Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress.

Chenopodium quinoa Willd. (quinoa), a member of the Amaranthaceae family, is an allotetraploid annual plant, endemic to South America. The plant of C. quinoa presents significant ecological plasticity with exceptional adaptability to several environmental stresses, including salinity. The resilience of quinoa to several abiotic stresses, as well as its nutritional attributes, have led to significant shifts in quinoa cultivation worldwide over the past century. This work first defines germination sensu stricto in quinoa where the breakage of the pericarp and the testa is followed by endosperm rupture (ER). Transcriptomic changes in early seed germination stages lead to unstable expression levels in commonly used reference genes that are typically stable in vegetative tissues. Noteworthy, no suitable reference genes have been previously identified specifically for quinoa seed germination under salt stress conditions. This work aims to identify these genes as a prerequisite step for normalizing qPCR data. To this end, germinating seeds from UDEC2 and UDEC4 accessions, with different tolerance to salt, have been analyzed under conditions of absence (0 mM NaCl) and in the presence (250 mM NaCl) of sodium chloride. Based on the relevant literature, six candidate reference genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Monensin sensitivity1 (MON1), Polypyrimidine tract-binding protein (PTB), Actin-7 (ACT7), Ubiquitin-conjugating enzyme (UBC), and 18S ribosomal RNA (18S), were selected and assessed for stability using the RefFinder Tool encompassing the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt in the evaluation. The data presented support the suitability of CqACT7 and CqUBC as reference genes for normalizing gene expression during seed germination under salinity stress. These recommended reference genes can be valuable tools for consistent qPCR studies on quinoa seeds.

Mitigating Salinity Stress in Quinoa (Chenopodium quinoa Willd.) with Biochar and Superabsorber Polymer Amendments.

In agriculture, soil amendments are applied to improve soil quality by increasing the water retention capacity and regulating the pH and ion exchange. Our study was carried out to investigate the impact of a commercial biochar (Bc) and a superabsorbent polymer (SAP) on the physiological and biochemical processes and the growth performance of Chenopodium quinoa (variety ICBA-5) when exposed to high salinity. Plants were grown for 25 days under controlled greenhouse conditions in pots filled with a soil mixture with or without 3% Bc or 0.2% SAP by volume before the initiation of 27 days of growth in hypersaline conditions, following the addition of 300 mM NaCl. Without the Bc or soil amendments, multiple negative effects of hypersalinity were detected on photosynthetic CO2 assimilation (Anet minus 70%) and on the production of fresh matter from the whole plant, leaves, stems and roots (respectively, 55, 46, 64 and 66%). Moreover, increased generation of reactive oxygen species (ROS) was indicated by higher levels of MDA (plus 142%), antioxidant activities and high proline levels (plus 311%). In the pots treated with 300 mM NaCl, the amendments Bc or SAP improved the plant growth parameters, including fresh matter production (by 10 and 17%), an increased chlorophyll content by 9 and 13% and Anet in plants (by 98 and 115%). Both amendments (Bc and SAP) resulted in significant salinity mitigation effects, decreasing proline and malondialdehyde (MDA) levels whilst increasing both the activity of enzymatic antioxidants and non-enzymatic antioxidants that reduce the levels of ROS. This study confirms how soil amendments can help to improve plant performance and expand the productive range into saline areas.

Salt and genotype impact on plant physiology and root proteome variations in tomato.

To evaluate the genotypic variation of salt stress response in tomato, physiological analyses and a proteomic approach have been conducted in parallel on four contrasting tomato genotypes. After a 14 d period of salt stress in hydroponic conditions, the genotypes exhibited different responses in terms of plant growth, particularly root growth, foliar accumulation of Na(+), and foliar K/Na ratio. As a whole, Levovil appeared to be the most tolerant genotype while Cervil was the most sensitive one. Roma and Supermarmande exhibited intermediary behaviours. Among the 1300 protein spots reproducibly detected by two-dimensional electrophoresis, 90 exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. A common set of proteins (nine spots), up- or down-regulated by salt-stress whatever the genotype, was detected. But the impact of the tomato genotype on the proteome variations was much higher than the salt effect: 33 spots that were not variable with salt stress varied with the genotype. The remaining number of variable spots (48) exhibited combined effects of the genotype and the salt factors, putatively linked to the degrees of genotype tolerance. The carbon metabolism and energy-related proteins were mainly up-regulated by salt stress and exhibited most-tolerant versus most-sensitive abundance variations. Unexpectedly, some antioxidant and defence proteins were also down-regulated, while some proteins putatively involved in osmoprotectant synthesis and cell wall reinforcement were up-regulated by salt stress mainly in tolerant genotypes. The results showed the effect of 14 d stress on the tomato root proteome and underlined significant genotype differences, suggesting the importance of making use of genetic variability.

Thylakoid proteome variation of Eutrema salsugineum in response to drought and salinity combined stress.

It is well known that plant responses to stress involve different events occurring at different places of the cell/leaf and at different time scales in relation with the plant development. In fact, the organelles proteomes include a wide range of proteins that could include a wide range of proteins showing a considerable change in cellular functions and metabolism process. On this basis, a comparative proteomics analysis and fluorescence induction measurements were performed to investigate the photosynthetic performance and the relative thylakoid proteome variation in Eutrema salsugineum cultivated under salt stress (200 mM NaCl), water deficit stress (PEG) and combined treatment (PEG + NaCl) as a hyperosmotic stress. The obtained results showed a significant decrease of plant growth under drought stress conditions, with the appearance of some toxicity symptoms, especially in plants subjected to combined treatment. Application of salt or water stress alone showed no apparent change in the chlorophyll a fluorescence transients, primary photochemistry (fluorescence kinetics of the O-J phase), the PQ pool state (J-I phase changes), (Fv/Fm) and (Fk/Fj) ratios. However, a considerable decrease of all these parameters was observed under severe osmotic stress (PEG + NaCl). The thylakoid proteome analysis revealed 58 proteins showing a significant variation in their abundance between treatments (up or down regulation). The combined treatment (PEG + NaCl) induced a decrease in the expression of the whole PSII core subunit (D1, D2, CP43, CP47, PsbE and PsbH), whereas the OEC subunits proteins remained constant. An increase in the amount of PsaD, PsaE, PsaF, PsaH, PsaK and PsaN was detected under drought stress (PEG5%). No significant change in the accumulation of Cyt b6 and Cyt f was observed. Some regulated proteins involved in cellular redox homeostasis were detected (glutamine synthetase, phosphoglycerate kinase, transketolase), and showed a significant decrease under the combined treatment. Some oxidative stress related proteins were significantly up-regulated under salt or drought stress and could play a crucial role in the PSI photoprotection and the control of ROS production level.

Post-stress restorative response of two quinoa genotypes differing in their salt resistance after salinity release.

Soil salinity is one of the most important environmental factors that adversely affect plant growth and productivity. Quinoa emerges as a good food candidate due to its exceptional nutritive value, and its adaptability to various abiotic stresses. This high quinoa potential was investigated in the present study by evaluating the impact of salinity and post-stress restorative processes, in order to test how a pulse of saline water affects the growth and survival of two quinoa genotypes differing in salt resistance, Kcoito (salt sensitive) and UDEC-5 (salt resistant). Plants established in non-saline nutrient solution (hydroponic system) were exposed to a pulse of 0, 100 and 300 mM NaCl salinity for three weeks followed by four weeks in nutrient solution. Both genotypes survived exposure to salinity pulses. After stress removal, only the salt resistant variety UDEC-5 presented a significant stimulation of growth above the level of the non-pulsed treatment. Furthermore, the two varieties showed different responses in physiological, biochemical and antioxidant parameters. Again, the salinity release was highly controlled in pulsed UDEC-5 and more targeted as in Kcoito. In a win-win situation, the NaCl remaining in the tissues was used from UDEC-5 to optimize water uptake (osmotic force), to release vacuolar nutrients to enhance indirectly photosynthesis and to reduce ionic burden. This straightforward adjustment was accompanied by priming-effects such as a high proline accumulation and a balanced oxidative stress defense to scavenge remaining toxic reactive oxygen species (ROS), to stabilize enzymes and to be poised and to reduce lipid peroxidation and membrane damage. It can be concluded, that both species can tolerate short periods of exposure to saline conditions and this gives some flexibility of transient or permanent irrigation with saline water. However, taken together all of these markers indicate that only UDEC-5 quinoa can utilize salinity pulses in the applied range to enhance, growth, their antioxidant defense and water relations even above the level of non-pulsed plants.

Photosynthetic performance of quinoa (Chenopodium quinoa Willd.) after exposure to a gradual drought stress followed by a recovery period.

Drought is an abiotic scourge, one of the major environmental stress factors that adversely affect plant growth and photosynthesis machinery through a disruption of cell organelles, arrangement thylakoid membranes and the electron transport chain. Herein, we probed the effect of drought stress on photosynthetic performance of Chenopodium quinoa Willd. Beforehand, plants were subjected to water deficit (as 15% Field Capacity, FC) for one (D-1W) or two weeks (D-2W), and were then re-watered at 95% FC for 2 weeks. Light and electron microscopy analysis of leaves showed no apparent changes in mesophyll cell organization and chloroplast ultrastructure after one week of drought stress, while a swelling of thylakoids and starch accumulation were observed after the prolonged drought (D-2W). The latter induced a decrease in both PSI and PSII quantum yields which was accompanied by an increase in F0 (minimum fluorescence) and a decline in Fm (maximum fluorescence). Drought stress influenced the fluorescence transients, where the major changes at the OJIP prompt FI level were detected in the OJ and IP phases. Prolonged drought induced a decrease in chl a fluorescence at IP phase which was readjusted and established back after re-watering and even more an increase was observed after 2 weeks of recovery. The maximum quantum yield of primary photochemistry (φPo) was unaffected by the different drought stress regimes. Drought induced an increase in the ABS/RC and DI0/RC ratios which was concurrent to a stable φPo (maximum quantum yield of PSII primary photochemistry). A substantial decrease in PI(ABS) was detected especially, during severe drought stress (D-2W) suggesting a drop in the PSII efficiency and the level of electron transport through the plastoquinone pool (PQ pool) towards oxidized PSI RCs (P700+). The immunoblot analysis of the main PSII proteins revealed considerable changes in the D1, D2, CP47, OEC, PsbQ and LHCII proteins under drought. These changes depend on the stress duration and recovery period. The main message of this investigation is the elevated recovery capacities of PSII and PSI photochemical activities after re-watering.

Comparative proteomic approach to study the salinity effect on the growth of two contrasting quinoa genotypes.

The aim of this study was to investigate the effect of NaCl salinity (0, 100 and 300 mM) on the individual response of the quinoa varieties Kcoito (Altiplano Ecotype) and UDEC-5 (Sea-level Ecotype) with physiological and proteomic approaches. Leaf protein profile was performed using two dimensional gel electrophoresis (2-DE). UDEC-5 showed an enhanced capacity to withstand salinity stress compared to Kcoito. In response to salinity, we detected overall the following differences between both genotypes: Toxicity symptoms, plant growth performance, photosynthesis performance and intensity of ROS-defense. We found a mirroring of these differences in the proteome of each genotype. Among the 700 protein spots reproducibly detected, 24 exhibited significant abundance variations between samples. These proteins were involved in energy and carbon metabolism, photosynthesis, ROS scavenging and detoxification, stress defense and chaperone functions, enzyme activation and ATPases. A specific set of proteins predominantly involved in photosynthesis and ROS scavenging showed significantly higher abundance under high salinity (300 mM NaCl). The adjustment was accompanied by a stimulation of various metabolic pathways to balance the supplementary demand for energy or intermediates. However, the more salt-resistant genotype UDEC-5 presented a beneficial and significantly higher expression of nearly all stress-related altered enzymes than Kcoito.

Comparative analysis of salt stress, duration and intensity, on the chloroplast ultrastructure and photosynthetic apparatus in Thellungiella salsuginea.

Salinity is one of the most important abiotic stress affecting plant growth and productivity worldwide. Photosynthesis, together with cell growth, is among the primary process affected by salinity. Here, we report the effects of salt stress on photosynthesis in the model halophyte Thellungiella salsuginea. Plants were grown in hydroponic system and then treated for 2 weeks with different NaCl concentrations (0, 100, 200 and 400 mM). Leaf analysis using both photonic and transmission electron microscopes showed some changes in mesophyll cell organization, including shape and dimension. Under high NaCl concentration (400 mM) a swelling of thylakoids and starch accumulation was also observed. The obtained results also showed a change in the photosynthetic efficiency of both photosystems (PSI and PSII), depending on both NaCl concentrations and duration of the stress treatment. Under moderate salinity (100 and 200 mM NaCl) no significant variation was observed in PSI and PSII yield parameters. Chlorophyll a fluorescence transient showed some variations in OJ, JI and IP phases under salt stress depending also on NaCl levels and the duration of stress. Under high salinity PSII donor side was affected as well as quantum yield of PSI which also showed a donor side limitation. A significant decrease on quantum yields Y(I) and Y(II) under high salt treatment (400 mM NaCl) for prolonged period of time (15 days) was observed. The decrease of these parameters was quantitatively compensated by a corresponding increase of energy thermal dissipation Y(NPQ) in photosystem II and a increase in the Y(ND) in PSI. Analysis of derived parameters from the OJIP transient curve revealed that ABS/RC decreased under NaCl treatment by reason of the increase in size of antenna of active reaction centers. An increase in the performance index PI(ABS), a slight decrease in the rate of DIO/RC, TRO/RC and the level of electron transport per PSII RC (ETO/RC) were observed during the first days of salt stress treatment reflecting a high PSII efficiency.

Combined effects of NaCl and Cd2+ stress on the photosynthetic apparatus of Thellungiella salsuginea.

Plants show complex responses to abiotic stress while, the effect of the stress combinations can be different to those seen when each stress is applied individually. Here, we report on the effects of salt and/or cadmium on photosynthetic apparatus of Thellungiella salsuginea. Our results showed a considerable reduction of plant growth with some symptoms of toxicity, especially with cadmium treatment. The structural integrity of both photosystems (PSI and PSII) was mostly maintained under salt stress. Cadmium induced a considerable decrease of both PSI and PSII quantum yields and the electron transport rate ETR(I) and ETR(II) paralleled by an increase of non-photochemical quenching (NPQ). In addition, cadmium alone affects the rate of primary photochemistry by an increase of fluorescence at O-J phase and also the photo-electrochemical quenching at J-I phase. A positive L-band appeared with (Cd) treatment as an indicator of lower PSII connectivity, and a positive K-band reflecting the imbalance in number of electrons at donor and acceptor side. In continuity to our previous studies which showed that NaCl supply reduced Cd2+ uptake and limited its accumulation in shoot of divers halophyte species, here as a consequence, we demonstrated the NaCl-induced enhancement effect of Cd2+ toxicity on the PSII activity by maintaining the photosynthetic electron transport chain as evidenced by the differences in ψO, φEo, ABS/RC and TR0/RC and by improvement of performance index PI(ABS), especially after short time of treatment. A significant decrease of LHCII, D1 and CP47 amounts was detected under (Cd) treatment. However, NaCl supply alleviates the Cd2+ effect on protein abundance including LHCII and PSII core complex (D1 and CP47).

Does Salicylic Acid (SA) Improve Tolerance to Salt Stress in Plants? A Study of SA Effects On Tomato Plant Growth, Water Dynamics, Photosynthesis, and Biochemical Parameters.

Environmental stresses such as salinity directly impact crop growth, and by extension, world food supply and societal prosperity. It is estimated that over 800 million hectares of land throughout the world are salt-affected. In arid and semi-arid regions, salt concentration can be close to that in the seawater. Hence, there are intensive efforts to improve plant tolerance to salinity and other environmental stressors. Salicylic acid (SA) is an important signal molecule for modulating plant responses to stress. In the present study, we examined, on multiple plant growth related endpoints, whether SA applied through the rooting medium could mitigate the adverse effects of salinity on tomato (Solanum lycopersicum) cv. Marmande. The latter is a hitherto understudied tomato plant from the above perspective; it is a classic variety that produces the large ribbed tomatoes in the Mediterranean and consumed worldwide. We found salt stress negatively affected the growth of cv. Marmande tomato plants. However, the SA-treated plants had greater shoot and root dry mass, leaf area compared to untreated plants when exposed to salt stress. Application of SA restores photosynthetic rates and photosynthetic pigment levels under salt (NaCl) exposure. Leaf water, osmotic potential, stomatal conductance transpiration rate, and biochemical parameters were also ameliorated in SA-treated plants under saline stress conditions. Overall, these data illustrate that SA increases cv. Marmande tomato growth by improving photosynthesis, regulation and balance of osmotic potential, induction of compatible osmolyte metabolism, and alleviating membrane damage. We suggest salicylic acid might be considered as a potential growth regulator to improve tomato plant salinity stress resistance, in the current era of global climate change.

Effect of salinity and calcium on tomato fruit proteome.

Salinity is a major abiotic stress that adversely affects plant growth and productivity. The physiology of the tomato in salty and nonsalty conditions has been extensively studied, providing an invaluable base to understand the responses of the plants to cultural practices. However few data are yet available at the proteomic level looking for the physiological basis of fruit development, under salt stress. Here, we report the effects of salinity and calcium on fruit proteome variations of two tomato genotypes (Cervil and Levovil). Tomato plants were irrigated with a control solution (3 dSm(-1)) or with saline solutions (Na or Ca+Na at 7.6 dSm(-1)). Tomato fruits were harvested at two ripening stages: green (14 days post-anthesis) and red ripe. Total proteins were extracted from pericarp tissue and separated by two-dimensional gel electrophoresis. Among the 600 protein spots reproducibly detected, 53 spots exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. Most of the identified proteins were involved in carbon and energy metabolism, salt stress, oxidative stress, and proteins associated with ripening process. Overall, there was a large variation on proteins abundance between the two genotypes that can be correlated to salt treatment or/and fruit ripening stage. The results showed a protective effect of calcium that limited the impact of salinization on metabolism, ripening process, and induced plant salt tolerance. Collectively, this work has improved our knowledge about salt and calcium effect on tomato fruit proteome.

Salt-stress induced physiological and proteomic changes in tomato (Solanum lycopersicum) seedlings.

Soil salinity is one of the major abiotic stress limiting crop productivity and the geographical distribution of many important crops worldwide. To gain a better understanding of the salinity stress responses at physiological and molecular level in cultivated tomato (Solanum lycopersicum. cv. Supermarmande), we carried out a comparative physiological and proteomic analysis. The tomato seedlings were cultivated using a hydroponic system in the controlled environment growth chamber. The salt stress (NaCl) was applied (0, 50, 100, 150 and 200 mM), and maintained for 14 days. Salt treatment induced a plant growth reduction estimated as fresh-dry weight. Photosynthetic pigments (chlorophyll a, b) content of NaCl-treated tomato plants was significantly decreased as the salinity level increased. Proline accumulation levels in leaf and root tissues increased significantly with increasing NaCl concentration. Relative electrolyte leakage known as an indicator of membrane damage caused by salt stress was increased proportionally according to the NaCl concentrations. Roots of control and salt-stressed plants were also sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis (2-DGE). Several proteins showed up- and downregulation during salt stress. MALDI-TOF/MS analysis and database searching of some of the identified proteins indicated that the proteins are known to be in a wide range of physiological processes, that is, energy metabolism, ROS (reactive oxygen species) scavenging and detoxification, protein translation, processing and degradation, signal transduction, hormone and amino acid metabolism, and cell wall modifications. All proteins might work cooperatively to reestablish cellular homeostasis under salt stress, water deficiency, and ionic toxicity.

[Salt tolerance of Setaria verticillata L.: a short-cycle poaceae]. / Tolérance à la salinité d'une poaceae à cycle court: la sétaire (Setaria verticillata L.).

The responses of growth, development, and nutrition to salt stress are examined in short-cycle Setaria verticillata. For these, two experiments are led. The first intended to study the effects of various concentrations of NaCl on the parameters of growth and nutrition during the vegetative phase. Fifteen-day-old platelets were grown on commercial peat irrigated with pure NaCl solutions (0 to 300 mM). After three weeks of culture, the plants were collected and divided into roots and shoots. The fresh and dry matter masses of the various bodies are given. The second experiment was intended to study the effect of different concentrations of NaCl on crop plants until maturity. The culture was led under the same conditions as the preceding one, but for three months until the end of the cycle (production and maturation of the seeds). At harvest, the plants were separated in roots, shoots, and grains. During all the development cycle, Setaria vertillata was very sensitive to salinity. The concentration of NaCl that caused an important reduction of dry weight production was about 75 mM. Dry matter deposition was more diminished in roots than shoots. The reduction of the production of growth observed seems associated with a higher accumulation of Na(+) in shoots and with a deficit alimentation of organs in K(+). During the reproductive phase, salt affects the components of the output and induces variability on the level of the production of biomass as significant as that noted during the phase of vegetative growth. Lastly, the capacity of germination of seeds was strongly dependent on the salt concentration of the culture medium of the plants mothers, a total loss of viability appearing on crop plants collected in the presence of NaCl 300 mM.