Plant biostimulants as natural alternatives to synthetic auxins in strawberry production: physiological and metabolic insights.

The demand for high-quality strawberries continues to grow, emphasizing the need for innovative agricultural practices to enhance both yield and fruit quality. In this context, the utilization of natural products, such as biostimulants, has emerged as a promising avenue for improving strawberry production while aligning with sustainable and eco-friendly agricultural approaches. This study explores the influence of a bacterial filtrate (BF), a vegetal-derived protein hydrolysate (PH), and a standard synthetic auxin (SA) on strawberry, investigating their effects on yield, fruit quality, mineral composition and metabolomics of leaves and fruits. Agronomic trial revealed that SA and BF significantly enhanced early fruit yield due to their positive influence on flowering and fruit set, while PH treatment favored a gradual and prolonged fruit set, associated with an increased shoot biomass and sustained production. Fruit quality analysis showed that PH-treated fruits exhibited an increase of firmness and soluble solids content, whereas SA-treated fruits displayed lower firmness and soluble solids content. The ionomic analysis of leaves and fruits indicated that all treatments provided sufficient nutrients, with heavy metals within regulatory limits. Metabolomics indicated that PH stimulated primary metabolites, while SA and BF directly affected flavonoid and anthocyanin biosynthesis, and PH increased fruit quality through enhanced production of beneficial metabolites. This research offers valuable insights for optimizing strawberry production and fruit quality by harnessing the potential of natural biostimulants as viable alternative to synthetic compounds.

Using the Maize Nested Association Mapping (NAM) Population to Partition Arbuscular Mycorrhizal Effects on Drought Stress Tolerance into Hormonal and Hydraulic Components.

In this study, a first experiment was conducted with the objective of determining how drought stress alters the radial water flow and physiology in the whole maize nested association mapping (NAM) population and to find out which contrasting maize lines should be tested in a second experiment for their responses to drought in combination with an arbuscular mycorrhizal (AM) fungus. Emphasis was placed on determining the role of plant aquaporins and phytohormones in the responses of these contrasting maize lines to cope with drought stress. Results showed that both plant aquaporins and hormones are altered by the AM symbiosis and are highly involved in the physiological responses of maize plants to drought stress. The regulation by the AM symbiosis of aquaporins involved in water transport across cell membranes alters radial water transport in host plants. Hormones such as IAA, SA, ABA and jasmonates must be involved in this process either by regulating the own plant-AM fungus interaction and the activity of aquaporins, or by inducing posttranscriptional changes in these aquaporins, which in turns alter their water transport capacity. An intricate relationship between root hydraulic conductivity, aquaporins and phytohormones has been observed, revealing a complex network controlling water transport in maize roots.

Optical topometry and machine learning to rapidly phenotype stomatal patterning traits for maize QTL mapping.

Stomata are adjustable pores on leaf surfaces that regulate the tradeoff of CO2 uptake with water vapor loss, thus having critical roles in controlling photosynthetic carbon gain and plant water use. The lack of easy, rapid methods for phenotyping epidermal cell traits have limited discoveries about the genetic basis of stomatal patterning. A high-throughput epidermal cell phenotyping pipeline is presented here and used for quantitative trait loci (QTL) mapping in field-grown maize (Zea mays). The locations and sizes of stomatal complexes and pavement cells on images acquired by an optical topometer from mature leaves were automatically determined. Computer estimated stomatal complex density (SCD; R2 = 0.97) and stomatal complex area (SCA; R2 = 0.71) were strongly correlated with human measurements. Leaf gas exchange traits were genetically correlated with the dimensions and proportions of stomatal complexes (rg = 0.39-0.71) but did not correlate with SCD. Heritability of epidermal traits was moderate to high (h2 = 0.42-0.82) across two field seasons. Thirty-six QTL were consistently identified for a given trait in both years. Twenty-four clusters of overlapping QTL for multiple traits were identified, with univariate versus multivariate single marker analysis providing evidence consistent with pleiotropy in multiple cases. Putative orthologs of genes known to regulate stomatal patterning in Arabidopsis (Arabidopsis thaliana) were located within some, but not all, of these regions. This study demonstrates how discovery of the genetic basis for stomatal patterning can be accelerated in maize, a C4 model species where these processes are poorly understood.

Short-Term Exposure to High Atmospheric Vapor Pressure Deficit (VPD) Severely Impacts Durum Wheat Carbon and Nitrogen Metabolism in the Absence of Edaphic Water Stress.

Low atmospheric relative humidity (RH) accompanied by elevated air temperature and decreased precipitation are environmental challenges that wheat production will face in future decades. These changes to the atmosphere are causing increases in air vapor pressure deficit (VPD) and low soil water availability during certain periods of the wheat-growing season. The main objective of this study was to analyze the physiological, metabolic, and transcriptional response of carbon (C) and nitrogen (N) metabolism of wheat (Triticum durum cv. Sula) to increases in VPD and soil water stress conditions, either alone or in combination. Plants were first grown in well-watered conditions and near-ambient temperature and RH in temperature-gradient greenhouses until anthesis, and they were then subjected to two different water regimes well-watered (WW) and water-stressed (WS), i.e., watered at 50% of the control for one week, followed by two VPD levels (low, 1.01/0.36 KPa and high, 2.27/0.62 KPa; day/night) for five additional days. Both VPD and soil water content had an important impact on water status and the plant physiological apparatus. While high VPD and water stress-induced stomatal closure affected photosynthetic rates, in the case of plants watered at 50%, high VPD also caused a direct impairment of the RuBisCO large subunit, RuBisCO activase and the electron transport rate. Regarding N metabolism, the gene expression, nitrite reductase (NIR) and transport levels detected in young leaves, as well as determinations of the δ15N and amino acid profiles (arginine, leucine, tryptophan, aspartic acid, and serine) indicated activation of N metabolism and final transport of nitrate to leaves and photosynthesizing cells. On the other hand, under low VPD conditions, a positive effect was only observed on gene expression related to the final step of nitrate supply to photosynthesizing cells, whereas the amount of 15N supplied to the roots that reached the leaves decreased. Such an effect would suggest an impaired N remobilization from other organs to young leaves under water stress conditions and low VPD.

A Microbial-Based Biostimulant Enhances Sweet Pepper Performance by Metabolic Reprogramming of Phytohormone Profile and Secondary Metabolism.

Microbial-based biostimulants can improve crop productivity by modulating cell metabolic pathways including hormonal balance. However, little is known about the microbial-mediated molecular changes causing yield increase. The present study elucidates the metabolomic modulation occurring in pepper (Capsicum annuum L.) leaves at the vegetative and reproductive phenological stages, in response to microbial-based biostimulants. The arbuscular mycorrhizal fungi Rhizoglomus irregularis and Funneliformis mosseae, as well as Trichoderma koningii, were used in this work. The application of endophytic fungi significantly increased total fruit yield by 23.7% compared to that of untreated plants. Multivariate statistics indicated that the biostimulant treatment substantially altered the shape of the metabolic profile of pepper. Compared to the untreated control, the plants treated with microbial biostimulants presented with modified gibberellin, auxin, and cytokinin patterns. The biostimulant treatment also induced secondary metabolism and caused carotenoids, saponins, and phenolic compounds to accumulate in the plants. Differential metabolomic signatures indicated diverse and concerted biochemical responses in the plants following the colonization of their roots by beneficial microorganisms. The above findings demonstrated a clear link between microbial-mediated yield increase and a strong up-regulation of hormonal and secondary metabolic pathways associated with growth stimulation and crop defense to environmental stresses.

Elucidating the Possible Involvement of Maize Aquaporins in the Plant Boron Transport and Homeostasis Mediated by Rhizophagus irregularis under Drought Stress Conditions.

Boron (B) is an essential micronutrient for higher plants, having structural roles in primary cell walls, but also other functions in cell division, membrane integrity, pollen germination or metabolism. Both high and low B levels negatively impact crop performance. Thus, plants need to maintain B concentration in their tissues within a narrow range by regulating transport processes. Both active transport and protein-facilitated diffusion through aquaporins have been demonstrated. This study aimed at elucidating the possible involvement of some plant aquaporins, which can potentially transport B and are regulated by the arbuscular mycorrhizal (AM) symbiosis in the plant B homeostasis. Thus, AM and non-AM plants were cultivated under 0, 25 or 100 µM B in the growing medium and subjected or not subjected to drought stress. The accumulation of B in plant tissues and the regulation of plant aquaporins and other B transporters were analyzed. The benefits of AM inoculation on plant growth (especially under drought stress) were similar under the three B concentrations assayed. The tissue B accumulation increased with B availability in the growing medium, especially under drought stress conditions. Several maize aquaporins were regulated under low or high B concentrations, mainly in non-AM plants. However, the general down-regulation of aquaporins and B transporters in AM plants suggests that, when the mycorrhizal fungus is present, other mechanisms contribute to B homeostasis, probably related to the enhancement of water transport, which would concomitantly increase the passive transport of this micronutrient.

Radial water transport in arbuscular mycorrhizal maize plants under drought stress conditions is affected by indole-acetic acid (IAA) application.

Drought stress is one of the most devastating abiotic stresses, compromising crop growth, reproductive success and yield. The arbuscular mycorrhizal (AM) symbiosis has been demonstrated to be beneficial in helping the plant to bear with water deficit. In plants, development and stress responses are largely regulated by a complex hormonal crosstalk. Auxins play significant roles in plant growth and development, in responses to different abiotic stresses or in the establishment and functioning of the AM symbiosis. Despite these important functions, the role of indole-3acetic acid (IAA) as a regulator of root water transport and stress response is not well understood. In this study, the effect of exogenous application of IAA on the regulation of root radial water transport in AM plants was analyzed under well-watered and drought stress conditions. Exogenous IAA application affected root hydraulic parameters, mainly osmotic root hydraulic conductivity (Lo), which was decreased in both AM and non-AM plants under water deficit conditions. Under drought, the relative apoplastic water flow was differentially regulated by IAA application in non-AM and AM plants. The effect of IAA on the internal cell component of root water conductivity suggests that aquaporins are involved in the IAA-dependent inhibition of this water pathway.

Elucidating the Possible Involvement of Maize Aquaporins and Arbuscular Mycorrhizal Symbiosis in the Plant Ammonium and Urea Transport under Drought Stress Conditions.

This study investigates the possible involvement of maize aquaporins which are regulated by arbuscular mycorrhizae (AM) in the transport in planta of ammonium and/or urea under well-watered and drought stress conditions. The study also aims to better understand the implication of the AM symbiosis in the uptake of urea and ammonium and its effect on plant physiology and performance under drought stress conditions. AM and non-AM maize plants were cultivated under three levels of urea or ammonium fertilization (0, 3 µM or 10 mM) and subjected or not to drought stress. Plant aquaporins and physiological responses to these treatments were analyzed. AM increased plant biomass in absence of N fertilization or under low urea/ ammonium fertilization, but no effect of the AM symbiosis was observed under high N supply. This effect was associated with reduced oxidative damage to lipids and increased N accumulation in plant tissues. High N fertilization with either ammonium or urea enhanced net photosynthesis (AN) and stomatal conductance (gs) in plants maintained under well-watered conditions, but 14 days after drought stress imposition these parameters declined in AM plants fertilized with high N doses. The aquaporin ZmTIP1;1 was up-regulated by both urea and ammonium and could be transporting these two N forms in planta. The differential regulation of ZmTIP4;1 and ZmPIP2;4 with urea fertilization and of ZmPIP2;4 with NH4+ supply suggests that these two aquaporins may also play a role in N mobilization in planta. At the same time, these aquaporins were also differentially regulated by the AM symbiosis, suggesting a possible role in the AM-mediated plant N homeostasis that deserves future studies.

The arbuscular mycorrhizal symbiosis regulates aquaporins activity and improves root cell water permeability in maize plants subjected to water stress.

Studies have suggested that increased root hydraulic conductivity in mycorrhizal roots could be the result of increased cell-to-cell water flux via aquaporins. This study aimed to elucidate if the key effect of the regulation of maize aquaporins by the arbuscular mycorrhizal (AM) symbiosis is the enhancement of root cell water transport capacity. Thus, water permeability coefficient (Pf ) and cell hydraulic conductivity (Lpc ) were measured in root protoplast and intact cortex cells of AM and non-AM plants subjected or not to water stress. Results showed that cells from droughted-AM roots maintained Pf and Lpc values of nonstressed plants, whereas in non-AM roots, these values declined drastically as a consequence of water deficit. Interestingly, the phosphorylation status of PIP2 aquaporins increased in AM plants subjected to water deficit, and Pf values higher than 12 µm s-1 were found only in protoplasts from AM roots, revealing the higher water permeability of AM root cells. In parallel, the AM symbiosis increased stomatal conductance, net photosynthesis, and related parameters, showing a higher photosynthetic capacity in these plants. This study demonstrates a better performance of AM root cells in water transport under water deficit, which is connected to the shoot physiological performance in terms of photosynthetic capacity.

Elevated ozone reduces photosynthetic carbon gain by accelerating leaf senescence of inbred and hybrid maize in a genotype-specific manner.

Exposure to elevated tropospheric ozone concentration ([O3 ]) accelerates leaf senescence in many C3 crops. However, the effects of elevated [O3 ] on C4 crops including maize (Zea mays L.) are poorly understood in terms of physiological mechanism and genetic variation in sensitivity. Using free air gas concentration enrichment, we investigated the photosynthetic response of 18 diverse maize inbred and hybrid lines to season-long exposure to elevated [O3 ] (~100 nl L-1 ) in the field. Gas exchange was measured on the leaf subtending the ear throughout the grain filling period. On average over the lifetime of the leaf, elevated [O3 ] led to reductions in photosynthetic CO2 assimilation of both inbred (-22%) and hybrid (-33%) genotypes. There was significant variation among both inbred and hybrid lines in the sensitivity of photosynthesis to elevated [O3 ], with some lines showing no change in photosynthesis at elevated [O3 ]. Based on analysis of inbred line B73, the reduced CO2 assimilation at elevated [O3 ] was associated with accelerated senescence decreasing photosynthetic capacity and not altered stomatal limitation. These findings across diverse maize genotypes could advance the development of more O3 tolerant maize and provide experimental data for parameterization and validation of studies modeling how O3 impacts crop performance.

Enhanced Drought Stress Tolerance by the Arbuscular Mycorrhizal Symbiosis in a Drought-Sensitive Maize Cultivar Is Related to a Broader and Differential Regulation of Host Plant Aquaporins than in a Drought-Tolerant Cultivar.

The arbuscular mycorrhizal (AM) symbiosis has been shown to improve maize tolerance to different drought stress scenarios by regulating a wide range of host plants aquaporins. The objective of this study was to highlight the differences in aquaporin regulation by comparing the effects of the AM symbiosis on root aquaporin gene expression and plant physiology in two maize cultivars with contrasting drought sensitivity. This information would help to identify key aquaporin genes involved in the enhanced drought tolerance by the AM symbiosis. Results showed that when plants were subjected to drought stress the AM symbiosis induced a higher improvement of physiological parameters in drought-sensitive plants than in drought-tolerant plants. These include efficiency of photosystem II, membrane stability, accumulation of soluble sugars and plant biomass production. Thus, drought-sensitive plants obtained higher physiological benefit from the AM symbiosis. In addition, the genes ZmPIP1;1, ZmPIP1;3, ZmPIP1;4, ZmPIP1;6, ZmPIP2;2, ZmPIP2;4, ZmTIP1;1, and ZmTIP2;3 were down-regulated by the AM symbiosis in the drought-sensitive cultivar and only ZmTIP4;1 was up-regulated. In contrast, in the drought-tolerant cultivar only three of the studied aquaporin genes (ZmPIP1;6, ZmPIP2;2, and ZmTIP4;1) were regulated by the AM symbiosis, resulting induced. Results in the drought-sensitive cultivar are in line with the hypothesis that down-regulation of aquaporins under water deprivation could be a way to minimize water loss, and the AM symbiosis could be helping the plant in this regulation. Indeed, during drought stress episodes, water conservation is critical for plant survival and productivity, and is achieved by an efficient uptake and stringently regulated water loss, in which aquaporins participate. Moreover, the broader and contrasting regulation of these aquaporins by the AM symbiosis in the drought-sensitive than the drought-tolerant cultivar suggests a role of these aquaporins in water homeostasis or in the transport of other solutes of physiological importance in both cultivars under drought stress conditions, which may be important for the AM-induced tolerance to drought stress.

Differential CO2 effect on primary carbon metabolism of flag leaves in durum wheat (Triticum durum Desf.).

C sink/source balance and N assimilation have been identified as target processes conditioning crop responsiveness to elevated CO2 . However, little is known about phenology-driven modifications of C and N primary metabolism at elevated CO2 in cereals such as wheat. Here, we examined the differential effect of elevated CO2 at two development stages (onset of flowering, onset of grain filling) in durum wheat (Triticum durum, var. Sula) using physiological measurements (photosynthesis, isotopes), metabolomics, proteomics and (15) N labelling. Our results show that growth at elevated CO2 was accompanied by photosynthetic acclimation through a lower internal (mesophyll) conductance but no significant effect on Rubisco content, maximal carboxylation or electron transfer. Growth at elevated CO2 altered photosynthate export and tended to accelerate leaf N remobilization, which was visible for several proteins and amino acids, as well as lysine degradation metabolism. However, grain biomass produced at elevated CO2 was larger and less N rich, suggesting that nitrogen use efficiency rather than photosynthesis is an important target for improvement, even in good CO2 -responsive cultivars.

Carbon balance, partitioning and photosynthetic acclimation in fruit-bearing grapevine (Vitis vinifera L. cv. Tempranillo) grown under simulated climate change (elevated CO2, elevated temperature and moderate drought) scenarios in temperature gradient greenhouses.

Although plant performance under elevated CO2 has been extensively studied in the past little is known about photosynthetic performance changing simultaneously CO2, water availability and temperature conditions. Moreover, despite of its relevancy in crop responsiveness to elevated CO2 conditions, plant level C balance is a topic that, comparatively, has received little attention. In order to test responsiveness of grapevine photosynthetic apparatus to predicted climate change conditions, grapevine (Vitis vinifera L. cv. Tempranillo) fruit-bearing cuttings were exposed to different CO2 (elevated, 700ppm vs. ambient, ca. 400ppm), temperature (ambient vs. elevated, ambient +4°C) and irrigation levels (partial vs. full irrigation). Carbon balance was followed monitoring net photosynthesis (AN, C gain), respiration (RD) and photorespiration (RL) (C losses). Modification of environment (13)C isotopic composition (δ(13)C) under elevated CO2 (from -10.30 to -24.93‰) enabled the further characterization of C partitioning into roots, cuttings, shoots, petioles, leaves, rachides and berries. Irrespective of irrigation level and temperature, exposure to elevated CO2 induced photosynthetic acclimation of plants. C/N imbalance reflected the inability of plants grown at 700ppm CO2 to develop strong C sinks. Partitioning of labeled C to storage organs (main stem and roots) did not avoid accumulation of labeled photoassimilates in leaves, affecting negatively Rubisco carboxylation activity. The study also revealed that, after 20 days of treatment, no oxidative damage to chlorophylls or carotenoids was observed, suggesting a protective role of CO2 either at current or elevated temperatures against the adverse effect of water stress.

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen-fixing alfalfa.

The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both (15) N2 and (13) C-depleted CO2 on exclusively nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root-derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N2 -fixing machinery, availability of Cnew and Nnew strongly diminished in the plants due to their replacement by C and N assimilated during the post-labelling period. In summary, our study indicated that during the first week of regrowth, root-derived C and N remobilization did not overcome C- and N-limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re-established.

Increased photosynthetic acclimation in alfalfa associated with arbuscular mycorrhizal fungi (AMF) and cultivated in greenhouse under elevated CO2.

Medicago sativa L. (alfalfa) can exhibit photosynthetic down-regulation when grown in greenhouse conditions under elevated atmospheric CO2. This forage legume can establish a double symbiosis with nitrogen fixing bacteria and arbuscular mycorrhizal fungi (AMF), which may increase the carbon sink effect of roots. Our aim was to assess whether the association of alfalfa with AMF can avoid, diminish or delay the photosynthetic acclimation observed in previous studies performed with nodulated plants. The results, however, showed that mycorrhizal (M) alfalfa at the end of their vegetative period had lower carbon (C) discrimination than non-mycorrhizal (NM) controls, indicating photosynthetic acclimation under ECO2 in plants associated with AMF. Decreased C discrimination was due to the acclimation of conductance, since the amount of Rubisco and the expression of genes codifying both large and small subunits of Rubisco were similar or slightly higher in M than in NM plants. Moreover, M alfalfa accumulated a greater amount of soluble sugars in leaves than NM plants, thus favoring a down-regulation effect on photosynthetic rates. The enhanced contents of sugars in leaves coincided with a reduced percentage of arbuscules in roots, suggesting decreased sink of carbohydrates from shoots to roots in M plants. The shorter life cycle of alfalfa associated with AMF in comparison with the NM controls may also be related to the accelerated photosynthetic acclimation in M plants. Further research is needed to clarify to what extent this behavior could be extrapolated to alfalfa cultivated in the field and subjected to periodic cutting of shoots under climatic change scenarios.

Harvest index combined with impaired N availability constrains the responsiveness of durum wheat to elevated CO2 concentration and terminal water stress.

Despite its relevance, few studies to date have analysed the role of harvest index (HI) in the responsiveness of wheat (Triticum spp.) to elevated CO2 concentration ([CO2]) under limited water availability. The goal of the present work was to characterise the role of HI in the physiological responsiveness of durum wheat (Triticum durum Desf.) exposed to elevated [CO2] and terminal (i.e. during grain filling) water stress. For this purpose, the performance of wheat plants with high versus low HI (cvv. Sula and Blanqueta, respectively) was assessed under elevated [CO2] (700µmolmol-1 vs 400µmolmol-1 CO2) and terminal water stress (imposed after ear emergence) in CO2 greenhouses. Leaf carbohydrate build-up combined with limitations in CO2 diffusion (in droughted plants) limited the responsiveness to elevated [CO2] in both cultivars. Elevated [CO2] only increased wheat yield in fully watered Sula plants, where its larger HI prevented an elevated accumulation of total nonstructural carbohydrates. It is likely that the putative shortened grain filling period in plants exposed to water stress also limited the responsiveness of plants to elevated [CO2]. In summary, our study showed that even under optimal water availability conditions, only plants with a high HI responded to elevated [CO2] with increased plant growth, and that terminal drought constrained the responsiveness of wheat plants to elevated [CO2].

Harvest index, a parameter conditioning responsiveness of wheat plants to elevated CO2.

The expansion of the world's population requires the development of high production agriculture. For this purpose, it is essential to identify target points conditioning crop responsiveness to predicted [CO2]. The aim of this study was to determine the relevance of ear sink strength in leaf protein and metabolomic profiles and its implications in photosynthetic activity and yield of durum wheat plants exposed to elevated [CO2]. For this purpose, a genotype with high harvest index (HI) (Triticum durum var. Sula) and another with low HI (Triticum durum var. Blanqueta) were exposed to elevated [CO2] (700 µmol mol(-1) versus 400 µmol mol(-1) CO2) in CO2 greenhouses. The obtained data highlighted that elevated [CO2] only increased plant growth in the genotype with the largest HI; Sula. Gas exchange analyses revealed that although exposure to 700 µmol mol(-1) depleted Rubisco content, Sula was capable of increasing the light-saturated rate of CO2 assimilation (Asat) whereas, in Blanqueta, the carbohydrate imbalance induced the down-regulation of Asat. The specific depletion of Rubisco in both genotypes under elevated [CO2], together with the enhancement of other proteins in the Calvin cycle, revealed that there was a redistribution of N from Rubisco towards RuBP regeneration. Moreover, the down-regulation of N, NO3 (-), amino acid, and organic acid content, together with the depletion of proteins involved in amino acid synthesis that was detected in Blanqueta grown at 700 µmol mol(-1) CO2, revealed that inhibition of N assimilation was involved in the carbohydrate imbalance and consequently with the down-regulation of photosynthesis and growth in these plants.

Photosynthetic and molecular markers of CO2-mediated photosynthetic downregulation in nodulated alfalfa.

Elevated CO2 leads to a decrease in potential net photosynthesis in long-term experiments and thus to a reduction in potential growth. This process is known as photosynthetic downregulation. There is no agreement on the definition of which parameters are the most sensitive for detecting CO2 acclimation. In order to investigate the most sensitive photosynthetic and molecular markers of CO2 acclimation, the effects of elevated CO2, and associated elevated temperature were analyzed in alfalfa plants inoculated with different Sinorhizobium meliloti strains. Plants (Medicago sativa L. cv. Aragón) were grown in summer or autumn in temperature gradient greenhouses (TGG). At the end of the experiment, all plants showed acclimation in both seasons, especially under elevated summer temperatures. This was probably due to the lower nitrogen (N) availability caused by decreased N2-fixation under higher temperatures. Photosynthesis measured at growth CO2 concentration, rubisco in vitro activity and maximum rate of carboxylation were the most sensitive parameters for detecting downregulation. Severe acclimation was also related with decreases in leaf nitrogen content associated with declines in rubisco content (large and small subunits) and activity that resulted in a drop in photosynthesis. Despite the sensitivity of rubisco content as a marker of acclimation, it was not coordinated with gene expression, possibly due to a lag between gene transcription and protein translation.

Alfalfa forage digestibility, quality and yield under future climate change scenarios vary with Sinorhizobium meliloti strain.

Elevated CO(2) may decrease alfalfa forage quality and in vitro digestibility through a drop in crude protein and an enhancement of fibre content. The aim of the present study was to analyse the effect of elevated CO(2), elevated temperature and Sinorhizobium meliloti strains (102F78, 102F34 and 1032 GMI) on alfalfa yield, forage quality and in vitro dry matter digestibility. This objective is in line with the selection of S. meliloti strains in order to maintain high forage yield and quality under future climate conditions. Plants inoculated with the 102F34 strain showed more DM production than those inoculated with 1032GMI; however, these strains did not show significant differences with 102F78 plants. Neutral or acid detergent fibres were not enhanced in plants inoculated with the 102F34 strain under elevated CO(2) or temperature and hence, in vitro dry matter digestibility was unaffected. Crude protein content, an indicator of forage quality, was negatively related to shoot yield. Plants inoculated with 102F78 showed a similar shoot yield to those inoculated with 102F34, but had higher crude protein content at elevated CO(2) and temperature. Under these climate change conditions, 102F78 inoculated plants produced higher quality forage. However, the higher digestibility of plants inoculated with the 102F34 strain under any CO(2) or temperature conditions makes them more suitable for growing under climate change conditions. In general, elevated CO(2) in combination with high temperature (Climate Change scenario) reduced IVDMD and CP content and enhanced fibre content, which means that animal production will be negatively affected.

Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.).

Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by N(ase) activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with N(ase) inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity. This decrease in N(ase) activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation in the nodule could also be associated with an osmoregulatory response to drought and might function as a protective agent against ROS. In droughted nodules, the decrease in N(2) fixation was caused by an increase in oxygen resistance that was induced in the nodule. This was a mechanism to avoid oxidative damage associated with reduced respiration activity and the consequent increase in oxygen content. This study highlighted that even though drought had a direct effect on leaves, the deleterious effects of drought on nodules also conditioned leaf responsiveness.