Heliotropium thermophilum adapts to high soil temperature in natural conditions due to its highly active antioxidant system protecting its photosynthetic machinery.

Heliotropium thermophilum (Boraginaceae) plants have strong antioxidant properties. This study investigated the effectiveness of the antioxidant system in protecting the photosynthetic machinery of H. thermophilum . Plants were obtained from Kizildere geothermal area in Buharkent district, Aydin, Turkey. Plants in the geothermal area that grew at 25-35°C were regarded as the low temperature group, while those that grew at 55-65°C were regarded as the high temperature group. We analysed the physiological changes of these plants at the two temperature conditions at stage pre-flowering and flowering. We meaured the effect of high soil temperature on water potential, malondialdehyde, cell membrane stability, and hydrogen peroxide analysis to determine stress levels on leaves and roots. Changes in antioxidant enzyme activities, ascorbate and chlorophyll content, chlorophyll fluorescence, photosynthetic gas exchange parameters, and photosynthetic enzymes (Rubisco and invertase) activities were also determined. Our results showed minimal changes to stress levels, indicating that plants were tolerant to high soil temperatures. In general, an increase in antioxidant enzyme activities, ascorbat levels, and all chlorophyll fluorescence parameters except for non-photochemical quenching (NPQ) and F v /F m were observed. The pre-flowering and flowering stages were both characterised by decreased NPQ, despite F v /F m not changing. Additionally, there was a rise in the levels of photosynthetic gas exchange parameters, Rubisco, and invertase activities. High temperature did not affect photosynthetic yield because H. thermophilum was found to stimulate antioxidant capacity, which reduces oxidative damage and maintains its photosynthetic machinery in high temperature conditions and therefore, it is tolerant to high soil temperature.

The combined application of rutin and silicon alleviates osmotic stress in maize seedlings by triggering accumulation of osmolytes and antioxidants' defense mechanisms.

Silicon (Si) has been shown to improve plant defenses against a variety of stresses. However, how rutin (Rut) affects stress factors is yet to be fully explored. Moreover, their combined role in osmotic stress response remains unclear. The current study was performed to determine how the use of Rut and Si, both separately and in combination, improved the physiological resilience of maize seedlings to two levels of osmotic stress (induced by polyethylene glycol (PEG) 6000). We aimed to enhance osmotic stress tolerance with the simultaneous use of Rut and Si. First, we selected the best water status and the lowest membrane damage enhancing concentration of Rut (60 ppm) and Si (1 mM) to research their tolerance and resistance to osmotic stress (moderate: 10% PEG, severe: 15% PEG). The application of Rut and Si separately and together reduced oxidative stress by decreasing the reactive oxygen species and improved the relative water content, osmoprotectants (proline, total soluble sugar, and glycine-betaine), ascorbate level, and some antioxidant defense-related enzyme activities and their gene expression in maize seedlings under osmotic stress. However, these effects were more promising under moderate stress. As a result, findings from the study indicate the synergistic effect of combined Rut and Si on osmotic stress tolerance in maize seedlings. Overall, the combination of Rut and Si was more effective than independent Rut and Si in reducing osmotic stress in maize seedlings. Here, it was clear that Rut played an active role in alleviating stress. This combined application can be useful for developing drought tolerance in crops for the agriculture sector.

Increased dehydrin level decreases leaf rolling grade by altering the reactive oxygen species homeostasis and abscisic acid content in maize subjected to osmotic stress.

Dehydrins (DHNs) are stress proteins involved in the development of protective reactions in plants against dehydration. The relationship between DHNs and morphological responses such as leaf rolling in plants exposed to water deficit is not well known. In this study, we detected how variations in DHN levels affect the leaf rolling response in maize exposed to osmotic stress in relation to the antioxidant system and ABA level. In this context, we altered the DHN levels in maize seedlings by treatment with bio-regulators (salicylic acid and abscisic acid) under PEG6000-free and PEG6000-induced osmotic stress. When the DHN levels were increased by the bio-regulators (25 µM SA and 100 µM ABA), the relative expression level of the Zea mays dehydrin COR410 gene increased in the seedlings, while reactive oxygen species (ROS) and leaf rolling grade decreased. Moreover, induction of DHNs caused increases in the antioxidant enzyme activity and content of antioxidant substances, and very high amounts of endogenous abscisic acid. When DHN level was suppressed by a bio-regulator (200 µM SA) in the maize seedlings, dehydrin COR410 expression level decreased, while ROS and the leaf rolling grade increased. Moreover, the antioxidant enzyme activity and content of antioxidant substances decreased in the seedlings, while the amount of abscisic acid increased. Taken all together, an increase in DHN level by bio-regulator treatment can stimulate the antioxidant system, enable abscisic acid regulation, and thus reduce leaf rolling through decreased ROS levels. The results also indicated that DHNs may be involved in the signal pathways inducing expression of some genes related to leaf rolling response, possibly by modulating ROS levels, in maize seedlings exposed to osmotic stress.

The antioxidant defense and glyoxalase systems contribute to the thermotolerance of Heliotropium thermophilum.

This study focused on the impact of the antioxidant defence and glyoxalase systems on extreme heat tolerance of the thermophilic plant Heliotropium thermophilum L. For this purpose, plants were exposed to 20, 40, 60 and 80±5°C soil temperature gradually for 15days under laboratory conditions. Our results showed that the hydrogen peroxide and superoxide levels of H. thermophilum were lower at 40±5°C and higher at 80±5°C compared with plants grown at 20±5°C. Some antioxidant enzyme activities tended to increase in plants at 40, 60 and 80±5°C compared with those at 20±5°C and the protein contents responsible for the antioxidant enzymes were in parallel with these enzyme activities. The contents of both reduced and oxidised ascorbate and glutathione rose with increasing temperature. Methylglyoxal level was lower at 40±5°C and higher at 80±5°C compared with plants grown at 20±5°C. Glyoxalase activities highly increased with rising of soil temperature from 20±5°C to 80±5°C. The results of this study suggest that differential modulations of enzymatic antioxidants and the increase in non-enzymatic antioxidants and glyoxalase activities can contribute to the development of the thermotolerance of H. thermophilum through the detoxification of reactive oxygen species and methylglyoxal.

Role of abscisic acid, osmolytes and heat shock factors in high temperature thermotolerance of Heliotropium thermophilum.

Heliotropium thermophilum can survive at a soil temperature of 65 °C in natural and laboratory conditions, but the mechanism of survival at high soil temperatures is not completely known. The objective of this study was to determine whether changes in abscisic acid (ABA), osmolytes and heat shock factors (HSFs) levels have an effective role in the development of thermotolerance in H. thermophilum at high temperatures. Soil temperature at which the thermophilic plant could live was gradually increased in laboratory conditions and the effects of four different temperatures (20 ± 5 °C: low, 40 ± 5 °C: mild, 60 ± 5 °C: medium, 80 ± 5 °C: extreme heat) were observed for 15 days. The results showed that the content of thiobarbituric acid reactive substances (TBARS) did not significantly change in extreme heat, whereas the leaf water potential and stomatal conductivity decreased. ABA biosynthesis, accumulation of osmolyte compounds including proline and total soluble sugars, and the expression levels of heat shock transcription factor A4A (HSFA4A), heat shock transcription factor A3 (HSFA3), and heat shock factor (HSF4) genes significantly increased with increase of soil temperature from 20 ± 5 °C to 80 ± 5 °C. In conclusion, we observed that H. thermophilum is an extreme thermophile. This plant can adjust osmotic activity to effectively take water through the osmolytes accumulation, reducing water loss by ABA-mediated stomatal closing and survive at high soil temperatures by stimulating the increased transcription level of HSFs.

SOS5 gene-abscisic acid crosstalk and their interaction with antioxidant system in Arabidopsis thaliana under salt stress.

SOS5 locus, encodes cell wall adhesion protein under salt stress conditions in plants, and it is required for normal cell expansion as well as for sustaining cell wall integrity and structure. However, it is still unknown how this gene locus-ABA cross-talk and interacts with the antioxidant mechanism under salt stress conditions. For this purpose, the study focused on mutant sos5-1 plant treated with ABA under NaCl stress and observed its growth and development as well as stomatal aperture, lipid peroxidation, proline, hydrogen peroxide (H2O2) and ABA contents, and some antioxidant enzyme activities. In addition, the expression levels of ABA related genes have been analysed by RT-PCR after stress application. According to findings, sos5-1 mutant plants treated with ABA under salt stress resulted in eliminated cellular damage compared to those which are solely exposed to salt stress; other observations include closing of stomata, decreased H2O2 content, increased amount of proline, and similarity with the wild type due to induced antioxidant enzyme activities. Besides, both ABA biosynthetic and inducible gene expressions of the mutant plant under salt stress were lower compared to the control, and catabolism gene expression was higher. As a result, SOS5 gene in synergy with ABA, scavenge the ROS by stimulating antioxidant system, leads to an increase in stress related gene expressions and thus contributes to salinity tolerance. This study is significant in the way that it shows how SOS5 gene locus, under salt stress conditions, interacts with antioxidant system in sustaining cell wall integrity.

Heliotropium thermophilum, an extreme heat tolerant species, promises plants about adaptation to high soil temperature conditions.

To understand high temperature tolerance, Heliotropium thermophilum, a flowering plant thriving in a geothermal field with a soil temperature ranging between 55 and 65 °C, was grown in controlled laboratory conditions and two different soil temperatures were applied to the plants. One of them was the control group (CT 25 ± 3 °C) and the other was the high temperature group (HT 60 ± 4 °C). Water potential, dry weight, cell membrane injury (CMI), lipid peroxidation, hydrogen peroxide, chlorophylls, carotenoids, flavonoids, anthocyanins, proline and total soluble sugar contents were measured. Contents of total soluble sugars, phenolics, flavonoids, anthocyanins, proline were found to be higher in HT group than CT while CMI was opposite. Moreover, no difference was determined in water potential, dry weight, lipid peroxidation, total chlorophyll and carotenoids between CT and HT. H. thermophilum plants adapted to high temperature under laboratory conditions through changing membrane lipid saturation, accumulating osmotically active compounds to save water or increase its uptake and inducing antioxidants such as phenolic compounds to keep reactive oxygen species under control. In conclusion, this study showed that H. thermophilum plant was highly resistant to high soil temperature under optimized laboratory conditions. Moreover, a plant that can withstand 60 °C for a long period of time up to 60 days under laboratory conditions was reported for the first time.

Fast determination of anthocyanins and free pelargonidin in fruits, fruit juices, and fruit wines by high-performance liquid chromatography using a core-shell column.

Anthocyanins are water-soluble pigments that are liable for colors ranging from red to blue of most fruits, vegetables, and flowers. A novel and fast method was developed for the determination of five anthocyanins and free pelargonidin by high-performance liquid chromatography coupled to photodiode array detection. A 10% formic acid and acetonitrile mixture was employed as mobile phase in the gradient elution mode. Mobile phase composition, column temperature, flow rate, injection volume, and column conditioning time were optimized by employing a stepwise strategy. Using a C18 core-shell column (100 × 4.6 mm, 2.7 µm), the separation of six analytes was accomplished in less than 9.5 min with a run-to-run analysis time of 19 min. The developed method was validated with respect to linearity (r > 0.9999), limit of detection, limit of quantification, intra-/interday precision (<2%), accuracy (98.6-104.4%), and specificity. Afterwards, the method was applied to the determination of anthocyanins present in 15 different samples including fruits, fruit juices, and fruit wines.

Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants.

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na(+)/H(+)) antiporter that transports Na(+) into the vacuole and exports H(+) into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na(+)/H(+) antiporter that exports Na(+) to the extracellular space and imports H(+) into the plant cell. Plants rely on these enzymes either to keep Na(+) out of the cell or to sequester Na(+) into vacuoles to avoid the toxic level of Na(+) in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops.

Current advances in the investigation of leaf rolling caused by biotic and abiotic stress factors.

Leaf rolling is known as a typical response to water deficit in numerous species such as rice, maize, wheat and sorghum. However, it results not only from the water deficit but also from other abiotic stress factors such as salt, temperature, heavy metals and UV radiation. In addition to the abiotic factors, herbivores, viruses, bacteria and fungi are biotic factors of leaf rolling. Leaf rolling is an effective protective mechanism from the effects of high light levels in agricultural fields and protects leaves of unirrigated plants from photodamage. The rolling reduces effective leaf area and transpiration, and thus is a potentially useful drought avoidance mechanism in dry areas. The current review focuses on the recent progress in understanding leaf rolling in relation to abiotic and biotic stress factors, the role of signal molecules, and the mechanisms of gene regulation.

Scavenging of reactive oxygen species in apoplastic and symplastic areas of rolled leaves in Ctenanthe setosa under drought stress.

The correspondence among apoplastic and symplastic antioxidant status, stomatal conductance and water potential was investigated during leaf rolling in Ctenanthe setosa (Rosc.) Eichler (Marantaceae) under drought stress. Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate form). In the leaf symplast, the highest changes were found in catalase (CAT) and guaiacol peroxidase (GPX) activities when compared to score 1 during leaf rolling. No significant change was observed in superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities in the symplast of leaf during the rolling. The same phenomenon was also present in the symplast of petiole except APX activity. In the leaf apoplast, the highest increase occurred in APX and GPX activities, whilst a slight increase in CAT and SOD activities. In the apoplast of petiole, the highest increment was found only in GPX activity, while there were small increases in SOD, APX and CAT activities. Hydrogen peroxide content increased up to score 3 in the apoplast and symplast of leaf and petiole but then slightly decreased. Also, superoxide production increased in the leaf and petiole apoplast but its quantity in the apoplast was much more than that of the symplast. On the other hand, NAD(P)H oxidase activity increased in the leaf but no change was observed in the petiole. In conclusion, as a result of water deficit during leaf rolling antioxidant enzymes are induced to scavenging of ROS produced in symplast and apoplast.

The relationship between leaf rolling and ascorbate-glutathione cycle enzymes in apoplastic and symplastic areas of Ctenanthe setosa subjected to drought stress.

The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities in the leaf (GR and DHAR) and leaf rolling and GSSG. These results showed that in apoplastic and symplastic areas, ASC-GSH cycle enzymes leading ROS detoxification may have a role in controlling leaf rolling.

The Relationship between Leaf Rolling and Ascorbate-Glutathione Cycle Enzymes in Apoplastic and Symplastic Areas of Ctenanthe setosa Subjected to Drought Stress

The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities ...