High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2, by constitutive and inducible expression of tobacco <Emphasis Type="Italic">osmotin</Emphasis>

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.     

Chemistry of costunolide and biological activity of the derived lactones

Costunolide and its derived C-16 germacranolides on oxidation with selenium dioxide-t-butyl hydroperoxide afforded two melampolides, an aldehydolactone and the corresponding hydroxylactone, in each case. Structures were assigned to these melampolides on the basis of spectral data and chemical correlation. The aldehydolactones were significantly more active root promotors than their parent lactones. Costunolide and related germacranolides underwent cyclization on treatment with iodine and pyridinium chlorochromate to afford interesting products. (?)-β-Frullanolide has been synthesized and shown to be biologically more active when compared with its parent trans-lactone.     

Gender differences in the modulation of cardiac gene expression by dietary conjugated linoleic acid isomers

In an earlier study, we showed that dietary conjugated linoleic acid (CLA) isomers can exert differential effects on heart function in male and female rats, but the underlying mechanisms for these actions are not known. Cardiomyocyte Ca2+ cycling is a key event in normal cardiac contractile function and defects in Ca2+ cycling are associated with cardiac dysfunction and heart disease. We therefore hypothesized that abnormalities in the sarcolemmal (SL) and sarcoplasmic reticulum (SR)-mediated regulation of intracellular Ca2+ contribute to altered cardiac contractile function of male and female rats owing to dietary CLA isomers. Healthy male and female Sprague-Dawley rats were fed different CLA isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. We determined the mRNA levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 2a, ryanodine receptor, phospholamban, calsequestrin, Na+-Ca2+-exchanger (NCX), and L-type Ca2+ channel in the left ventricle (LV) by RT-PCR. The SR function was assessed by measurement of Ca2+-uptake and -release. Significant gender differences were seen in the LV NCX, L-type Ca2+ channel, and ryanodine receptor mRNA expression levels in control male and female rats. Dietary CLA isomers in the various forms induced changes in the mRNA levels of SERCA 2a, NCX, and L-type Ca2+ channel in the LV of both male and female hearts. Whereas protein contents of the Ca2+ cycling proteins were altered, changes in SR Ca2+-uptake and -release were also detected in both male and female rats in response to dietary CLA. The results of this study demonstrate that long-term dietary supplementation can modulate cardiac gene expression and SR function in a gender-related manner and may, in part, contribute to altered cardiac contractility.     

Nitric oxide is involved in salicylic acid-induced flowering of Lemna aequinoctialis Welw.

Salicylic acid (SA) is a well-known inducer of flowering in Lemna under both non-inductive and inductive photoperiod conditions. However, the underlying mechanism is not well understood. Herein, we report for the first time that nitric oxide (NO) is partially involved in SA-induced flowering in L. aequinoctialis (Syn. L. paucicostata Hegelm.). Our results demonstrated that SA-induced flowering is significantly reduced by exogenous application of NO scavengers; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and methylene blue, nitric oxide synthase inhibitors; N-ω-nitro-l-arginine and N-ω-nitro-l-arginine-methyl ester hydrochloride, and nitrate reductase inhibitor; sodium tungstate in two strains of Lemna viz. 6746 and LP₆. Altogether our present findings shed a light on the new role of NO in SA-induced flowering and open interesting directions that need further investigation.     

A multi-step phosphorelay two-component system impacts on tolerance against dehydration stress in common wheat

Wheat is an important staple crop, and its productivity is severely constrained by drought stress (DS). An understanding of the molecular basis of drought tolerance is necessary for genetic improvement of wheat for tolerance to DS. The two-component system (TCS) serves as a common sensor-regulator coupling mechanism implicated in the regulation of diverse biological processes (including response to DS) not only in prokaryotes, but also in higher plants. In the latter, TCS generally consists of two signalling elements, a histidine kinase (HK) and a response regulator (RR) associated with an intermediate element called histidine phosphotransferase (HPT). Keeping in view the possible utility of TCS in developing water use efficient (WUE) wheat cultivars, we identified and characterized 62 wheat genes encoding TCS elements in a silico study; these included 7 HKs, 45 RRs along with 10 HPTs. Twelve of the 62 genes showed relatively higher alterations in the expression under drought. The quantitative RT-PCR (qRT-PCR)-based expression analysis of these 12 TCS genes was carried out in wheat seedlings of a drought sensitive (HD2967) and a tolerant (Dharwar Dry) cultivar subjected to either dehydration stress or cytokinin treatment. The expression of these 12 genes under dehydration stress differed in sensitive and tolerant genotypes, even though for individual genes, both showed either up-regulation or down-regulation. In response to the treatment of cytokinin, the expression of type-A RR genes was higher in the tolerant genotype, relative to that in the sensitive genotype, the situation being reverse for the type-B RRs. These results have been discussed in the context of the role of TCS elements in drought tolerance in wheat.     

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

Background

Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6).

Results

Efficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. Additionally, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.

Conclusion

The bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1014) contains supplementary material, which is available to authorized users.     

Metformin is synthetically lethal with glucose withdrawal in cancer cells

Comment on: Menendez JA, et al. Cell Cycle 2012; 11: 2782-92.In a recent issue of Cell Cycle, Menendez and colleagues proposed a novel concept, that metformin is synthetically lethal with glucose withdrawal in cancer cells.¹ Historically, synthetic lethality has focused on how tumor cells are responsive to certain agents that only harbor specific constitutive epigenetic or genetic lesions.² More recent data from several groups have uncovered that altered tumor microenvironment could be used to confer synthetic lethality to specific drugs, defined as “contextual synthetic lethality,” that is microenvironment-mediated. For example, hypoxia-induced HR (homologous repair) defect has been shown to be synthetically lethal to PARP inhibition, while PARP inhibition, per se, did not alter HR inhibition or function, thus providing a prime example of “contextual synthetic lethality.”³ In this report, Menendez et al. have elegantly connected the glucose-deprived tumor microenvironment in primary tumors as a synthetic lethal partner to metformin. Metformin is a FDA-approved drug to treat diabetic patients that is gaining momentum as a repurposing drug for cancer treatment.⁴ Using several different breast cancer cells with and without oncogenic activation, the authors have shown that the glucose-rich conditions of the in vitro experiments dictates the use of very high concentrations of metformin, which are not applicable to glucose-starved in vivo conditions. While other reports have alluded to the effect of glucose withdrawal in killing genetically compromised cells to therapeutic effect of metformin in vitro,⁵ Menendez et al have provided a logical explanation for the use of very high concentrations of metformin to achieve anticancer effects in vitro in the high glucose-rich environment used in these experiments, which are clinically not applicable in vivo in patients.Based on these findings, it can be envisaged that in the tumor microenvironment, where the cancer cells are under extreme nutritional and hypoxic stress (a niche for cancer stem cells), metformin treatment could favor synthetic lethality and hence effectively can attenuate tumor growth. The tumor microenvironment thus enables the bioenergetic switch in favor of glycolysis and dependence on glucose and glutamine as a rapid source of nutrition. While the authors’ data clearly depicts how metformin eliminates the tolerance of the breast cancer cells to fluctuations in glucose concentrations, it is important to understand how the availability of other dominant sources of energy, such as glutamine, might participate in this scenario. It is plausible that subtype of breast cancers, i.e., basal vs luminal, might depend on different energy sources, albeit to a different extent.⁶ This is important, because tumor cells often acquire metabolic adaptability toward available preferred energy source to adapt well to nutritional stress via autophagy and altered metabolism.⁷ Along these lines, the authors rationalize the therapeutic targeting of the cancer stem cells by metformin through its synthetic lethal activity to the hyperglycotic phenotype often seen in CSC to sustain their stemness.⁸ Further characterization of how metformin treatment alters the metabolic nodes in cancer stem cells and/or p53-null cells would explain the underpinning mechanisms for increased susceptibility of these indolent and aggressive cancer cells toward metformin.It is well documented that metformin, by inhibiting complex I of respiratory chain in mitochondria (ETCI), induces a decrease in the ATP levels, and that glucose depletion also decreases ATP levels, albeit to varying levels. Therefore, it is possible that simultaneous targeting of both pathways (glycolytic pathway and OXPHOS) caused ATP depletion below a critical threshold, resulting in cell death. This concept is supported by the elegant study⁹ highlighting the effectiveness of combination of glycolysis inhibition by 2-DG and metformin in several preclinical models exhibiting anti-tumor effects, including MB-MDA231 used in this study.Since recent studies indicate that inhibiting glucose uptake with small-molecule inhibitors led to a decline in cylcin E2 and p-RB levels,¹⁰ it is a possibility that cell cycle inhibitor levels are also regulated under glucose withdrawal conditions, sensitizing cells to cytotoxic effects of metformin in breast cancer cells.Considering data from several studies, a view that metformin treatment has pleotropic effects on several signaling pathways under glucose-free conditions seems a practical possibility. Overall, this work offers several new insights into glucose-dependent mechanisms underpinning the mode of action of metformin as a viable therapeutic strategy.