmental factors i.e. air temperature, light intensity, air vapor pressure deficit, and minimum night-time temperature. Ozone exposure caused a negative impact on AGB, SB and LB. The O3 sensitivity of sugarcane may be related to its high gsto (∼535 mmol H2O m−2 s−1). As sugarcane is adapted to hot climate conditions, gsto was restricted when the current minimum air temperature (Tmin) was below ∼14 °C and the minimum night-time air temperature of the previous day (Tnmin) was below ∼7.5 °C. The flux-based index (PODy) performed better than the exposure-based indices in estimating O3 effect on biomass losses. We recommend a y threshold of 2 nmol m−2 s−1 to incorporate O3 effects on both AGB and SB and 1 nmol m−2 s−1 on LB. In order not to exceed 4% reduction in the growth of these two sugarcane genotypes, we recommend the following critical levels: 1.09 and 1.04 mmol m−2 POD2 for AGB, 0.91 and 0.96 mmol m−2 POD2 for SB, and 3.00 and 2.36 mmol m−2 POD1 for LB of IACSP95-5000 and IACSP94-2094, respectively.

Bárbara Baêsso Moura, Yasutomo Hoshika, Rafael Vasconcelos Ribeiro& Elena Paoletti

This study aimed to evaluate the photosynthetic and antioxidant responses of Jatropha curcas plants exposed to heat stress applied simultaneously in both shoots and roots and solely in each tissue. Thirty-day-old plants were transferred to two growth chambers, where they were subjected to 27 or 42 °C. In each growth chamber, the root system was isolated and then subjected to 27 or 42 °C. Then, four groups of plants were formed according to shoot/root temperature: 27/27; 27/42; 42/27; and 42/42 °C. Plants under 27/27 °C were considered as control and plants were exposed to each thermal treatment for 12 h. The leaf CO2 assimilation rate, stomatal conductance, and mesophyll conductance were decreased by high temperature, regardless of plant tissue. However, primary photochemistry given by effective quantum yield of PSII, electron transport rate, photochemical and non-photochemical quenchings, and maximum electron transport rate driving RuBP regeneration revealed that high shoot temperature was more deleterious for photosynthesis than high root temperature. J. curcas presented efficient mechanisms to avoid photo-damage and photo-inhibition through increases in non-photochemical quenching, maintenance of the maximum quantum yield of PSII, and increases in total superoxide dismutase, ascorbate peroxidase, and catalase activities when shoots were exposed to high temperature, regardless of root temperature. In general, our data suggest that J. curcas present an efficient antioxidant protection system in photosynthetic tissues and acclimation of PSII avoiding photo-damage and photo-inhibition under heat stress. On the other hand, high root temperature inhibits antioxidant metabolism, leading to higher oxidative damage in root tissues and affecting primary photochemistry in leaf tissues.

Evandro Nascimento Silva, Joaquim Albenisio Gomes Silveira, Rafael Vasconcelos Ribeiro, Jarvys Êxoda Oliveira & Ricardo Araújo Cardoso

Eucalyptus plant is one of the most economic important forest species explored worldwide and in some countries its cultivation is expanding to regions with lower temperatures, including Brazil. This study aimed to compare the photochemical response of 18 Eucalyptus species, one Corimbia and one hybrid subjected to low temperature using two chilling treatments. Six-month-old plants were exposed to (1) gradual chilling (20–0 °C in four steps of 5 °C for 10 min) and abrupt chilling (20−5 °C in one step). Based on chlorophyll fluorescence parameters, eucalyptus species responded differently depending on how chilling was applied. E. tereticornis presented the best performance under chilling, regardless how it was imposed. The effective quantum yield of photosystem II was the best parameter to identify variation in photochemical responses of eucalyptus species to chilling. It was also observed that E. brassiana, C. citriodora, E. grandis, E. gunni, and E. resinifera were sensitive to the way chilling was applied, leading to differential photochemical response.

Flávia Camila Schimpl, Rafael Vasconcelos Ribeiro, Luciano Pereira, Haroldo Silva Rodrigues & Paulo Mazzafera

Under field conditions, plants are exposed to cycles of dehydration and rehydration during their lifespan. In this study, we hypothesized that sugarcane plants previously exposed to cycles of water deficits will perform better than plants that have never faced water deficits when both are subjected to low water availability. Sugarcane plants were grown in a nutrient solution and exposed to one (1WD), two (2WD) or three (3WD) water deficit cycles. As the reference, plants were grown in a nutrient solution without adding polyethylene glycol. Under water deficits, leaf gas exchange was significantly reduced in 1WD and 2WD plants. However, 3WD plants showed similar CO2 assimilation and lower stomatal conductance compared to the reference plants, with increases in intrinsic water-use efficiency. Abscisic acid concentrations were lower in 3WD plants than in 1WD plants. Our data revealed root H2O2 concentration as an important chemical signal, with the highest root H2O2 concentrations found in 3WD plants. These plants presented higher root dry matter and root:shoot ratios compared to the reference plants, as well as higher biomass production when water was available. Our data suggest that sugarcane plants were able to store information from previous stressful events, with plant performance improving under water deficits. In addition, our findings provide a new perspective for increasing drought tolerance in sugarcane plants under nursery conditions.

Fernanda Castro Correia Marcos, Neidiquele Maria Silveira, João B. Mokochinski, Alexandra C. H. F. Sawaya, Paulo Eduardo Ribeiro Marchiori, Eduardo C. Machado, Gustavo M. Souza, Marcos Guimarães de Andrade Landell & Rafael Vasconcelos Ribeiro

Morpho‐physiological changes caused by particular plant growth‐promoting rhizobacteria were evaluated in sugarcane plants under varying water availability. Under well‐watered conditions, we have found one rhizobacteria isolate (IAC‐RBcr5) able to enhance root dry matter and photosynthesis of sugarcane plants. The IAC‐RBcr5 genome was sequenced and high similarity was found with Pseudomonas putida GB‐1. Based on increased root system size of inoculated plants, we hypothesised that sugarcane plants inoculated with IAC‐RBcr5 would have improved performance under water deficit. Although IAC‐RBcr5 had improved plant leaf CO2 assimilation under water shortage, inoculation caused reduction of biomass accumulation in sugarcane. The negative influence of water deficit on shoot growth rate and root traits such as volume, area, diameter, length and specific root area was higher in plants treated with IAC‐RBcr5 as compared to non‐inoculated ones. However, rhizobacteria‐induced improvements in leaf and root proline contents would represent a strategy for storing carbon and nitrogen during low water availability and helping both organisms to resume their metabolism after rehydration. In conclusion, we found and identified a rhizobacterium able to improve growth and photosynthesis of sugarcane plants. Such benefit for plant growth was lost under low water availability as a likely consequence of increased carbon‐energy demand by rhizobacteria and their sensitivity to drought.

Pedro Eduardo Rampazzo, Fernanda Castro Correia Marcos, M.A.P. Cipriano, Paulo Eduardo Ribeiro Marchiori, S. S. Freitas, Eduardo Caruso Machado, L.C. Nascimento, M. Brocchi & Rafael Vasconcelos Ribeiro