Profiling of conserved orthologs and miRNAs for understanding their role in salt tolerance mechanism of Sesuvium portulacastrum L.

BACKGROUND: Sesuvium portulacastrum is a facultative halophyte capable of thriving in a saline environment. Despite molecular studies conducted to unravel its salt adaptation mechanism, there is a paucity of information on the role of salt-responsive orthologs and microRNAs (miRNAs) in this halophyte. Here, we searched the orthology to identify salt-responsive orthologs and miRNA targets of Sesuvium using the Arabidopsis genome. METHODS: The relative fold change of orthologs, conserved miRNAs, and miRNA targets of Sesuvium was analyzed under 100 mM (LS) and 250 mM NaCl (HS) treatment at 24 h using qRT-PCR. The comparison between the expression of Sesuvium orthologs and Arabidopsis orthologs (Arabidopsis eFP browser database) was used to identify differentially expressed genes. RESULTS: Upon salt treatment, we found that SpCIPK3 (1.95-fold in LS and 2.90-fold in HS) in Sesuvium roots, and SpNHX7 (1.61-fold in LS and 6.39-fold in HS) and, SpSTPK2 (2.54-fold in LS and 7.65-fold in HS) in Sesuvium leaves were upregulated in a salt concentration-specific manner. In Arabidopsis, these genes were either downregulated or did not show significant variation, implicating its significance in the halophytic nature of Sesuvium. Furthermore, miRNAs like miR394a, miR396a, and miR397a exhibited a negative correlation with their targets-Frigida interacting protein 1, Cysteine proteinases superfamily protein, and Putative laccase, respectively under different salt treatments. CONCLUSION: The study revealed that the high salt tolerance in Sesuvium is associated with distinct transcriptional reprogramming, hence, to gain holistic mechanistic insights, global-scale profiling is required.

Multifaceted roles of silicon in mitigating environmental stresses in plants.

Food security relies on plant productivity and plant's resilience to climate change driven environmental stresses. Plants employ diverse adaptive mechanisms of stress-signalling pathways, antioxidant defense, osmotic adjustment, nutrient homeostasis and phytohormones. Over the last few decades, silicon has emerged as a beneficial element for enhancing plant growth productivity. Silicon ameliorates biotic and abiotic stress conditions by regulating the physiological, biochemical and molecular responses. Si-uptake and transport are facilitated by specialized Si-transporters (Lsi1, Lsi2, Lsi3, and Lsi6) and, the differential root anatomy has been shown to reflect in the varying Si-uptake in monocot and dicot plants. Silicon mediates a number of plant processes including osmotic, ionic stress responses, metabolic processes, stomatal physiology, phytohormones, nutrients and source-sink relationship. Further studies on the transcriptional and post-transcriptional regulation of the Si transporter genes are required for better uptake and transport in spatial mode and under different stress conditions. In this article, we present an account of the availability, uptake, Si transporters and, the role of Silicon to alleviate environmental stress and improve plant productivity.

FT-IR profiling reveals differential response of roots and leaves to salt stress in a halophyte Sesuvium portulacastrum (L.) L.

In a halophyte, Sesuvium portulacastrum (L.) L., we have applied Fourier Transform InfraRed (FT-IR) spectroscopy to detect the corresponding changes associated with salt-induced physiological changes under long- term treatment with 0, 100 and 500 mM NaCl. FT-IR profiles showed changes in chemical composition and functional groups of proteins, lipids and carbohydrates due to salt treatments, evident as differential FT-IR profiles in both roots and leaves specific to these metabolites. Further, the Principle Component Analysis (PCA) was applied to identify the main sources of variation in FT-IR data due to differential treatment. In PCA, the PC1 showed 85.55% and PC2 showed 18.18% variability in data and confirmed differential response of root and leaves to salt treatment in Sesuvium. The results suggest that FT-IR spectrometry can be used to study stress-induced metabolic changes in plants in relation to their salt tolerance.

Evaluation of Spinacia oleracea (L.) for phytodesalination and augmented production of bioactive metabolite, 20-hydroxyecdysone.

In this study, adaptive features of Spinacia oleracea to different levels of salinity, its use in desalination and production of 20-Hydroxyecdysone were studied. Plants showed survival up to EC 12 dS/m with reduced growth as compared with control. Net photosynthesis rate, transpiration, stomatal conductance, and water use efficiency of salt treated plants declines with increasing salinity stress. Higher antioxidant enzyme activities and compatible solutes accumulation were observed in salt treated plants as function of osmotic adjustment. Significant Na+ sequestration and Na/K ratio were noted with increase in salt stress in comparison to the control. Since the plant accumulates a bioactive, secondary metabolite 20-Hydroxyecdysone (20E), we observed significant 20E content in plants grown at EC 4-12 dS/m in comparison to control. Furthermore, a preliminary field experiment, showed significant reduction in the soil electrical conductivity by 1.8 ds/m after 90 days of plant growth with Na+ sequestration in plant biomass. Subsequent to this growth period, the phytodesalinized soil supported the significant growth of a glycophyte (rice). Our results suggest that S. oleracea can adapt to saline conditions with antioxidant defense and osmotic adjustment. The plant can be used as a potential candidate for desalination and also for enhanced production of 20-Hydroxyecdysone.

The vacuolar proton pyrophosphatase gene (SbVPPase) from the Sorghum bicolor confers salt tolerance in transgenic Brahmi [Bacopa monnieri (L.) Pennell].

Plants overcome the effect of Na+ toxicity either by excluding Na+ at the plasma membrane or by sequestering them into the vacuoles. Influx of Na+ ions into the plant vacuoles is usually driven by H+ generated by vacuolar-type H+-ATPase as well as vacuolar proton pyrophosphatse (VPPase). In the present study, we have developed Bacopa monnieri transgenics via Agrobacterium tumefaciens containing the recombinant vector pCAMBIA2300-SbVPPase gene. Transformants were produced using nodal explants. Transformants were confirmed by PCR and DNA blot analysis. qPCR analysis showed higher transcript levels of SbVPPase compared to untransformed control (UC). Higher VPPase activity was recorded in transgenics compared to UC. Under 150 mM salt stress, transgenic shoots showed enhanced Na+ accumulation with better biomass production, increased glycine betaine content, and total soluble sugar levels than UC. Transgenic shoots showed 2.9-3.8-folds lower levels of malondialdehyde content indicating lesser membrane damage. Increase in antioxidant enzyme activities (1.4-3.2-folds) was observed in transgenics compared to UC. Transgenics also displayed 7.3-9.0-folds enhanced accumulation of the medicinally important compound bacoside A. Increased biomass production, accumulation of Na+, osmolytes (glycine betaine, sugars etc.), and elevated antioxidant enzyme activities indicate better osmotic adjustment in transgenics by compartmentalization of Na+ into the vacuoles under salt stress conditions. Thus, overexpression of SbVPPase in Bacopa alleviated salt stress by sequestering Na+.

Temporal and spatial changes in ion homeostasis, antioxidant defense and accumulation of flavonoids and glycolipid in a halophyte Sesuvium portulacastrum (L.) L.

Salinity is an important environmental constraint limiting plant productivity. Understanding adaptive responses of halophytes to high saline environments may offer clues to manage and improve salt stress in crop plants. We have studied physiological, biochemical and metabolic changes in a perennial, fast growing halophyte, Sesuvium portulacastrum under 0 mM (control), 150 mM (low salt, LS) and 500 mM (high salt, HS) NaCl treatments. The changes in growth, relative water content, cation, osmolyte accumulation, H2O2 and antioxidant enzyme activity (SOD, CAT and APX) were observed under different treatment conditions. A positive correlation was revealed for sodium ion accumulation with malondialdehyde (r2 = 0.77), proline (r2 = 0.88) and chlorophyll content (r2 = 0.82) under salt treatment while a negative correlation was observed with relative tissue water content (r2 = -0.73). The roots and leaves showed contrasting accumulation of potassium and sodium ions under LS treatment. Temporal and spatial study of sodium and potassium ion content indicated differential accumulation pattern in roots and leaves, and, high potassium levels in root. Higher H2O2 content was recorded in roots than leaves and the antioxidant enzyme activities also showed significant induction under salt treatment conditions. Gene expression profiling of sodium transporters, Sodium proton exchanger (NHX3), Vacuolar ATPase (vATPase) and Salt overly sensitive1 (SOS1) showed up regulation under salt stress after 6-24 hr of NaCl treatment. Metabolite changes in the salt stressed leaves showed increased accumulation of flavonoids (3,5-dihydroxy-6,4'-dimethoxy-flavone-7-O-[α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranoside], and3,5-dihydroxy-6,3',4'-trimethoxy-flavone-7-O-[α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranoside] in both LS and HS treatments, while a glycolipid, 1-O-linolenyl-2-O-(palmitoyl)-3-O-galactopyranosyl glycerol, accumulated more in LS over HS treatments and control. The results suggest that differential spatial and temporal cation levels in roots and leaves, and accumulation of flavanoid and glycolipid could be responsible for salt adaptation of S. portulacastrum.

Looking at Halophytic Adaptation to High Salinity Through Genomics Landscape.

Soil salinity is an important stress factor that limits plant growth and productivity. For a given plant species, it is critical to sense and respond to salt stimuli followed by activation of multitude of mechanisms for plants to survive. Halophytes, the wonders of saline soils, have demonstrated ability to withstand and reproduce in at least 200 mM NaCl concentration, which makes them an ideal system to study mechanism of salt adaptation for imparting salt tolerance in glycophytes. Halophytes and salt sensitive glycophytes adapt different defense strategies towards salinity stress. These responses in halophytes are modulated by a well orchestrated network of signaling pathways, including calcium signaling, reactive oxygen species and phytohormones. Moreover, constitutive expression of salt stress response related genes, which is only salt inducible in glycophytes, maintains salt tolerance traits in halophytes. The focus of this review is on the adaptive considerations of halophytes through the genomics approaches from the point of view of sensing and signaling components involved in mediating plant responses to salinity.

Salt responsive physiological, photosynthetic and biochemical attributes at early seedling stage for screening soybean genotypes.

Salt stress affects all the stages of plant growth however seed germination and early seedling growth phases are more sensitive and can be used for screening of crop germplasm. In this study, we aimed to find the most effective indicators of salt tolerance for screening ten genotypes of soybean (SL-295, Gujosoya-2, PS-1042, PK-1029, ADT-1, RKS-18, KDS-344, MAUS-47, Bragg and PK-416). The principal component analysis (PCA) resulted in the formation of three different clusters, salt sensitive (SL-295, Gujosoya-2, PS-1042 and ADT-1), salt tolerant (MAUS-47, Bragg and PK-416) and moderately tolerant/sensitive (RKS-18, PK-1029 and KDS-344) suggesting that there was considerable genetic variability for salt tolerance in the soybean genotypes. Subsequently, genotypes contrasting in salt tolerance were analyzed for their physiological traits, photosynthetic efficiency and mitochondrial respiration at seedling and early germination stages under different salt (NaCl) treatments. It was found that salt mediated increase in AOX-respiration, root and shoot K+/Na+ ratio, improved leaf area and water use efficiency were the key determinants of salinity tolerance, which could modulate the net photosynthesis (carbon assimilation) and growth parameters (carbon allocation). The results suggest that these biomarkers could be can be useful for screening soybean genotypes for salt tolerance.

Comparative analysis of MazEF and HicAB toxin-antitoxin systems of the cyanobacterium, Anabaena sp. PCC7120.

Anabaena PCC7120 has two annotated toxin-antitoxin systems: MazEF and HicAB. Overexpression of either of the toxins severely inhibited the growth of Escherichia coli BL21(plysS)(DE3). Of the two Anabaena toxins, MazF exhibited higher toxicity than HicA as evidenced by (i) 100-fold lower viability upon overexpression of MazF compared to HicA; (ii) complete loss of cell viability within 1 h of induction of MazF expression, as against >103 colony forming units mL-1 in case of HicA; (iii) inability to maintain the MazF overexpressing plasmid in E. coli cells; and (iv) neutralisation of the toxin was effective at the molar ratio of 1:1.9 for MazF:MazE and 13:1 for HicA:HicB, indicating higher antitoxin requirement for neutralisation of MazF. The growth inhibitory effect of MazF was found to be higher in lag phase cultures compared to mid-logarithmic phase cultures of E. coli, while the reverse was true for HicA. The results suggest possible distinct roles for MazEF and HicAB systems of Anabaena.

Response of Ajowan to water stress induced by polyethylene glycol (PEG) 6000 during seed germination and seedling growth.

Seed germination and subsequent metabolic changes in Ajowan (Trachyspermum ammi L.) (NRCSS AA-2) seedlings was studied under water limiting conditions, imposed by increasing concentrations of polyethylene glycol (PEG 6000). Five water stress conditions (0, -0.05, -0.1, -0.15 and -0.2 MPa) were created in the laboratory in a completely randomized design. The results revealed that water stress (-0.2 MPa) significantly reduced seed germination components like final germination percent (80%) radical (64%) and plumule (63%) length, fresh (63%) and dry (74%) weight of seedlings and vigor index (SVI) by 92% over control. Decrease in osmotic potential resulted in decreased protein content (56%) with concomitant increase in total sugars (55%) at -0.2 MPa as compared to control. Significant increase in free proline and glycine betaine content by 1.5 to 2 folds was observed at the highest water stress condition. The seedlings exhibited increased activity of superoxide dismutase and peroxidase under stressed condition. In the present study, it was found that Ajowan was a moderately drought tolerant species at laboratory level.

Investigation of arsenic accumulation and tolerance potential of Sesuvium portulacastrum (L.) L.

Sesuvium portulacastrum (L.) L., a facultative halophyte, is considered a suitable candidate for the phytoremediation of metals. An investigation of As accumulation and tolerance was conducted in Sesuvium plants upon exposure to As(V) (100-1000 µM) for 30 d. Plants demonstrated a good growth even after prolonged exposure (30 d) to high As(V) concentrations (1000 µM) and a significant As accumulation (155 µg g⁻¹ dry weight) with a bioaccumulation factor of more than ten at each concentration. The results of shoot and root dry weight, malondialdehyde accumulation, photosynthetic pigments, and total soluble proteins demonstrated that plants did not experience significant toxicity even at 1000 µM As(V) after 30 d. However, metabolites (total non-protein thiols and cysteine) and enzymes (serine acetyltransferase, cysteine synthase and γ-glutamylcysteine synthetase) of thiol metabolism, in general, remained either unaffected or showed slight decline. Hence, plants tolerated high As(V) concentrations without an involvement of thiol metabolism as a major component. Taken together, the results indicate that plants are potential As accumulator and may find application in the re-vegetation of As contaminated sites.

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of "Soma" drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 µM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog's) medium fortified with either IAA or NAA (0.5-2.0 µM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 µM 2,4-D+5 µM BA. However the maximum cerpegin content (470 µg/g dry weight) was recorded in dried callus derived on MS+10 µM IAA+5 µM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.

Somatic embryogenesis in sisal ( Agave sisalana Perr. ex. Engelm).

A protocol has been developed for somatic embryogenesis and plant regeneration of sisal (Agave sisalana Perr. ex. Engelm). Embryogenic callus cultures were initiated from young shoots raised in vitro from the stem portion of the bulbil on medium supplemented with 1-2 mg l(-1) kinetin (KN) and 0.2-0.5 mg l(-1) alpha-naphthaleneacetic acid plus KN or 1-1.5 mg l(-1 )benzylaminopurine (BAP) or 0.25-0.5 mg l(-1 )2,4-dichlorophenoxyacetic acid plus BAP or 0.5-1.0 mg l(-1) KN. Embryos at various developmental stages (globular-, heart- or torpedo-shaped) produced mature and germinating embryos on being transferred to a new medium containing 0-0.25 mg l(-1 )KN. After 28 days, a maximum of 76% germinated embryos was obtained on a medium supplemented with 0.1 mg l(-1) KN. The capacity for embryogenesis remained constant in the callus upon subculturing on the same medium for more than 48 months. Histological observations showed a distinct multicellular origin for most of the somatic embryos as they developed from epidermal, sub-epidermal and inside callus cells, while a few of them originated from a superficial callus cell. Plantlets regenerated from embryos were transferred to the field where their survival rate was 100%.

In vitro plant regeneration from callus of niger (Guizotia abyssinica Cass.) cv. Sahyadri.

Callus formation was achieved with root, hypocotyl, and cotyledon explants of niger (Guizotia abyssinica Cass.) cultivar Sahyadri on Murashige and Skoog medium containing 0.5 mg l-1 ß-indoleacetic acid + 1.5 mg l-1 6-benzylaminopurine (BAP). Hypocotyl and cotyledon-derived calli when transferred onto a medium with 0.5 mg l-1 BAP produced an average of 12-32 shoots/ callus culture. The callus retained its potential for shoot regeneration for more than 19 months. The shoots formed an extensive root system and were transferred to pots kept in a greenhouse, where the survival rate was 98%. The plantlets flowered in vitro if transfer to fresh medium or to soil was delayed by 40-50 days. All regenerants were diploid with 2n=30.