Interactive Temperature and CO2 Rise, Salinity, Drought, and Bacterial Inoculation Alter the Content of Fatty Acids, Total Phenols, and Oxalates in the Edible Halophyte Salicornia ramosissima

Citation: Mesa-Marín, J.; Mateos-Naranjo, E.; Carreiras, J.; Feijão, E.; Duarte, B.; Matos, A.R.; Betti, M.; Del Rio, C.; Romero-Bernal, M.; Montaner, J.; et al. Interactive Temperature and CO2Rise, Salinity, Drought, and Bacterial Inoculation Alter the Content of Fatty Acids, Total Phenols, and Oxalates in the Edible Halophyte Salicornia ramosissima.Plants 2023,12, 1395. https://doi.org/10.3390/ plants12061395 Academic Editors: Milagros Bueno, María del Pilar Cordovilla and Manuel Melendo Luque Received: 30 December 2022 Revised: 8 March 2023 Accepted: 18 March 2023 Published: 21 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Interactive Temperature and CO2Rise, Salinity, Drought, and Bacterial Inoculation Alter the Content of Fatty Acids, Total Phenols, and Oxalates in the Edible Halophyte Salicornia ramosissima Jennifer Mesa-Marín1,*, Enrique Mateos-Naranjo 1, João Carreiras 2, Eduardo Feijão2, Bernardo Duarte 2,3 , Ana Rita Matos 3,4 , Marco Betti 5, Carmen Del Rio 6, Marina Romero-Bernal 6, Joan Montaner 6,7 and Susana Redondo-Gómez 1 1Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain 2MARE—Marine and Environmental Sciences Centre, ARNET—Aquatic Research Infrastructure Network Associated Laboratory, Faculdade de Ciências, Universidade de Lisbon, Campo Grande, 1749-016 Lisbon, Portugal 3Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal 4BioISI—Biosystems and Integrative Sciences Institute, Plant Functional Genomics Group, Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal 5Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain 6Institute of Biomedicine of Seville (IBiS), Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, 41013 Seville, Spain 7Department of Neurology, Hospital Universitario Virgen Macarena, 41009 Seville, Spain *Correspondence: author [email protected] Abstract: In this work, we studied the combined effect of increased temperature and atmospheric CO 2 , salt and drought stress, and inoculation with plant-growth-promoting rhizobacteria (PGPR) on the growth and some nutritional parameters of the edible halophyte Salicornia ramosissima. We found that the increase in temperature and atmospheric CO 2 , combined with salt and drought stresses, led to important changes in S. ramosissima fatty acids (FA), phenols, and oxalate contents, which are compounds of great importance for human health. Our results suggest that the S. ramosissima lipid profile will change in a future climate change scenario, and that levels of oxalate and phenolic compounds may change in response to salt and drought stress. The effect of inoculation with PGPR depended on the strains used. Some strains induced the accumulation of phenols in S. ramosissima leaves at higher temperature and CO 2 while not altering FA profile but also led to an accumulation of oxalate under salt stress. In a climate change scenario, a combination of stressors (temperature, salinity, drought) and environmental conditions (atmospheric CO 2, PGPR) will lead to important changes in the nutritional profiles of edible plants. These results may open new perspectives for the nutritional and economical valorization of S. ramosissima. Keywords: climate change; nutritional quality; plant-growth-promoting rhizobacteria; biofertilizer; gas chromatography; spectrophotometry 1. Introduction The study of halophytes has increased recently since these crops are an option for sustainable agriculture in marginal environments. The cultivation of conventional crops is facing severe limitations such as scarcity of good quality water and soil salinization and degradation due to climate change and intensive agriculture [ 1 ]. Halophytes subsist and reproduce under soil salinities of more than 200 mmol L −1 NaCl. In contrast to Plants 2023,12, 1395. https://doi.org/10.3390/plants12061395 https://www.mdpi.com/journal/plants Plants 2023,12, 1395 2 of 18 glycophytes, halophytes are extremely productive under saline irrigation and are able to grow in adverse environments [ 2 ]. They are good candidates as cash crops because they may be domesticated through conventional breeding programs, and they still can achieve high, economically lucrative yields [3–6]. The halophyte Salicornia ramosissima J. Woods (Chenopodiaceae) produces succulent shoots and is highly appreciated as a gourmet vegetable [ 4 , 6 ]. S. ramosissima shoots are consumed boiled, sautéed, or fresh in salads for their umami flavor and crunchiness. They are also processed into beverages such as beer [ 7 – 9 ]. Thus, attention started to be paid to the nutritional profile of this “sea asparagus” [ 10 ] a decade ago. Among other properties, it has been found that Salicornia members have a high content of polyunsaturated fatty acids (PUFAs) and polyphenols, which are beneficial for human consumption because of their antioxidant properties [ 7 , 9 ]. In halophytes, it is known that these molecules are accumulated to higher concentrations in unstressed conditions, as compared to glycophytes, to cope with salinity-induced oxidative stress [ 11 ]. In S. ramosissima, the most abundant PUFAs are linolenic acid (omega-3) and linoleic acid (omega-6), which are essential FAs for humans because the human body is not able to synthesize them [ 12 ]. Regarding total phenol content, Salicornia members [ 13 ] record the smallest values in polyphenols compared to other chenopods such as Halimione or Atriplex [ 14 ]. Even so, Ventura et al. [ 15 ] found up to 1.2 mg GAE g −1 FW in S. persica, while the lower limit of other non-halophytic leafy vegetables rated as rich in phenolic compounds is >0.5 mg GAE g −1 FW [ 11 ]. On the other hand, care should be taken for Salicornia ingestion because of its high salt and oxalate content. Salt excess in the human diet is a major risk factor, especially for hypertension, while oxalic acid binds blood calcium by forming crystals of insoluble calcium oxalate, leading to blood hypocalcemia and the formation of renal stones [ 7 ]. Members of Chenopodiaceae family are known to contain a high oxalate content [ 7 , 16 ]. They use oxalic acid for the maintenance of the ionic equilibrium, especially for high-capacity calcium regulation [ 17 ] and also as a protection mechanism against herbivory, as the ingestion of large amounts of oxalic acid may cause animal death by blood pH disruption [ 6 ]. Lastly, the capacity of halophytes for heavy metal resistance and detoxification has been linked to complexation reactions with oxalic acid [18–20]. Altogether, S. ramosissima may be an alternative cash crop to exploit less arable lands which are unsuitable for conventional crops. However, climate change should be considered as a source of abiotic stress, as it is causing the rise of temperatures and CO 2 and some associated events such as droughts [ 21 ]. Research is needed in order to predict the physiological and biochemical response of S. ramosissima and assess its eligibility for breeding programs and human consumption. To date, it is known that S. ramosissima has an optimal growth at low salinity (200 mmol L −1 NaCl), although it tolerates high salinity ( 600 mmol L−1NaCl ) [ 22 ]. Generally, an elevated atmospheric CO 2 concentration ( 700 ppm ) has a plant-growth-promoting effect in C3 plants, and this includes an improvement in S. ramosissima plant physiological performance by increasing net photosynthetic rate (A N ) and water-use efficiency (iWUE), promoting the accumulation of osmoprotective compounds and the modulation of enzyme antioxidant machinery [ 23 , 24 ]. Finally, we know that short-term, extreme temperature events have a negative impact on S. ramosissima fitness, as revealed by lower antioxidant enzyme activity and the decrease in its photosynthetic efficiency [ 25 ]. These shifts may lead to variations in S. ramosissima chemical composition and, consequently, in its nutritional value. However, research in the nutritional profile of halophytes under abiotic stress is very scarce. To date, it has mostly focused on revealing the salt effect as an approach to their domestication and irrigation with saline water. For example, Maciel et al. [ 26 ] revealed that S. ramosissima showed higher levels of omega-3 FA when produced in marine aquaponics, compared with wild populations. In parallel, Lima et al. [ 27 ] found the greatest amounts of phenolic compounds in S. ramosissima shoots when they grew in intermediate levels of salinity (between 110 and 200 mmol L−1NaCl ). Nevertheless, nothing is known about the response of FA, polyphenols, and oxalates in S. ramosissima under raised temperature and CO2, or drought. Plants 2023,12, 1395 3 of 18 Lastly, the use of specific halotolerant plant-growth-promoting bacteria (PGPB) associated with halophytes could be an important practice for crop production under adverse conditions [ 28 – 30 ]. Some authors have found that the use of PGPB in S. bigelovii resulted in increased shoot growth and seed yield [ 31 – 36 ]. In S. ramosissima, bacterial inoculation has been applied only to studying seed germination response [ 37 ] and plant phytoremediation capacity [ 38 ]. From a nutritional perspective, the effect of PGPB has never been assessed in S. ramosissima. Thus, the objective of this work is to gain insight on the aforementioned questions and to elucidate the effect of simultaneous temperature and CO 2 rise, salinity, and drought, together with PGPB inoculation, on S. ramosissima nutritional value, with an emphasis on its content in FA, polyphenols, and oxalates. The coexistence of stressors, despite its logistical complexity, reflect the environmental reality in a more reliable way, as climatic events in nature occur jointly. Based on the bibliography, we hypothesize that high salinity, high temperature, and drought may increase S. ramosissima content in FA, phenols, and oxalates to counteract stress. Moreover, we hypothesize that elevated CO 2 and PGPR inoculation may improve plant fitness and, therefore, cushion nutrient disruption. This knowledge may open new perspectives for the nutritional and economical valorization of S. ramosissima. 2. Results 2.1. Effect of Combined Temperature, CO2, Irrigation, and Biofertilization on S. ramosissima Shoots Fatty Acid Content Data obtained for saturated palmitic (C16:0) and stearic acid (C18:0), monounsaturated chloroplastidial trans-hexadecenoic acid (C16:1t) and oleic acid (C18:1), and polyunsaturated linoleic (C18:2) and linolenic acid (C18:3) were analyzed at the end of the experiment (Figure 1). The FA profile in S. ramosissima leaves was dominated by PUFAs (approximately 65%, Table 1), which are represented by linoleic and linolenic acid, followed by saturated FAs (SFAs) (approximately 25%, Table 1) and palmitic and stearic acid. The monounsaturated FAs (MUFAs) group was represented by trans-hexadecenoic and oleic acid (approximately 9%, Table 1). The FA profile was clearly affected by the increment of temperature and CO 2 (p< 0.001, Table 2), as mostly evidenced by an increase in the percentage of C16:0 (6%), C18:1 (40%), and C18:2 (8%) and a decrease in C18:3 (24% on average). This was confirmed by the increase in the 18:2/18:3, PUFA/MUFA, and Omega 6 indexes and the decrease in the Omega 3 index (Table 1). Differences among irrigation treatments were significant only for C18:3 content (p< 0.05, Table 2), which was slightly reduced by drought, whereas PGPR inoculation did not produce changes in S. ramosissima FA profile (p> 0.05, Table 2). The interaction of parameters did not show generalized significant differences on FA profile (p> 0.05, Table 2). Plants 2023,12, 1395 4 of 18 Table 1. Fatty acids for S. ramosissima shoots after 30 days of the experiment: saturated FA (SFA), monounsaturated FA (MUFAs), polyunsaturated FA (PUFA), and indexes of lipid nutritional quality: oxidizability (Cox), oxidative susceptibility (OS), hypocholesterolemic/hypercholesterolemic (h/H), peroxidizability (PI), atherogenic index (AI), thrombogenic index (TI), and omega 3 and omega 6. Values are means ±S.E. (n= 5). Irrigation Optimal (171 mmol NaCl L−1) T and CO2400 ppm +4 ◦C, 700 ppm Inoculation C12345C12345 SFA 23.8 ±0.5 25.7 ±2.3 23.7 ±0.4 24.3 ±0.5 24.7 ±0.3 24.4 ±0.4 27.0 ±0.3 25.8 ±0.6 26.1 ±0.6 25.7 ±0.4 26.6 ±1.6 26.1 ±0.2 MUFA 8.6 ±0.7 7.5 ±1.1 10.0 ±0.4 10.2 ±0.6 10.5 ±0.9 9.9 ±0.3 10.6 ±0.9 12.4 ±1.0 11.4 ±0.7 11.5 ±0.9 15.9 ±1.5 12.9 ±0.7 PUFA 67.7 ±0.7 66.8 ±2.7 66.3 ±0.6 65.5 ±0.4 64.7 ±0.8 65.7 ±0.6 62.3 ±1.2 61.8 ±1.4 62.5 ±1.0 62.8 ±0.8 57.5 ±3.0 61.0 ±0.6 PUFA/SFA 2.9 ±0.1 2.7 ±0.3 2.8 ±0.1 2.7 ±0.1 2.6 ±0.0 2.7 ±0.1 2.3 ±0.1 2.4 ±0.1 2.4 ±0.1 2.4 ±0.1 2.2 ±0.2 2.3 ±0.0 18:2/18:3 0.8 ±0.04 0.7 ±0.05 0.8 ±0.04 0.9 ±0.01 0.7 ±0.04 0.8 ±0.05 1.14 ±0.12 0.87 ±0.03 1.06 ±0.07 1.00 ±0.03 1.10 ±0.06 1.27 ±0.13 Cox 11.4 ±0.2 11.4 ±0.5 11.0 ±0.2 10.7 ±0.1 11.0 ±0.1 10.9 ±0.2 9.8 ±0.3 10.2 ±0.2 10.0 ±0.2 10.1 ±0.1 8.6 ±0.9 9.5 ±0.2 OS 5171.4 ±100.5 5170.4 ±245.0 5005.4 ±87.3 4853.7 ±28.6 5017.9 ±70.4 4972.5 ±89.7 4441.5 ±158.5 4618.9 ±113.0 4499.2 ±105.0 4566.6 ±47.4 3852.1 ±444.8 4256.0 ±106.2 h/H 3.4 ±0.1 3.2 ±0.2 3.5 ±0.1 3.3 ±0.1 3.3 ±0.1 3.4 ±0.1 2.9 ±0.1 3.1 ±0.1 3.0 ±0.1 3.0 ±0.1 3.0 ±0.2 3.0 ±0.0 PI 83.3 ±2.1 83.7 ±4.2 80.6 ±1.9 76.8 ±0.5 83.1 ±1.6 80.2 ±2.1 67.9 ±3.3 75.1 ±1.6 70.0 ±2.3 71.9 ±0.5 59.6 ±8.8 65.4 ±2.4 AI 0.30 ±0.01 0.35 ±0.05 0.31 ±0.01 0.30 ±0.01 0.30 ±0.01 0.30 ±0.01 0.37 ±0.01 0.35 ±0.01 0.35 ±0.01 0.35 ±0.01 0.36 ±0.03 0.35 ±0.00 TI 0.2 ±0.01 0.19 ±0.03 0.18 ±0.01 0.19 ±0.00 0.18 ±0.00 0.2 ±0.01 0.25 ±0.01 0.21 ±0.01 0.23 ±0.01 0.22 ±0.00 0.38 ±0.14 0.25 ±0.01 Omega 3 38.5 ±1.3 39.2 ±2.4 36.6 ±1.1 34.5 ±0.3 38.1 ±0.9 36.5 ±1.3 29.6 ±2.0 33.2 ±1.0 30.5 ±1.3 31.5 ±0.4 22.7 ±5.7 27.3 ±1.5 Omega 6 29.1 ±0.8 27.5 ±1.3 29.7 ±0.6 31.1 ±0.3 26.7 ±1.0 29.2 ±1.0 32.8 ±1.3 28.7 ±0.7 32.0 ±1.0 31.3 ±0.8 34.9 ±2.9 33.7 ±1.2 Irrigation Salinity (510 mmol NaCl L−1) T and CO2400 ppm +4 ◦C, 700 ppm Inoculation C12345C12345 SFA 24.4 ±0.6 23.9 ±0.4 24.1 ±0.4 24.8 ±0.3 25.8 ±1.6 25.5 ±0.8 22.6 ±2.5 25.9 ±0.4 27.2 ±0.9 24.9 ±0.5 26.4 ±0.2 26.1 ±0.3 MUFA 9.2 ±0.8 9.4 ±0.5 9.9 ±0.5 11.7 ±0.3 11.3 ±1.1 9.7 ±1.7 21.5 ±8.5 12.1 ±0.5 13.4 ±1.4 11.1 ±0.8 14.4 ±1.5 13.0 ±1.0 PUFA 66.4 ±1.3 66.7 ±0.8 66.0 ±0.7 63.6 ±0.2 62.8 ±2.6 64.8 ±1.9 55.9 ±6.0 62.0 ±0.9 59.4 ±2.3 64.1 ±1.2 59.2 ±1.6 60.9 ±0.8 PUFA/SFA 2.7 ±0.1 2.8 ±0.1 2.7 ±0.1 2.6 ±0.0 2.5 ±0.2 2.6 ±0.1 2.5 ±0.1 2.4 ±0.1 2.2 ±0.1 2.6 ±0.1 2.2 ±0.1 2.3 ±0.0 18:2/18:3 0.9 ±0.1 0.8 ±0.08 0.9 ±0.03 0.9 ±0.02 0.9 ±0.10 0.8 ±0.03 1.14 ±0.10 0.91 ±0.03 1.05 ±0.11 0.99 ±0.06 1.07 ±0.08 0.99 ±0.05 Cox 10.9 ±0.3 11.1 ±0.3 10.7 ±0.1 10.4 ±0.1 10.4 ±0.6 10.8 ±0.3 8.9 ±0.9 10.1 ±0.2 9.6 ±0.5 10.3 ±0.3 9.5 ±0.4 9.8 ±0.1 OS 4955.5 ±157.5 5028.9 ±131.9 4855.1 ±69.3 4720.1 ±39.4 4711.4 ±281.8 4884.6 ±138.0 3992.4 ±444.1 4591.0 ±73.6 4310.4 ±236.7 4677.5 ±140.1 4263.3 ±171.3 4441.2 ±44.8 h/H 3.3 ±0.1 3.4 ±0.1 3.4 ±0.1 3.2 ±0.1 3.1 ±0.2 3.2 ±0.1 4.0 ±0.8 3.0 ±0.1 2.8 ±0.1 3.2 ±0.1 2.9 ±0.1 3.0 ±0.1 PI 78.4 ±3.1 80.5 ±3.0 76.1 ±1.2 75.9 ±1.1 76.3 ±5.1 78.2 ±1.8 66.3 ±4.3 74.0 ±1.1 69.1 ±3.8 73.6 ±2.6 68.6 ±2.5 71.1 ±1.1 AI 0.32 ±0.01 0.3 ±0.01 0.3 ±0.01 0.3 ±0.01 0.3 ±0.03 0.3 ±0.01 0.30 ±0.04 0.35 ±0.01 0.37 ±0.02 0.33 ±0.01 0.36 ±0.00 0.35 ±0.00 TI 0.19 ±0.01 0.2 ±0.01 0.2 ±0.01 0.2 ±0.01 0.2 ±0.03 0.2 ±0.01 0.21 ±0.02 0.22 ±0.01 0.25 ±0.03 0.21 ±0.01 0.24 ±0.01 0.23 ±0.00 Omega 3 35.6 ±1.9 36.7 ±1.8 34.1 ±0.8 33.6 ±0.5 34.0 ±3.0 35.6 ±1.1 26.5 ±3.5 32.5 ±0.7 29.5 ±2.5 32.4 ±1.6 28.8 ±1.9 30.7 ±0.6 Omega 6 30.8 ±1.1 30.0 ±1.2 31.9 ±0.6 29.9 ±0.3 28.8 ±0.6 29.1 ±1.2 29.4 ±3.2 29.5 ±0.6 29.8 ±0.6 31.6 ±0.7 30.3 ±0.5 30.2 ±1.1 Irrigation Drought T and CO2400 ppm +4 ◦C, 700 ppm Inoculation C12345C12345 SFA 24.2 ±0.6 25.3 ±1.2 24.8 ±0.1 28.5 ±2.4 24.9 ±0.3 24.4 ±0.5 26.0 ±0.5 25.9 ±0.7 26.8 ±0.6 26.6 ±0.7 26.3 ±0.5 25.5 ±0.5 MUFA 9.1 ±0.6 8.4 ±0.6 10.8 ±1.0 10.7 ±1.4 9.6 ±0.4 10.7 ±0.8 9.6 ±0.7 12.4 ±1.4 9.1 ±1.3 9.3 ±0.5 13.3 ±0.5 11.5 ±0.9 PUFA 66.7 ±0.9 66.3 ±1.6 64.4 ±1.0 60.9 ±3.6 65.5 ±0.7 64.9 ±1.1 64.4 ±0.9 61.7 ±2.1 51.0 ±3.3 64.0 ±1.0 60.4 ±0.9 63.0 ±1.3 PUFA/SFA 2.8 ±0.1 2.7 ±0.2 2.6 ±0.1 2.2 ±0.3 2.6 ±0.1 2.7 ±0.1 2.5 ±0.1 2.4 ±0.1 1.9 ±0.2 2.4 ±0.1 2.3 ±0.1 2.5 ±0.1 18:2/18:3 0.9 ±0.03 0.8 ±0.07 0.9 ±0.06 0.9 ±1.16 0.7 ±0.04 0.8 ±0.05 1.04 ±0.10 1.15 ±0.17 2.01 ±0.25 1.08 ±0.15 1.46 ±0.12 1.03 ±0.09 Cox 11.0 ±0.2 11.2 ±0.4 10.6 ±0.3 9.8 ±1.1 11.1 ±0.1 10.9 ±0.3 10.3 ±0.1 9.8 ±0.5 7.3 ±0.6 10.2 ±0.3 9.1 ±0.3 10.1 ±0.4 OS 4988.5 ±89.1 5069.5 ±190.4 4819.3 ±136.3 4459.0 ±503.6 5026.3 ±59.1 4951.7 ±131.6 4654.4 ±63.4 4415.0 ±248.2 3284.4 ±295.5 4620.1 ±166.6 4092.5 ±123.9 4571.8 ±174.8 h/H 3.3 ±0.1 3.2 ±0.1 3.3 ±0.0 2.7 ±0.3 3.2 ±0.0 3.3 ±0.1 3.0 ±0.1 3.0 ±0.1 2.4 ±0.2 2.9 ±0.1 3.0 ±0.1 3.0 ±0.1 PI 78.9 ±1.5 81.5 ±3.9 77.6 ±2.4 70.4 ±10.7 81.9 ±1.3 81.0 ±2.5 71.3 ±2.2 69.2 ±4.4 43.2 ±5.9 70.6 ±3.9 61.4 ±2.5 71.9 ±3.5 AI 0.3 ±0.01 0.3 ±0.02 0.3 ±0.00 0.4 ±0.05 0.3 ±0.01 0.3 ±0.01 0.35 ±0.01 0.35 ±0.01 0.46 ±0.04 0.36 ±0.01 0.36 ±0.01 0.34 ±0.01 TI 0.2 ±0.01 0.2 ±0.02 0.2 ±0.01 0.3 ±0.12 0.2 ±0.00 0.2 ±0.01 0.22 ±0.01 0.24 ±0.03 0.38 ±0.04 0.23 ±0.02 0.27 ±0.02 0.22 ±0.01 Omega 3 36.0 ±0.9 37.7 ±2.2 34.7 ±1.7 31.1 ±6.3 37.6 ±0.8 36.7 ±1.6 31.8 ±1.2 29.6 ±2.9 17.8 ±2.7 31.5 ±2.3 24.8 ±1.5 31.4 ±2.2 Omega 6 30.7 ±0.4 28.6 ±0.8 29.6 ±0.7 29.8 ±2.8 27.9 ±0.8 28.2 ±0.9 32.6 ±1.7 32.1 ±1.0 33.2 ±0.8 32.6 ±1.5 35.6 ±0.7 31.6 ±1.0 Plants 2023,12, 1395 5 of 18 Table 2. Generalized linear model (GLM) significance as p-values for FA and phenol and oxalate content of S. ramosissima shoots under conditions of T/CO 2 , irrigation, and PGPR inoculation (as categorical variables) and their interaction (p* < 0.05, p** < 0.01, p*** < 0.001). Treatment FA 16:0 FA 18:0 FA 16:1t FA 18:1 FA 18:2 FA 18:3 Phenols Oxalate T/CO20.0000 *** 0.0590 0.0000 *** 0.0000 *** 0.0000 *** 0.0000 *** 0.4109 0.0000 *** Irrigation 0.1267 0.7633 0.0000 *** 0.1763 0.2043 0.0221 ** 0.0000 *** 0.0000 *** Inoculation 0.5454 0.3962 0.5359 0.0315 ** 0.2587 0.0869 0.0966 0.0000 *** T/CO2×Irrigation 0.4179 0.0871 0.0005 *** 0.0262 ** 0.0037 ** 0.1464 0.0000 *** 0.3307 T/CO2×Inoculation 0.4104 0.6916 0.0002 *** 0.0740 0.0011 ** 0.0137 ** 0.0075 ** 0.0000 *** Irrigation ×Inoculation 0.1679 0.3482 0.1805 0.4864 0.5135 0.1859 0.0000 *** 0.0000 *** T/CO2×Irrigation ×Inoculation 0.1088 0.7653 0.0174 * 0.3431 0.7529 0.0858 0.0000 *** 0.0000 *** Plants 2023, 12, x FOR PEER REVIEW 4 of 18 Figure 1. Relative content of FA in S. ramosissima shoots, as expressed by percentage, after 30 days under irrigation (optimal 171 mmol L−1 NaCl, salinity 510 mmol L−1 NaCl, and drought) and inoculation (control non-inoculated—C, and biofertilizers 1 to 5) treatments in (A) 25/14 °C 400 ppm CO2 chamber and (B) 29/18 °C 700 ppm CO2 chamber. Values are means ± S.E. (n = 5) (GLM: T/CO2 × Irrigation × Inoculation, p > 0.05). Table 1. Fatty acids for S. ramosissima shoots after 30 days of the experiment: saturated FA (SFA), monounsaturated FA (MUFAs), polyunsaturated FA (PUFA), and indexes of lipid nutritional quality: oxidizability (Cox), oxidative susceptibility (OS), hypocholesterolemic/hypercholesterolemic (h/H), peroxidizability (PI), atherogenic index (AI), thrombogenic index (TI), and omega 3 and omega 6. Values are means ± S.E. (n = 5). Irrigation Optimal (171 mmol NaCl L−1) T and CO2 400 ppm +4 °C, 700 ppm Inoculation C 1 2 3 4 5 C 1 2 3 4 5 SFA 23.8 ± 0.5 25.7 ± 2.3 23.7 ± 0.4 24.3 ± 0.5 24.7 ± 0.3 24.4 ± 0.4 27.0 ± 0.3 25.8 ± 0.6 26.1 ± 0.6 25.7 ± 0.4 26.6 ± 1.6 26.1 ± 0.2 MUFA 8.6 ± 0.7 7.5 ± 1.1 10.0 ± 0.4 10.2 ± 0.6 10.5 ± 0.9 9.9 ± 0.3 10.6 ± 0.9 12.4 ± 1.0 11.4 ± 0.7 11.5 ± 0.9 15.9 ± 1.5 12.9 ± 0.7 PUFA 67.7 ± 0.7 66.8 ± 2.7 66.3 ± 0.6 65.5 ± 0.4 64.7 ± 0.8 65.7 ± 0.6 62.3 ± 1.2 61.8 ± 1.4 62.5 ± 1.0 62.8 ± 0.8 57.5 ± 3.0 61.0 ± 0.6 PUFA/SFA 2.9 ± 0.1 2.7 ± 0.3 2.8 ± 0.1 2.7 ± 0.1 2.6 ± 0.0 2.7 ± 0.1 2.3 ± 0.1 2.4 ± 0.1 2.4 ± 0.1 2.4 ± 0.1 2.2 ± 0.2 2.3 ± 0.0 18:2/18:3 0.8 ± 0.04 0.7 ± 0.05 0.8 ± 0.04 0.9 ± 0.01 0.7 ± 0.04 0.8 ± 0.05 1.14 ± 0.12 0.87 ± 0.03 1.06 ± 0.07 1.00 ± 0.03 1.10 ± 0.06 1.27 ± 0.13 Cox 11.4 ± 0.2 11.4 ± 0.5 11.0 ± 0.2 10.7 ± 0.1 11.0 ± 0.1 10.9 ± 0.2 9.8 ± 0.3 10.2 ± 0.2 10.0 ± 0.2 10.1 ± 0.1 8.6 ± 0.9 9.5 ± 0.2 OS 5171.4 ± 100.5 5170.4 ± 245.0 5005.4 ± 87.3 4853.7 ± 28.6 5017.9 ± 70.4 4972.5 ± 89.7 4441.5 ± 158.5 4618.9 ± 113.0 4499.2 ± 105.0 4566.6 ± 47.4 3852.1 ± 444.8 4256.0 ± 106.2 h/H 3.4 ± 0.1 3.2 ± 0.2 3.5 ± 0.1 3.3 ± 0.1 3.3 ± 0.1 3.4 ± 0.1 2.9 ± 0.1 3.1 ± 0.1 3.0 ± 0.1 3.0 ± 0.1 3.0 ± 0.2 3.0 ± 0.0 PI 83.3 ± 2.1 83.7 ± 4.2 80.6 ± 1.9 76.8 ± 0.5 83.1 ± 1.6 80.2 ± 2.1 67.9 ± 3.3 75.1 ± 1.6 70.0 ± 2.3 71.9 ± 0.5 59.6 ± 8.8 65.4 ± 2.4 AI 0.30 ± 0.01 0.35 ± 0.05 0.31 ± 0.01 0.30 ± 0.01 0.30 ± 0.01 0.30 ± 0.01 0.37 ± 0.01 0.35 ± 0.01 0.35 ± 0.01 0.35 ± 0.01 0.36 ± 0.03 0.35 ± 0.00 TI 0.2 ± 0.01 0.19 ± 0.03 0.18 ± 0.01 0.19 ± 0.00 0.18 ± 0.00 0.2 ± 0.01 0.25 ± 0.01 0.21 ± 0.01 0.23 ± 0.01 0.22 ± 0.00 0.38 ± 0.14 0.25 ± 0.01 Omega 3 38.5 ± 1.3 39.2 ± 2.4 36.6 ± 1.1 34.5 ± 0.3 38.1 ± 0.9 36.5 ± 1.3 29.6 ± 2.0 33.2 ± 1.0 30.5 ± 1.3 31.5 ± 0.4 22.7 ± 5.7 27.3 ± 1.5 Omega 6 29.1 ± 0.8 27.5 ± 1.3 29.7 ± 0.6 31.1 ± 0.3 26.7 ± 1.0 29.2 ± 1.0 32.8 ± 1.3 28.7 ± 0.7 32.0 ± 1.0 31.3 ± 0.8 34.9 ± 2.9 33.7 ± 1.2 Figure 1. Relative content of FA in S. ramosissima shoots, as expressed by percentage, after 30 days under irrigation (optimal 171 mmol L −1 NaCl, salinity 510 mmol L −1 NaCl, and drought) and inoculation (control non-inoculated—C, and biofertilizers 1 to 5) treatments in ( A ) 25/14 ◦ C 400 ppm CO 2 chamber and ( B ) 29/18 ◦ C 700 ppm CO 2 chamber. Values are means ± S.E. (n= 5) (GLM: T/CO2×Irrigation ×Inoculation, p> 0.05). On the other hand, our results (Table 1) showed that the edible shoots of S. ramosissima had a health-promoting lipid content and that they were altered by temperature and CO 2 rise. AI and TI values were low and consistent among all treatments (0.2–0.3), slightly increasing at higher temperature and atmospheric CO 2 (0.2–0.4), as did h/H values (2.5–4). Regarding lipid oxidation, OS values were high at approximately 4400–5200 but dropped with raised temperature and atmospheric CO 2 to 3200–4700, as did Cox values, which dropped from 11 to 9, approximately. The PI value followed the same pattern, dropping from 70–80 to 60–75. Plants 2023,12, 1395 6 of 18 2.2. Effect of Combined Temperature, CO 2 , Irrigation, and Biofertilization on Total Phenol Content in S. ramosissima Shoots Total phenol content at the end of the experiment in S. ramosissima shoots is represented in Figure 2. In non-biofertilized plants, concentration of total phenols ranged from 5 to 9 mg GAE g−1DW . Increases in temperature and CO 2 levels did not cause significant differences in phenol content (p> 0.05). At optimal temperature/CO 2 , salinity or drought also did not cause significant differences in phenol content (p> 0.05). However, at +4 ◦ C and 700 ppm atmospheric CO 2 , saline irrigation induced a decrease in phenol content (p< 0.05) in S. ramosissima leaves, whereas drought induced a slight increase. The combination of all the parameters studied, including biofertilization, had a significant effect on the phenol content of S. ramosissima leaves (Table 2, GLM: T/CO 2× Irrigation × Inoculation, p< 0.001). Biofertilization with PGPR produced a phenol increase under drought in both optimal and raised T/CO 2 . In particular, a very high phenol content was detected in S. ramosissima plants inoculated with rhizobia inoculum number 4, reaching 15 mg GAE g −1 shoot DW, a 65% increase over its respective non-inoculated control. Plants 2023, 12, x FOR PEER REVIEW 6 of 18 2.2. Effect of Combined Temperature, CO2, Irrigation, and Biofertilization on Total Phenol Content in S. ramosissima Shoots Total phenol content at the end of the experiment in S. ramosissima shoots is represented in Figure 2. In non-biofertilized plants, concentration of total phenols ranged from 5 to 9 mg GAE g−1 DW. Increases in temperature and CO2 levels did not cause significant differences in phenol content (p > 0.05). At optimal temperature/CO2, salinity or drought also did not cause significant differences in phenol content (p > 0.05). However, at +4 °C and 700 ppm atmospheric CO2, saline irrigation induced a decrease in phenol content (p < 0.05) in S. ramosissima leaves, whereas drought induced a slight increase. The combination of all the parameters studied, including biofertilization, had a significant effect on the phenol content of S. ramosissima leaves (Table 2, GLM: T/CO2 × Irrigation × Inoculation, p < 0.001). Biofertilization with PGPR produced a phenol increase under drought in both optimal and raised T/CO2. In particular, a very high phenol content was detected in S. ramosissima plants inoculated with rhizobia inoculum number 4, reaching 15 mg GAE g−1 shoot DW, a 65% increase over its respective non-inoculated control. Figure 2. Total phenol content in S. ramosissima shoots, as expressed by mg GAE g−1 DW, after 30 days under irrigation (optimal 171 mmol L−1 NaCl, salinity 510 mmol L−1 NaCl, and drought) and inoculation (control non-inoculated—C, and biofertilizers 1 to 5) treatments in (A) 25/14 °C 400 ppm CO2 chamber and (B) 29/18 °C 700 ppm CO2 chamber. Values are means ± S.E. (n = 3). Different letters indicate means that are significantly different from each other (GLM: T/CO2 × Irrigation × Inoculation, p < 0.001). “T/CO2 × Irrig × Inoc” in the upper right corner of the panels indicates triple interaction significant effects (p *** < 0.001). 2.3. Effect of Combined Temperature, CO2, Irrigation, and Biofertilization on Oxalate Concentration in S. ramosissima Shoots Oxalic acid concentration in S. ramosissima leaves at the end of the experiment is shown in Figure 3. In non-biofertilized plants, oxalic acid ranged from 25 to 40 µmol g−1 FW. Temperature/CO2 rise and drought did not cause significant differences in oxalate content (p > 0.05). However, saline irrigation (510 mmol L−1 NaCl) caused a decrease in oxalate content, which was more pronounced at +4 °C and 700 ppm CO2 (p < 0.05). The combination of all the parameters studied, including biofertilization, had a significant effect on the oxalate concentration of S. ramosissima leaves (Table 2, GLM: T/CO2 × Irrigation × Inoculation, p < 0.001). Rhizobacteria biofertilization produced a significant increase in Figure 2. Total phenol content in S. ramosissima shoots, as expressed by mg GAE g −1 DW, after 30 days under irrigation (optimal 171 mmol L −1 NaCl, salinity 510 mmol L −1 NaCl, and drought) and inoculation (control non-inoculated—C, and biofertilizers 1 to 5) treatments in ( A ) 25/14 ◦ C 400 ppm CO 2 chamber and ( B ) 29/18 ◦ C 700 ppm CO 2 chamber. Values are means ± S.E. (n= 3). Different letters indicate means that are significantly different from each other (GLM: T/CO 2× Irrigation × Inoculation, p< 0.001). “T/CO 2× Irrig × Inoc” in the upper right corner of the panels indicates triple interaction significant effects (p*** < 0.001). 2.3. Effect of Combined Temperature, CO2, Irrigation, and Biofertilization on Oxalate Concentration in S. ramosissima Shoots Oxalic acid concentration in S. ramosissima leaves at the end of the experiment is shown in Figure 3. In non-biofertilized plants, oxalic acid ranged from 25 to 40 µ mol g −1 FW. Temperature/CO 2 rise and drought did not cause significant differences in oxalate content (p> 0.05). However, saline irrigation (510 mmol L −1 NaCl) caused a decrease in oxalate content, which was more pronounced at +4 ◦ C and 700 ppm CO 2 (p< 0.05). The combination of all the parameters studied, including biofertilization, had a significant effect on the oxalate concentration of S. ramosissima leaves (Table 2, GLM: T/CO 2× Irrigation × Inoculation, p< 0.001). Rhizobacteria biofertilization produced a significant increase in oxalate levels under salinity, being more evident at +4 ◦ C and 700 ppm CO 2 . Plants inoculated with the 5 PGPR consortia showed significantly higher oxalate content than the non-inoculated plants (p< 0.05), especially consortium number 4, which increased the leaf Plants 2023,12, 1395 7 of 18 oxalate content up to 2.6 × at 515 mmol L −1 NaCl and 1.7 × at optimal 171 mmol L −1 NaCl compared to non-inoculated S. ramosissima. Plants 2023, 12, x FOR PEER REVIEW 7 of 18 oxalate levels under salinity, being more evident at +4 °C and 700 ppm CO2. Plants inoculated with the 5 PGPR consortia showed significantly higher oxalate content than the non-inoculated plants (p < 0.05), especially consortium number 4, which increased the leaf oxalate content up to 2.6x at 515 mmol L−1 NaCl and 1.7x at optimal 171 mmol L−1 NaCl compared to non-inoculated S. ramosissima. Figure 3. Oxalate content in S. ramosissima shoots, as expressed by µmol oxalate g−1 FW, after 30 days under irrigation (optimal 171 mmol L−1 NaCl, salinity 510 mmol L−1 NaCl, and drought) and inoculation (control non-inoculated—C, and biofertilizers 1 to 5) treatments in (A) 25/14 °C 400 ppm CO2 chamber and (B) 29/18 °C 700 ppm CO2 chamber. Values are means ± S.E. (n = 3). Different letters indicate means that are significantly different from each other (GLM: T/CO2 × Irrigation × Inoculation, p < 0.001). “T/CO2 × Irrig × Inoc” in the upper right corner of the panels indicates triple interaction significant effects (p *** < 0.001). 3. Discussion We analyzed the combined effect of increased temperature and CO2, salt and drought treatments, and PGPR inoculation for 30 days on the S. ramosissima shoot nutrient content. As an edible cash crop, the contents of FAs, oxalate, and phenolic compounds in S. ramosissima shoots are very important from a nutritional point of view but also from a physiological perspective as part of a complex mechanism for abiotic stress tolerance. FA profile, besides its nutritional relevance, has been suggested as a useful biomarker for abiotic stress in halophytes species, such as Spartina maritima, Spartina patens, Halimione portulacoides, and Sarcocornia fruticosa, and their levels are highly related to the photosynthetic functioning of these species [39,40]. We found that S. ramosissima leaves were dominated by PUFAs (especially by linolenic and linoleic acid), followed by SFAs (mostly palmitic acid), in agreement with the results obtained for other Salicornia species such as S. europaea [41] and S. bigelovii [42]. On the other hand, Isca et al. [43], who studied for the first time the lipid profile of S. ramosissima, found a greater proportion of SFAs rather than PUFAs. In agreement with these results are also the data obtained by Radwan et al. [44] for S. fruticosa. Such saturated FA profiles are common in halophytic plants and may be related to salt tolerance mechanisms [9], probably through a decrease in membrane permeability to NaCl. However, Elsebaie et al. [45] reported oleic MUFA as the most abundant in S. fruticosa (more than 50% of FA). Altogether, it seems that the differences observed in the fatty acid composition in Salicornia are mainly related to the species and morphological zone of the plant under study, besides environmental conditions [43]. In our work, an increase in temperature by 4 ºC and atmospheric CO2 to 700 ppm caused a clear impact on the FA profile of S. ramosissima shoots, leading to the loss of unsaturation Figure 3. Oxalate content in S. ramosissima shoots, as expressed by µ mol oxalate g −1 FW, after 30 days under irrigation (optimal 171 mmol L −1 NaCl, salinity 510 mmol L −1 NaCl, and drought) and inoculation (control non-inoculated—C, and biofertilizers 1 to 5) treatments in ( A ) 25/14 ◦ C 400 ppm CO 2 chamber and ( B ) 29/18 ◦ C 700 ppm CO 2 chamber. Values are means ± S.E. (n= 3). Different letters indicate means that are significantly different from each other (GLM: T/CO 2× Irrigation × Inoculation, p< 0.001). “T/CO 2× Irrig × Inoc” in the upper right corner of the panels indicates triple interaction significant effects (p*** < 0.001). 3. Discussion We analyzed the combined effect of increased temperature and CO 2 , salt and drought treatments, and PGPR inoculation for 30 days on the S. ramosissima shoot nutrient content. As an edible cash crop, the contents of FAs, oxalate, and phenolic compounds in S. ramosissima shoots are very important from a nutritional point of view but also from a physiological perspective as part of a complex mechanism for abiotic stress tolerance. FA profile, besides its nutritional relevance, has been suggested as a useful biomarker for abiotic stress in halophytes species, such as Spartina maritima,Spartina patens,Halimione portulacoides, and Sarcocornia fruticosa, and their levels are highly related to the photosynthetic functioning of these species [ 39 , 40 ]. We found that S. ramosissima leaves were dominated by PUFAs (especially by linolenic and linoleic acid), followed by SFAs (mostly palmitic acid), in agreement with the results obtained for other Salicornia species such as S. europaea [41] and S. bigelovii [ 42 ]. On the other hand, Isca et al. [ 43 ], who studied for the first time the lipid profile of S. ramosissima, found a greater proportion of SFAs rather than PUFAs. In agreement with these results are also the data obtained by Radwan et al. [ 44 ] for S. fruticosa. Such saturated FA profiles are common in halophytic plants and may be related to salt tolerance mechanisms [ 9 ], probably through a decrease in membrane permeability to NaCl. However, Elsebaie et al. [ 45 ] reported oleic MUFA as the most abundant in S. fruticosa (more than 50% of FA). Altogether, it seems that the differences observed in the fatty acid composition in Salicornia are mainly related to the species and morphological zone of the plant under study, besides environmental conditions [ 43 ]. In our work, an increase in temperature by 4 º C and atmospheric CO 2 to 700 ppm caused a clear impact on the FA profile of S. ramosissima shoots, leading to the loss of unsaturation degree, as shown by the increase in the 18:2/18:3, PUFA/MUFA, and Omega 6 indexes and the decrease in the Omega 3 index. The same response was observed by Pérez-Romero et al. [ 23 ]. According to several reports, lipids may be involved in the PSII protection in the presence of salt Plants 2023,12, 1395 8 of 18 stress [ 46 – 48 ]. In photosynthetic tissues, linolenic acid is associated with the synthesis of galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which are fundamental for the correct function of photosynthesis [ 49 ]. Moreover, a direct action of ROS production during stress exposition has been described in the literature [ 50 , 51 ], which may lead to PUFA degradation by direct action in double bonds. These mechanisms may explain the decrease that we observed in S. ramosissima relative content of C18:3 upon a temperature and CO 2 rise. However, according to Duarte et al. [ 48 ], there was no correlation between the decrease in C18:3 and the photosynthetic apparatus performance in the halophyte Aster tripolium. We expected more evident changes in the FA profile of S. ramosissima under salinity and drought, as observed by Maciel et al. [ 26 ] in S. ramosissima grown in marine aquaponics, which had higher levels of n-3 FA compared with the wild populations. However, compared to the effect of temperature/CO 2 , we did not find significant differences in S. ramosissima FA profile with salinity or drought, and neither did other authors [ 15 , 43 ]. Trans-hexadecenoic acid (16:1t), a fatty acid that is present exclusively in the chloroplast [ 52 ], is generated by a membrane-bound desaturase acting on C16:0, which is esterified to phosphatidylglycerol (PG), which is the only phospholipid present in thylakoids. The decrease in the concentration of this fatty acid observed under drought indicates a reduction in the fluidity of the chloroplast membrane [ 53 ]. FA quality indexes showed that the increase in temperature and CO 2 predicted for the future atmosphere decreased the quality of FA, in terms of oxidative stability. That is, they are more prone to oxidation, and in terms of human health, they have a lower protective potential for the cardiovascular system [39]. Regarding the changes in phenolic compounds, only a few studies have analyzed total phenol content in S. ramosissima. The values here reported, from 5 to 9 mg GAE g −1 DW (4 to 14 mg GAE g −1 DW in inoculated plants), were similar to the values found by other authors for pot-grown S. ramosissima (9 to 13 mg GAE g −1 DW [ 27 ]). These concentrations seem to be greater for wild S. ramosissima populations. For example, Barreira et al. [9] detected up to 33 mg GAE g −1 DW, while Antunes et al. [ 54 ] found up to 40 mg GAE g−1DW and 24 mg GAE g−1DW for plants harvested in July and May, respectively. Duarte et al. [55] also found higher phenolic values in several halophytes during spring and summer, suggesting that it may be a strategy to cope with oxidative stress due to high light intensities and increased salinity during the summer. Moreover, some flavonoid production is boosted under high irradiation to reflect excessive solar energy. Our results are in agreement for the reported range of phenol content for Salicornia species, that is, from 1 to 50 mg GAE g −1 DW (S. herbacea [ 56 – 58 ], S. bigelovii [ 15 ] or S. europaea [ 14 ]). It seems logical that phenols may increase under salt stress conditions in order to help the plants to cope with salinity, as has been demonstrated by Wang et al. [ 59 ] for tobacco and also in other species such as Hyssopus officinalis [ 60 ] and Apera spica-venti [ 61 ]. In halophytes, several authors have reported the same pattern, as they observed up to a 1.5 × increase in total phenol content after irrigation with seawater [ 15 , 62 , 63 ]. In this study, we observed a slight but significant increase in phenol content as a response to saline irrigation in S. ramosissima. Despite the fact that this result appears to indicate an adaptation response to salinity, other authors observed the opposite effect, and Lima et al. [ 27 ] concluded that S. ramosissima cultivated with intermediate levels of salinity (110 and 200 mmol L −1 NaCl) showed higher amounts of phenolic compounds, while Kang et al. [ 57 ] reported higher levels of polyphenols in freshwater-cultivated S. herbacea than naturally-grown plants. The halophyte Haloxylon stocksii also showed a decrease in total phenol content after watering with media containing either 100 or 300 mmol L −1 NaCl, compared to control plants [ 64 ]. Therefore, it seems that there is not a common pattern of response to salinity conditions in different species of halophyte plants. This could be due to the fact that different systems may be used in each species in order to adapt to with salt stress. For example, our results showed a different response of phenol content to saline irrigation at ambient CO 2 and at elevated CO 2 . The decrease in phenol content at a higher atmospheric CO 2 concentration may probably be driven by the fitness improvement mediated by CO 2 in S. ramosissima [ 24 , 65 ]. Contrary Plants 2023,12, 1395 9 of 18 to that, in non-halophyte species, increased temperatures and/or CO 2 clearly induced the accumulation of total phenols in leaves as a defense mechanism, such as in Vallisneria spiralis [ 66 ], Alternanthera sessilis [ 67 ], chickpea [ 68 ], and garlic [ 69 ]. Production of these antioxidant compounds can be induced by other stress abiotic conditions rather than salinity, such as UV-C radiation, floods, or drought [ 70 ]. Accordingly, in our experiment, elevated temperature and drought increased phenol content in S. ramosissima shoots, probably as an antioxidant defense mechanism. Contrary to FA and phenols, low levels of oxalates are a desirable trait for human consumption. However, it should not be forgotten that oxalic acid is a basic plant mineral regulator. In rice, it has been suggested that oxalate accumulation has a genetic basis and does not depend on CO 2 and temperature conditions [ 71 ]. In Rumex obtusifolius, it has been observed that 600 ppm atmospheric CO 2 lowered the concentration of oxalate in leaves, compared to ambient CO 2 [ 72 ]. In our work, increases in temperature and atmospheric CO 2 resulted in lower levels of oxalate under saline irrigation. This effect may be due to complexation of free oxalic acid to maintain ionic equilibrium. In the same line, Khan et al. [ 73 ] found that non-saline controls of the halophyte Cressa cretica showed higher oxalate concentrations and that it was also related to plant population density. Accordingly, S. europaea showed a reduction of 80% of oxalate content in response to increasing NaCl concentration. Nevertheless, in a similar way to what we observed for phenolic compound content, there is not a consistent pattern in response to salinity for the levels of oxalates. Other authors have found out that the increase in salinity in the growth medium of halophytes resulted in higher oxalate content [64,74,75]. Finally, we found that the effect of bacterial inoculation in S. ramosissima depended greatly on the bacterial strains that were employed, as reported previously by other authors [ 76 ]. Moreover, we observed that bacterial properties are more important than their origin, as the consortia with more interesting effects in our experiment were number 3 and 4, which were originally isolated from the rhizosphere of halophytes Spartina maritima and Atriplex portulacoides, while consortium number 5, which was isolated from S. ramosissima, did not produce relevant responses. High and middle marsh area colonized by A. portulacoides is more prone to higher salinities and lower water availability, so it may be expected that bacteria from these sites might be adapted to counteract these effects. Other authors have proven that bacterial inoculation reduces the negative effects of salinity stress by means of phenol accumulation [ 77 ]. The rationale behind this pattern could be related to the host defense response, which is linked to the activation of secondary pathways associated with the onset of induced resistance, including the oxidation and polymerization of pre-existing phenols and the synthesis of new phenolic compounds via the activation of the phenylpropanoid pathway [ 78 ]. However, in our experiment, phenol accumulation after bacterial inoculation was evidenced only in plants subjected to drought. In contrast with other authors, we did not observe phenol accumulation at saline irrigation. Rueda-Puente et al. [35] observed that S. bigelovii seedlings at 250 mmol L−1 NaCl inoculated with K. pneumoniae increased their phenolic content by 19% compared to non-inoculated controls. Their results were in agreement with those of Dong et al. [ 79 ], who additionally reported that K. pneumoniae established endophytic populations in S. bigelovii . Then, perhaps bacteria that colonize inner plant tissues induce a greater host defense response and, consequently, a greater phenol accumulation. In this work, the bacteria used are salt-tolerant rhizobacteria, which naturally grow in soil and root surfaces at 510 mmol l−1NaCl. However, the loss of soil moisture may be a critical stress factor that induces some bacteria to colonize plant roots after seeking more appropriate living conditions. On the other hand, oxalate content increased with saline irrigation of S. ramosissima. It may be related to their ability to counteract salt stress and promote plant growth, which involves plant biomass increment and ion uptake and, therefore, the need to support the ionic homeostasis machinery. Altogether, consortia 3 and 4 greatly increased phenol and oxalate accumulation in response to saline irrigation and drought. 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