Open Access
Physiological, hormonal and metabolic responses of two alfalfa cultivars with contrasting responses to drought
(MDPI, 2019) Soba Hidalgo, David; Zhou, Bangwei; Arrese-Igor Sánchez, César; Munné Bosch, Sergi; Aranjuelo Michelena, Iker; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Alfalfa (Medicago sativa L.) is frequently constrained by environmental conditions such as drought. Within this context, it is crucial to identify the physiological and metabolic traits conferring a better performance under stressful conditions. In the current study, two alfalfa cultivars (San Isidro and Zhong Mu) with different physiological strategies were selected and subjected to water limitation conditions. Together with the physiological analyses, we proceeded to characterize the isotopic, hormone, and metabolic profiles of the different plants. According to physiological and isotopic data, Zhong Mu has a water-saver strategy, reducing water lost by closing its stomata but fixing less carbon by photosynthesis, and therefore limiting its growth under water-stressed conditions. In contrast, San Isidro has enhanced root growth to replace the water lost through transpiration due to its more open stomata, thus maintaining its biomass. Zhong Mu nodules were less able to maintain nodule N2 fixing activity (matching plant nitrogen (N) demand). Our data suggest that this cultivar-specific performance is linked to Asn accumulation and its consequent N-feedback nitrogenase inhibition. Additionally, we observed a hormonal reorchestration in both cultivars under drought. Therefore, our results showed an intra-specific response to drought at physiological and metabolic levels in the two alfalfa cultivars studied.
Open Access
Holm oak decline is determined by shifts in fine root phenotypic plasticity in response to belowground stress
(Wiley, 2022) Encinas-Valero, Manuel; Esteban Terradillos, Raquel; Hereş, Ana-Maria; Vivas, María; Fakhet, Dorra; Aranjuelo Michelena, Iker; Solla, Alejandro; Moreno, Gerardo; Curiel Yuste, Jorge; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Climate change and pathogen outbreaks are the two major causes of decline in Mediterranean holm oak trees (Quercus ilex L. subsp. ballota (Desf.) Samp.). Crown-level changes in response to these stressful conditions have been widely documented but the responses of the root systems remain unexplored. The effects of environmental stress over roots and its potential role during the declining process need to be evaluated. We aimed to study how key morphological and architectural root parameters and nonstructural carbohydrates of roots are affected along a holm oak health gradient (i.e. within healthy, susceptible and declining trees). Holm oaks with different health statuses had different soil resource-uptake strategies. While healthy and susceptible trees showed a conservative resource-uptake strategy independently of soil nutrient availability, declining trees optimized soil resource acquisition by increasing the phenotypic plasticity of their fine root system. This increase in fine root phenotypic plasticity in declining holm oaks represents an energy-consuming strategy promoted to cope with the stress and at the expense of foliage maintenance. Our study describes a potential feedback loop resulting from strong unprecedented belowground stress that ultimately may lead to poor adaptation and tree death in the Spanish dehesa.
Open Access
Genetic and isotope ratio mass spectrometric evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves
(Public Library of Science, 2017) Baslam, Marouane; Baroja Fernández, Edurne; Ricarte Bermejo, Adriana; Sánchez López, Ángela María; Aranjuelo Michelena, Iker; Bahaji, Abdellatif; Muñoz Pérez, Francisco José; Almagro Zabalza, Goizeder; Pujol, Pablo; Galarza, Regina; Teixidor, Pilar; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous 13CO2 pulse-chase based studies indicated that starch degradation does not operate in illuminated Arabidopsis leaves. Here we show that leaves of gwd, sex4, bam4, bam1/bam3 and amy3/isa3/lda starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown dpe1 plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown mex1/ pglct plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGlcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (δ13C) in starch of leaves of 13CO2 pulsed-chased WT and AGP lacking aps1 plants. Notably, the rate of increase of δ13C in starch of aps1 leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, δ13C decline in starch of aps1 leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.
Open Access
Onfield estimation of quality parameters in alfalfa through hyperspectral spectrometer data
(Elsevier, 2024) Gámez Guzmán, Angie Lorena; Vatter, Thomas; Santesteban García, Gonzaga; Araus, José Luis; Aranjuelo Michelena, Iker; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Alfalfa is a forage of vast importance around the world. In the past, near-infrared spectroscopy (NIRS) technique have been explored in the lab to determine quality traits such as fibre content in dried and ground material. During the last decade, portable hyperspectral devices have emerged as a tools for in-field prediction, of not only crop yield but also a large range of quality and physiological markers. The objective of this study was to estimate quality parameters in an alfalfa crop using hyperspectral data acquired from a full-range (350–2500 nm) spectrometer under field conditions. Reflected spectra were measured in single leaves as well as at the canopy level, then reflectance was related to target parameters such as biomass, leaf pigments, sugars, protein, and mineral contents. Due to their large effect on crop quality parameters, meteorological conditions and phenological stages were included as predictors in the models. We found that meteorological and phenological variables improved the accuracies and percentage of variance explained (R2) for most of the parameters evaluated. Based on R2 values, the best prediction models were obtained for biomass (0.71), sucrose (0.65), flavonoids (Flav) (0.56) and nitrogen (0.70) with normalized root mean squared errors of 0.196, 0.32, 0.087 and 0.08, respectively. These parameters were associated mainly with visible (VIS) (approx. 350–700 nm) and near infrared (NIR) (700–1250 nm) regions of the spectrum. Regarding mineral composition, the best prediction models were developed for P (0.51), B (0.50) and Zn (0.44), associated with the short-wave infra-red (SWIR) region (1250–2500 nm). The results of this study demonstrated the potential of hyperspectral techniques to be used as a base for performing initial evaluations in the field of quality traits in alfalfa crops.
Open Access
Soybean inoculated with one bradyrhizobium strain isolated at elevated [CO2] show an impaired C and N metabolism when grown at ambient [CO2]
(Frontiers Media, 2021) Soba Hidalgo, David; Aranjuelo Michelena, Iker; Gakière, Bertrand; Gilard, Françoise; Pérez López, Usue; Mena Petite, Amaia; Muñoz Rueda, Alberto; Lacuesta, Maite; Sanz Sáez, Álvaro; Ciencias; Zientziak
Soybean (Glycine max L.) future response to elevated [CO2] has been shown to differ when inoculated with B. japonicum strains isolated at ambient or elevated [CO2]. Plants, inoculated with three Bradyrhizobium strains isolated at different [CO2], were grown in chambers at current and elevated [CO2] (400 vs. 700 ppm). Together with nodule and leaf metabolomic profile, characterization of nodule N-fixation and exchange between organs were tested through 15N2-labeling analysis. Soybeans inoculated with SFJ14-36 strain (isolated at elevated [CO2]) showed a strong metabolic imbalance, at nodule and leaf levels when grown at ambient [CO2], probably due to an insufficient supply of N by nodules, as shown by 15N2-labeling. In nodules, due to shortage of photoassimilate, C may be diverted to aspartic acid instead of malate in order to improve the efficiency of the C source sustaining N2-fixation. In leaves, photorespiration and respiration were boosted at ambient [CO2] in plants inoculated with this strain. Additionally, free phytol, antioxidants, and fatty acid content could be indicate induced senescence due to oxidative stress and lack of nitrogen. Therefore, plants inoculated with Bradyrhizobium strain isolated at elevated [CO2] may have lost their capacity to form effective symbiosis at ambient [CO2] and that was translated at whole plant level through metabolic impairment.
Open Access
Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes
(Oxford University Press, 2018) Fernández San Millán, Alicia; Aranjuelo Michelena, Iker; Ancín Rípodas, María; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Agronomia, Bioteknologia eta Elikadura; Agronomía, Biotecnología y Alimentación; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.
Open Access
Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis
(Public Library of Science, 2015) Bahaji, Abdellatif; Sánchez López, Ángela María; Diego, Nuria de; Muñoz Pérez, Francisco José; Baroja Fernández, Edurne; Li, Jun; Ricarte Bermejo, Adriana; Baslam, Marouane; Aranjuelo Michelena, Iker; Almagro Zabalza, Goizeder; Humplik, Jan F.; Novák, Ondrej; Spíchal, Lukás; Dolezal, Karel; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIM010491.RI2; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.
Open Access
Elevated CO2 has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat
(Oxford University Press, 2020) Tcherkez, Guillaume; Ben Mariem, Sinda; Larraya Reta, Luis María; García Mina, José M.; Zamarreño, Ángel M.; Paradela, Alberto; Cui, Jing; Badeck, Franz-Werner; Meza, Diego; Rizza, Fulvia; Bunce, James; Han, Xue; Tausz-Posch, Sabine; Cattivelli, Luigi; Fangmeier, Andreas; Aranjuelo Michelena, Iker; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Gobierno de Navarra / Nafarroako Gobernua, PI040 TRIGOCLIM
While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.
Open Access
Functional analysis of the taproot and fibrous roots of Medicago truncatula: sucrose and proline catabolism primary response to water deficit
(Elsevier, 2019) Castañeda Presa, Verónica; Peña, Marlon de la; Azcárate Górriz, Lidia; Aranjuelo Michelena, Iker; González García, Esther; Ciencias; Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Root performance represents a target factor conditioning plant development under drought conditions. Moreover, recent root phenotyping studies remark relevant differences on functionality of the different root types. However, despite its relevance, the performance of different types of roots such as primary/taproot (tapR) and lateral/fibrous roots (fibR) under water stress conditions is largely unknown. In the current study, the impact of water stress on target C and N metabolism (namely sucrose and proline) processes were characterized in tapR and fibR of Medicago truncatula plants exposed to different water stress severity regimes (moderate versus severe). While both root types exhibit some common responses to face water stress, the study highlighted important physiological and metabolic differences between them. The tapR proved to have an essential role on carbon and nitrogen partitioning rather than just on storage. Moreover, this root type showed a higher resilience towards water deficit stress. Sucrose metabolization at sucrose synthase level was early blocked in this tissue together with a selective accumulation of some amino acids such as proline and branched chain amino adds, which may act as alternative carbon sources under water deficit stress conditions. The decline in respiration, despite the over-accumulation of carbon compounds, suggests a modulation at sucrose cleavage level by sucrose synthase and invertase. These data not only provide new information on the carbon and nitrogen metabolism modulation upon water deficit stress but also on the different role, physiology, and metabolism of the taproot and fibrous roots. In addition, obtained results highlight the fact that both root types show distinct performance under water deficit stress; this factor can be of great relevance to improve breeding programs for increasing root efficiency under adverse conditions.
Open Access
Does the response of Rubisco and photosynthesis to elevated [CO2] change with unfavourable environmental conditions?
(Oxford University Press, 2024-09-12) Ancín Rípodas, María; Gámez Guzmán, Angie Lorena; Jáuregui Mosquera, Iván; Galmes, J.; Sharwood, R. E.; Erice, G.; Ainsworth, E. A.; Tissue, D. T.; Sanz-Sáez, A.; Aranjuelo Michelena, Iker; Ciencias; Zientziak; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Climate change due to anthropogenic CO2 emissions affects plant performance globally. To improve crop resilience, we need to understand the effects of elevated CO2 concentration (e[CO2]) on CO2 assimilation and Rubisco biochemistry. However, the interactive effects of e[CO2] and abiotic stress are especially unclear. This study examined the CO2 effect on photosynthetic capacity under different water availability and temperature conditions in 42 different crop species, varying in functional group, photosynthetic pathway, and phenological stage. We analysed close to 3000 data points extracted from 120 published papers. For C-3 species, e[CO2] increased net photosynthesis and intercellular [CO2], while reducing stomatal conductance and transpiration. Maximum carboxylation rate and Rubisco in vitro extractable maximal activity and content also decreased with e[CO2] in C-3 species, while C-4 crops are less responsive to e[CO2]. The interaction with drought and/or heat stress did not significantly alter these photosynthetic responses, indicating that the photosynthetic capacity of stressed plants responded to e[CO2]. Moreover, e[CO2] had a strong effect on the photosynthetic capacity of grasses mainly in the final stages of development. This study provides insight into the intricate interactions within the plant photosynthetic apparatus under the influence of climate change, enhancing the understanding of mechanisms governing plant responses to environmental parameters.