SarCTAB: an efficient and cost-effective DNA isolation protocol from geophytes.

Geophytes are herbaceous plants that grow anew from underground buds and are excellent models to study storage organ formation. However, molecular studies involving geophytes are constrained due to the presence of a wide spectrum of polysaccharides and polyphenols that contaminate the genomic DNA. At present, several protocols exist for the extraction of genomic DNA from different plant species; however, isolating high-quality DNA from geophytes is challenging. Such challenges are further complexed by longer incubation time and multiple precipitation steps involved in existing DNA isolation methods. To overcome such problems, we aimed to establish a DNA extraction method (SarCTAB) which is an economical, quick, and sustainable way of DNA isolation from geophytes. We improved the traditional CTAB method by optimizing key ingredients such as sarcosine, ß-mercaptoethanol, and high molar concentration of sodium chloride (NaCl), which resulted in high concentration and good-quality DNA with lesser polysaccharides, proteins, and polyphenols. This method was evaluated to extract DNA from storage organs of six different geophytes. The SarCTAB method provides an average yield of 1755 ng/µl of high-quality DNA from 100 mg of underground storage tissues with an average standard purity of 1.86 (260/280) and 1.42 (260/230). The isolated genomic DNA performed well with Inter-simple sequence repeat (ISSR) amplification, restriction digestion with EcoRI, and PCR amplification of plant barcode genes viz. matK and rbcL. Also, the cost involved in DNA isolation was low when compared to that with commercially available kits. Overall, SarCTAB method works effectively to isolate high-quality genomic DNA in a cost-effective manner from the underground storage tissues of geophytes, and can be applied for next-generation sequencing, DNA barcoding, and whole genome bisulfite sequencing.

Nocardiopsis lucentensis and thiourea co-application mitigates arsenic stress through enhanced antioxidant metabolism and lignin accumulation in rice.

Arsenic (As) is a group-1 carcinogenic metalloid that threatens global food safety and security, primarily via its phytotoxicity in the staple crop rice. In the present study, ThioAC, the co-application of thiourea (TU, a non-physiological redox regulator) and N. lucentensis (Act, an As-detoxifying actinobacteria), was evaluated as a low-cost approach for alleviating As(III) toxicity in rice. To this end, we phenotyped rice seedlings subjected to 400 mg kg-1 As(III) with/without TU, Act or ThioAC and analyzed their redox status. Under As-stress conditions, ThioAC treatment stabilized photosynthetic performance, as indicated by 78 % higher total chlorophyll accumulation and 81 % higher leaf biomass, compared with those of As-stressed plants. Further, ThioAC improved root lignin levels (2.08-fold) by activating the key enzymes of lignin biosynthesis under As-stress. The extent of reduction in total As under ThioAC (36 %) was significantly higher than TU (26 %) and Act (12 %), compared to those of As-alone treatment, indicating their synergistic interaction. The supplementation of TU and Act activated enzymatic and non-enzymatic antioxidant systems, respectively, with a preference for young (TU) and old (Act) leaves. Additionally, ThioAC activated enzymatic antioxidants, specifically GR (∼3-fold), in a leaf-age specific manner and suppressed ROS-producing enzymes to near-control levels. This coincided with 2-fold higher induction of polyphenols and metallothionins in ThioAC-supplemented plants, resulting in improved antioxidant defence against As-stress. Thus, our findings highlighted ThioAC application as a robust, cost-effective ameliorative strategy, for achieving As-stress mitigation in a sustainable manner.

Maize roots and shoots show distinct profiles of oxidative stress and antioxidant defense under heavy metal toxicity.

Heavy metal accumulation in agricultural land causes crop production losses worldwide. Metal homeostasis within cells is tightly regulated. However, homeostasis breakdown leads to accumulation of reactive oxygen species (ROS). Overall plant fitness under stressful environment is determined by coordination between roots and shoots. But little is known about organ specific responses to heavy metals, whether it depends on the metal category (redox or non-redox reactive) and if these responses are associated with heavy metal accumulation in each organ or there are driven by other signals. Maize seedlings were subjected to sub-lethal concentrations of four metals (Zn, Ni, Cd and Cu) individually, and were quantified for growth, ABA level, and redox alterations in roots, mature leaves (L1,2) and young leaves (L3,4) at 14 and 21 days after sowing (DAS). The treatments caused significant increase in endogenous metal levels in all organs but to different degrees, where roots showed the highest levels. Biomass was significantly reduced under heavy metal stress. Although old leaves accumulated less heavy metal content than root, the reduction in their biomass (FW) was more pronounced. Metal exposure triggered ABA accumulation and stomatal closure mainly in older leaves, which consequently reduced photosynthesis. Heavy metals induced oxidative stress in the maize organs, but to different degrees. Tocopherols, polyphenols and flavonoids increased specifically in the shoot under Zn, Ni and Cu, while under Cd treatment they played a minor role. Under Cu and Cd stress, superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were induced in the roots, however ascorbate peroxidase (APX) activity was only increased in the older leaves. Overall, it can be concluded that root and shoot organs specific responses to heavy metal toxicity are not only associated with heavy metal accumulation and they are specialized at the level of antioxidants to cope with.

UTR-Dependent Control of Gene Expression in Plants.

Throughout their lives, plants sense many developmental and environmental stimuli, and activation of optimal responses against these stimuli requires extensive transcriptional reprogramming. To facilitate this activation, plant mRNA contains untranslated regions (UTRs) that significantly increase the coding capacity of the genome by producing multiple mRNA variants from the same gene. In this review we compare UTRs of arabidopsis (Arabidopsis thaliana) and rice (Oryza sativum) at the genome scale to highlight their complexity in crop plants. We discuss different modes of UTR-based regulation with emphasis on genes that regulate multiple plant processes, including flowering, stress responses, and nutrient homeostasis. We demonstrate functional specificity in genes with variable UTR length and propose future research directions.

Zinc-induced differential oxidative stress and antioxidant responses in Chlorella sorokiniana and Scenedesmus acuminatus.

Algae are frequently exposed to toxic metals, and zinc (Zn) is one of the major toxicants present. We exposed two green microalgae, Chlorella sorokiniana and Scenedesmus acuminatus, to sub-lethal concentrations (1.0 and 0.6mM) of Zn for seven days. Algal responses were analysed at the level of growth, oxidative stress, and antioxidants. Growth parameters such as cell culture yield and pigment content were less affected by Zn in C. sorokiniana, despite the fact that this alga accumulated more zinc than S. acuminatus. Also, C. sorokiniana, but not S. acuminatus, was able to acclimatize during long-term exposure to toxic concentrations of the test metals (specific growth rate (µ) was 0.041/day and total chlorophyll was 14.6mg/mL). Although, Zn induced oxidative stress in both species, C. sorokiniana experienced less stress than S. acuminatus. This could be explained by a higher accumulation of antioxidants in C. sorokiniana, where flavonoids, polyphenols, tocopherols, glutathione (GSH) and ascorbate (ASC) content increased. Moreover, antioxidant enzymes glutathione S transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD), peroxidase (POX) and ascorbate peroxidase (APX), showed increased activities in C. sorokiniana. In addition to, and probably also underlying, the higher Zn tolerance in C. sorokiniana, this alga also showed higher Zn biosorption capacity. Use of C. sorokiniana as a bio-remediator, could be considered.

Future Climate CO2 Levels Mitigate Stress Impact on Plants: Increased Defense or Decreased Challenge?

Elevated atmospheric CO2 can stimulate plant growth by providing additional C (fertilization effect), and is observed to mitigate abiotic stress impact. Although, the mechanisms underlying the stress mitigating effect are not yet clear, increased antioxidant defenses, have been held primarily responsible (antioxidant hypothesis). A systematic literature analysis, including "all" papers [Web of Science (WoS)-cited], addressing elevated CO2 effects on abiotic stress responses and antioxidants (105 papers), confirms the frequent occurrence of the stress mitigation effect. However, it also demonstrates that, in stress conditions, elevated CO2 is reported to increase antioxidants, only in about 22% of the observations (e.g., for polyphenols, peroxidases, superoxide dismutase, monodehydroascorbate reductase). In most observations, under stress and elevated CO2 the levels of key antioxidants and antioxidant enzymes are reported to remain unchanged (50%, e.g., ascorbate peroxidase, catalase, ascorbate), or even decreased (28%, e.g., glutathione peroxidase). Moreover, increases in antioxidants are not specific for a species group, growth facility, or stress type. It seems therefore unlikely that increased antioxidant defense is the major mechanism underlying CO2-mediated stress impact mitigation. Alternative processes, probably decreasing the oxidative challenge by reducing ROS production (e.g., photorespiration), are therefore likely to play important roles in elevated CO2 (relaxation hypothesis). Such parameters are however rarely investigated in connection with abiotic stress relief. Understanding the effect of elevated CO2 on plant growth and stress responses is imperative to understand the impact of climate changes on plant productivity.

High Salinity Induces Different Oxidative Stress and Antioxidant Responses in Maize Seedlings Organs.

Salinity negatively affects plant growth and causes significant crop yield losses world-wide. Maize is an economically important cereal crop affected by high salinity. In this study, maize seedlings were subjected to 75 mM and 150 mM NaCl, to emulate high soil salinity. Roots, mature leaves (basal leaf-pair 1,2) and young leaves (distal leaf-pair 3,4) were harvested after 3 weeks of sowing. Roots showed the highest reduction in biomass, followed by mature and young leaves in the salt-stressed plants. Concomitant with the pattern of growth reduction, roots accumulated the highest levels of Na(+) followed by mature and young leaves. High salinity induced oxidative stress in the roots and mature leaves, but to a lesser extent in younger leaves. The younger leaves showed increased electrolyte leakage (EL), malondialdehyde (MDA), and hydrogen peroxide (H2O2) concentrations only at 150 mM NaCl. Total antioxidant capacity (TAC) and polyphenol content increased with the increase in salinity levels in roots and mature leaves, but showed no changes in the young leaves. Under salinity stress, reduced ascorbate (ASC) and glutathione (GSH) content increased in roots, while total tocopherol levels increased specifically in the shoot tissues. Similarly, redox changes estimated by the ratio of redox couples (ASC/total ascorbate and GSH/total glutathione) showed significant decreases in the roots. Activities of enzymatic antioxidants, catalase (CAT, EC 1.11.1.6) and dehydroascorbate reductase (DHAR, EC 1.8.5.1), increased in all organs of salt-treated plants, while superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione-s-transferase (GST, EC 2.5.1.18) and glutathione reductase (GR, EC 1.6.4.2) increased specifically in the roots. Overall, these results suggest that Na(+) is retained and detoxified mainly in roots, and less stress impact is observed in mature and younger leaves. This study also indicates a possible role of ROS in the systemic signaling from roots to leaves, allowing leaves to activate their defense mechanisms for better protection against salt stress.

Nutritional Status as the Key Modulator of Antioxidant Responses Induced by High Environmental Ammonia and Salinity Stress in European Sea Bass (Dicentrarchus labrax).

Salinity fluctuation is one of the main factors affecting the overall fitness of marine fish. In addition, water borne ammonia may occur simultaneously with salinity stress. Additionally, under such stressful circumstances, fish may encounter food deprivation. The physiological and ion-osmo regulatory adaptive capacities to cope with all these stressors alone or in combination are extensively addressed in fish. To date, studies revealing the modulation of antioxidant potential as compensatory response to multiple stressors are rather lacking. Therefore, the present work evaluated the individual and combined effects of salinity challenge, ammonia toxicity and nutritional status on oxidative stress and antioxidant status in a marine teleost, European sea bass (Dicentrarchus labrax). Fish were acclimated to normal seawater (32 ppt), to brackish water (20 ppt and 10 ppt) and to hypo-saline water (2.5 ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20 mg/L representing 50% of 96h LC50 value for ammonia) for 12 h, 48 h, 84 h and 180 h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Results show that in response to decreasing salinities, oxidative stress indices such as xanthine oxidase activity, levels of hydrogen peroxide (H2O2) and lipid peroxidation (malondialdehyde, MDA) increased in the hepatic tissue of fasted fish but remained unaffected in fed fish. HEA exposure at normal salinity (32 ppt) and at reduced salinities (20 ppt and 10 ppt) increased ammonia accumulation significantly (84 h-180 h) in both feeding regimes which was associated with an increment of H2O2 and MDA contents. Unlike in fasted fish, H2O2 and MDA levels in fed fish were restored to control levels (84 h-180 h); with a concomitant increase in superoxide dismutase (SOD), catalase (CAT), components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase), ascorbate peroxidase (APX) activity and reduced ascorbate (ASC) content. On the contrary, fasted fish could not activate many of these protective systems and rely mainly on CAT and ASC dependent pathways as antioxidative sentinels. The present findings exemplify that in fed fish single factors and a combination of HEA exposure and reduced seawater salinities (upto 10 ppt) were insufficient to cause oxidative damage due to the highly competent antioxidant system compared to fasted fish. However, the impact of HEA exposure at a hypo-saline environment (2.5 ppt) also defied antioxidant defence system in fed fish, suggesting this combined factor is beyond the tolerance range for both feeding groups. Overall, our results indicate that the oxidative stress mediated by the experimental conditions were exacerbated during starvation, and also suggest that feed deprivation particularly at reduced seawater salinities can instigate fish more susceptible to ammonia toxicity.

High environmental ammonia elicits differential oxidative stress and antioxidant responses in five different organs of a model estuarine teleost (Dicentrarchus labrax).

We investigated oxidative status and antioxidant profile in five tissues (brain, liver, gills, muscle and kidney) of European sea bass (Dicentrarchus labrax) when exposed to high environmental ammonia (HEA, 20 mg/L~1.18 mM as NH4HCO3) for 12 h, 2 days, 3.5 days, 7.5 days and 10 days. Results show that HEA triggered ammonia accumulation and induced oxidative stress in all tissues. Unlike other organs, hydrogen peroxide (H2O2) and malondialdehyde (MDA) accumulation in liver were restored to control levels. This recovery was associated with a concomitant augmentation in superoxide dismutase (SOD), catalase (CAT), components of glutathione redox cycle (glutathione peroxidase GPX, glutathione reductase, reduced glutathione), ascorbate peroxidase activity and reduced ascorbate content. On the contrary, in brain during prolonged exposure many of these anti-oxidant enzymes were either unaffected or inhibited, which resulted in persistent over-accumulation of H2O2 and MDA. Branchial and renal tissue both involved in osmo-regulation, revealed an entirely dissimilar compensatory response; the former rely mainly on the ascorbate dependent defensive system while the glutathione catalytic pathway was activated in the latter. In muscle, GPX activity first rose (3.5 days) followed by a subsequent drop, counterbalanced by simultaneous increment of CAT. HEA resulted in a relatively mild oxidative stress in the muscle and kidney, probably explaining the modest anti-oxidative responses. Our findings exemplify that oxidative stress as well as antioxidant potential are qualitatively diverse amongst different tissues, thereby demonstrating that for biomonitoring studies the screening of adaptive responses at organ level should be preferred over whole body response.

Anti-oxidative defences are modulated differentially in three freshwater teleosts in response to ammonia-induced oxidative stress.

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH4HCO3) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h-48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Unlike in trout, H2O2 and MDA accumulation in carp and goldfish liver was restored to control levels (84 h-180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.

Ability of ellagic acid to alleviate osmotic stress on chickpea seedlings.

Seed germination and growth of seedlings are critical phases of plant life that are adversely affected by various environmental cues. Water availability is one of the main factors that limit the productivity of many crops. This study was conducted to assess the changes in the sensitivity of chickpea seedlings to osmotic stress by prior treatment of chickpea seeds with a low concentration (50 ppm) of ellagic acid. Ellagic acid was isolated and purified from Padina boryana Thivy by chromatographic techniques. After ellagic acid treatment, seeds were germinated for 10 days under different osmotic potentials (0, -0.2, -0.4, -0.6 and -0.8 MPa) of polyethylene glycol (PEG) solutions. Ellagic acid treatment accelerated the germination and seedling growth of chickpea under osmotic stress conditions. Consistent with the accelerated growth, ellagic acid-treated seedlings also showed a significant increase in the total antioxidant capacity (FRAP) as well as an increase in the compatible solutes (proline and glycine betaine) content. Additionally, treated seedlings revealed lower lipid peroxidation levels (MDA), electrolyte leakage (EL) and H2O2. Flavonoid and reduced glutathione (GSH) content, and the activity of antioxidant enzymes [catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), glutathione reductase (GR)] and enzymes of the shikimic acid pathway [phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS)] all showed a remarkable increase with ellagic acid pretreatment compared to untreated seedlings especially under mild osmotic stress values (-0.2 and -0.4 MPa). These results suggested that treatment with ellagic acid could confer an increased tolerance of chickpea seedlings to osmotic stress, through reducing levels of H2O2 and increasing antioxidant capacity.