Luciano Freschi

Nitric oxide and phytohormone interactions: current status and perspectives

Nitric oxide (NO) is currently considered a ubiquitous signal in plant systems, playing significant roles in a wide range of responses to environmental and endogenous cues. During the signaling events leading to these plant responses, NO frequently interacts with plant hormones and other endogenous molecules, at times originating remarkably complex signaling cascades. Accumulating evidence indicates that virtually all major classes of plant hormones may influence, at least to some degree, the endogenous levels of NO. In addition, studies conducted during the induction of diverse plant responses have demonstrated that NO may also affect biosynthesis, catabolism/conjugation, transport, perception, and/or transduction of different phytohormones, such as auxins, gibberellins, cytokinins, abscisic acid, ethylene, salicylic acid, jasmonates, and brassinosteroids. Although still not completely elucidated, the mechanisms underlying the interaction between NO and plant hormones have recently been investigated in a number of species and plant responses. This review specifically focuses on the current knowledge of the mechanisms implicated in NO–phytohormone interactions during the regulation of developmental and metabolic plant events. The modifications triggered by NO on the transcription of genes encoding biosynthetic/degradative enzymes as well as proteins involved in the transport and signal transduction of distinct plant hormones will be contextualized during the control of developmental, metabolic, and defense responses in plants. Moreover, the direct post-translational modification of phytohormone biosynthetic enzymes and receptors through S-nitrosylation will also be discussed as a key mechanism for regulating plant physiological responses. Finally, some future perspectives toward a more complete understanding of NO–phytohormone interactions will also be presented and discussed.

A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.

Nitrogen metabolism and translocation in soybean plants subjected to root oxygen deficiency

Although nitrate (NO3(-)) but not ammonium (NH4(+)) improves plant tolerance to oxygen deficiency, the mechanisms involved in this phenomenon are just beginning to be understood. By using gas chromatography-mass spectrometry, we investigated the metabolic fate of (15)NO3(-) and (15)NH4(+) in soybean plants (Glycine max L. Merril cv. IAC-23) subjected to root hypoxia. This stress reduced the uptake of (15)NO3(-) and (15)NH4(+) from the medium and decreased the overall assimilation of these nitrogen sources into amino acids in roots and leaves. Root (15)NO3(-) assimilation was more affected by hypoxia than that of (15)NH4(+), resulting in enhanced nitrite and nitric oxide release in the solution. However, (15)NO3(-) was translocated in substantial amounts by xylem sap and considerable (15)NO3(-) assimilation into amino acids also occurred in the leaves, both under hypoxia and normoxia. By contrast, (15)NH4(+) assimilation occurred predominantly in roots, resulting in accumulation of mainly (15)N-alanine in this tissue during hypoxia. Analysis of lactate levels suggested higher fermentation in roots from NH4(+)-treated plants compared to the NO3(-) treatment. Thus, foliar NO3(-) assimilation may be relevant to plant tolerance to oxygen deficiency, since it would economize energy expenditure by hypoxic roots. Additionally, the involvement of nitric oxide synthesis from nitrite in the beneficial effect of NO3(-) is discussed.

Nitric oxide signaling and its crosstalk with other plant growth regulators in plant responses to abiotic stress

Nitric oxide (NO) is a free radical molecule involved in an array of functions under physiological and adverse environmental conditions. As other free radical molecules, NO biological action depends on its cellular concentration, acting as a signal molecule when produced at low concentration or resulting in cellular damage when produced at sufficiently high levels to trigger nitro-oxidative stress. Over the last decade, significant progress has been made in characterizing NO metabolism and action mechanism, revealing that diverse biosynthetic routes can generate this free radical in plants and its action mainly occurs through posttranslational modification (nitration and S-nitrosylation) of target proteins. Intricate crosstalk networks between NO and other signaling molecules have been described involving phytohormones, other second messengers, and key transcription factors. This review will focus on our current understanding of NO interplay with phytohormones and other plant growth regulators under abiotic stress conditions.

Nitric Oxide, Ethylene, and Auxin Cross Talk Mediates Greening and Plastid Development in Deetiolating Tomato Seedlings.

Light-evoked cotyledon greening and chloroplast differentiation in deetiolating tomato seedlings are orchestrated by regulatory feedback loops involving phytochromes, nitric oxide, auxins, and ethylene. The transition from etiolated to green seedlings involves the conversion of etioplasts into mature chloroplasts via a multifaceted, light-driven process comprising multiple, tightly coordinated signaling networks. Here, we demonstrate that light-induced greening and chloroplast differentiation in tomato (Solanum lycopersicum) seedlings are mediated by an intricate cross talk among phytochromes, nitric oxide (NO), ethylene, and auxins. Genetic and pharmacological evidence indicated that either endogenously produced or exogenously applied NO promotes seedling greening by repressing ethylene biosynthesis and inducing auxin accumulation in tomato cotyledons. Analysis performed in hormonal tomato mutants also demonstrated that NO production itself is negatively and positively regulated by ethylene and auxins, respectively. Representing a major biosynthetic source of NO in tomato cotyledons, nitrate reductase was shown to be under strict control of both phytochrome and hormonal signals. A close NO-phytochrome interaction was revealed by the almost complete recovery of the etiolated phenotype of red light-grown seedlings of the tomato phytochrome-deficient aurea mutant upon NO fumigation. In this mutant, NO supplementation induced cotyledon greening, chloroplast differentiation, and hormonal and gene expression alterations similar to those detected in light-exposed wild-type seedlings. NO negatively impacted the transcript accumulation of genes encoding phytochromes, photomorphogenesis-repressor factors, and plastid division proteins, revealing that this free radical can mimic transcriptional changes typically triggered by phytochrome-dependent light perception. Therefore, our data indicate that negative and positive regulatory feedback loops orchestrate ethylene-NO and auxin-NO interactions, respectively, during the conversion of colorless etiolated seedlings into green, photosynthetically competent young plants.

Shedding light on ethylene metabolism in higher plants

Ethylene metabolism in higher plants is regulated by a wide array of endogenous and environmental factors. During most physiological processes, ethylene levels are mainly determined by a strict control of the rate-limiting biosynthetic steps responsible for the production of 1-aminocyclopropane-1-carboxylic acid (ACC) and its subsequent conversion to ethylene. Responsible for these reactions, the key enzymes ACC synthase and ACC oxidase are encoded by multigene families formed by members that can be differentially regulated at the transcription and post-translational levels by specific developmental and environmental signals. Among the wide variety of environmental cues controlling plant ethylene production, light quality, duration, and intensity have consistently been demonstrated to influence the metabolism of this plant hormone in diverse plant tissues, organs, and species. Although still not completely elucidated, the mechanisms underlying the interaction between light signal transduction and ethylene evolution appears to involve a complex network that includes central transcription factors connecting multiple signaling pathways, which can be reciprocally modulated by ethylene itself, other phytohormones, and specific light wavelengths. Accumulating evidence has indicated particular photoreceptors as essential mediators in light-induced signaling cascades affecting ethylene levels. Therefore, this review specifically focuses on discussing the current knowledge of the potential molecular mechanisms implicated in the light-induced responses affecting ethylene metabolism during the regulation of developmental and metabolic plant responses. Besides presenting the state of the art in this research field, some overlooked mechanisms and future directions to elucidate the exact nature of the light–ethylene interplay in higher plants will also be compiled and discussed.

Survival strategies of citrus rootstocks subjected to drought.

Two citrus rootstocks, Rangpur lime (RL) and Sunki Maravilha mandarin (SM), were analyzed either ungrafted or grafted with their reciprocal graft combinations or with shoot scions of two commercial citrus varieties: Valencia orange (VO) and Tahiti acid lime (TAL). All graft combinations were subjected to distinct watering regimes: well-watered, severe drought and rehydration. Growth and water relation parameters, gas exchange as well as sugar and hormone profiles were determined. Data indicated that RL adopted a dehydration avoidance strategy and maintained growth, whereas SM adopted a dehydration tolerance strategy focused on plant survival. Compared with RL, the leaves and roots of SM exhibited higher concentrations of abscisic acid and salicylic acid, which induced drought tolerance, and accumulation of carbohydrates such as trehalose and raffinose, which are important reactive oxygen species scavengers. SM rootstocks were able to transfer their survival strategy to the grafted shoot scions (RL, VO, TAL). Because of their contrasting survival strategies, RL reached the permanent wilting point more quickly than SM whereas SM recovered from prolonged droughts more efficiently than RL. This is one of the most complete studies of drought tolerance mechanisms in citrus crops and is the first to use reciprocal grafting to clarify scion/rootstock interactions.

Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond senescence-associated Chl degradation that, when compromised, affects isoprenoid and carbon metabolism which ultimately alters the fruits nutraceutical content.

Crassulacean Acid Metabolism in Epiphytic Orchids: Current Knowledge, Future Perspectives

1.1 Crassulacean Acid Metabolism (CAM) Crassulacean Acid Metabolism (CAM) is one of three photosynthetic assimilation pathways of atmospheric CO2, together with the photosynthetic pathways C3 and C4 (Silvera et al., 2010a). The CAM is characterized by the temporal separation between CO2 fixation and its assimilation into organic compounds. In CAM plants, CO2 is fixed during the dark period through the action of the enzyme phosphoenolpyruvate carboxylase (PEPC), which uses CO2 for carboxylation of phosphoenolpyruvate (PEP), giving rise to oxaloacetate (OAA). The OAA formed is converted into malate by the action of malate dehydrogenase (MDH). Then, this organic acid is transported to the vacuole along with H+ ions, causing the typical nocturnal acidification of CAM plants. During the light period, the decarboxylation of malate and refixation of the CO2 by the enzyme ribulose bisphosphate carboxylase oxygenase (RUBISCO C3 cycle) takes place in the cytosol, causing a decrease of acidity in the tissues (Herrera, 2009; Luttge, 2004; Silvera et al., 2010b) (Figure 1). The CAM pathway can be separated into four phases (Luttge, 2004; Osmond, 1978; Silvera et al., 2010b). Phase I is characterized by the opening of stomata during the night, the uptake and subsequent fixation of atmospheric CO2 by PEPC in the cytosol and the formation of organic acids, such as malate. Phase II consists of fixing CO2 by the enzyme RUBISCO and PEPC concurrently, a phase characterized essentially by the decrease in the activity of PEPC and the start of the activity of RUBISCO. Phase III consists of the reduction of stomatal opening, efflux of organic acids from the vacuole and subsequent decarboxylation of these acids. Finally, phase IV comprises the depletion in the stock of organic acids associated with an increase of stomata conductance. Due to nighttime fixation of atmospheric CO2, CAM plants exhibit greater water use efficiency (EUA) when compared with the photosynthetic pathways C3 and C4 (Herrera, 2009), given that CAM plants use 50 to 100 g of water per gram of CO2 fixed, while C3 plants use 400 to 500 g (Drennam & Nobel, 2000). The ratio of transpiration is 3to 10-fold lower in CAM plants than in C3 (Kluge & Ting, 1978). Besides the EUA, another advantage of CAM comprises mechanisms to minimize the damage caused by reactive oxygen species (ROS) (Sunagawa et al., 2010).

Interactions Between Nutrients and Crassulacean Acid Metabolism

CAM expression is under the control of an intricate signaling network involving numerous interconnected environmental cues strongly linked to nutrient accessibility for plants. Mineral nutrients are essential elements for conferring plant resistance to drought or salinity while both these abiotic stresses can disturb the nutritional relations and modulate the photosynthetic pathway of CAM plants. Hypothetical connections between CAM photosynthesis and mineral nitrogen metabolism have been suggested as possible mechanisms conferring physiological advantages for plant survival under severe environment conditions. Although the mineral nutrition of CAM plants has received relatively scarce attention, some studies have consistently demonstrated that different degrees of nutrient fertilization can influence CAM expression. In addition, the nutritional aspect of CAM regulation is frequently related to the action of other environmental factors, especially light and water availability. Among all the essential macronutrients, variation in nitrogen content usually shows the strongest correlation with CAM activity. Some evidence indicates that specific effects of nitrate and ammonium ions are crucial in the CAM modulation rather than an influence of nitrogen status itself. Therefore, future research is required to unravel the effects and the respective metabolic and signaling mechanisms of interaction between mineral nutrients and other environmental signals on CAM photosynthesis.