The role of microRNAs and other endogenous small RNAs in plant stress responses.

Crop yields are significantly reduced by biotic and abiotic stresses throughout the world. To reduce the damage caused by stress factors, plants have evolved sophisticated adaptive responses involving reprogramming gene expression at the transcriptional, post-transcriptional and post-translational levels. A better understanding of such processes will lead to new strategies to improve plant stress tolerance. Recently discovered endogenous small RNAs (microRNAs and small-interfering RNAs) have emerged as important players in plant stress responses. The observation that some of the small RNAs are up- or down-regulated in response to stress implies that these small RNAs have a role in stress tolerance. Stress-induced small RNAs might down-regulate their target genes, which may encode negative regulators of stress responses. Conversely, small RNAs down-regulated in response to stress cause the accumulation of their target mRNAs, which may contribute positively to the adaptation to stress. Here, we review the current status of small RNAs involved in biotic and abiotic stress regulatory networks.

Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA families.

MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) have emerged as important regulators of gene expression in higher eukaryotes. Recent studies indicate that genomes in higher plants encode lineage-specific and species-specific miRNAs in addition to the well-conserved miRNAs. Leguminous plants are grown throughout the world for food and forage production. To date the lack of genomic sequence data has prevented systematic examination of small RNAs in leguminous plants. Medicago truncatula, a diploid plant with a near-completely sequenced genome has recently emerged as an important model legume. We sequenced a small RNA library generated from M. truncatula to identify not only conserved miRNAs but also novel small RNAs, if any. Eight novel small RNAs were identified, of which four (miR1507, miR2118, miR2119 and miR2199) are annotated as legume-specific miRNAs because these are conserved in related legumes. Three novel transcripts encoding TIR-NBS-LRR proteins are validated as targets for one of the novel miRNA, miR2118. Small RNA sequence analysis coupled with the small RNA blot analysis, confirmed the expression of around 20 conserved miRNA families in M. truncatula. Fifteen transcripts have been validated as targets for conserved miRNAs. We also characterized Tas3-siRNA biogenesis in M. truncatula and validated three auxin response factor (ARF) transcripts that are targeted by tasiRNAs. These findings indicate that M. truncatula and possibly other related legumes have complex mechanisms of gene regulation involving specific and common small RNAs operating post-transcriptionally.

Optimization of in vitro culture media for improvement in yield of Navara ancient Indian medicinal rice.

Medicinally important ancient Navara rice (GI Kerala, India 2007) is a very short duration (60-70 days) variety with a yield of only 0.5 tonnes/hectare costing ~ Rs. 400/kg. It is used for indigenous treatment for chronic diseases by local and oral consumption. In this study, scutellum-derived calli were generated from mature Navara seeds and these were inoculated on different CIM-1 to CIM-5 media supplemented with 2.5 mg/l 2,4-dichlorophenoxyacetic acid. Regeneration of calli on different regeneration media RI, RII and RIII media were performed. Regeneration of 30-day-old calli on RI media showed 30%, for RII it showed no regeneration and on RIII media only 12% regeneration was obtained. The addition of glutamine and proline showed a 30-40% improvement in somatic embryogenesis. The 74-88% callus induction frequency was obtained on CIM-1 to CIM-5. The fresh weight (mg) of 30-day-old calli is CIM-2 < CIM-3 < CIM-4 < CIM-1 << CIM-5 and corresponding size shows CIM-2-CIM-3 < CIM-5 < CIM-1 < CIM-4. A negative correlation between the callus fresh weight and the regeneration efficiency was observed. In CIM-5, 20-25 days 3.4-fold increase and 25-30 days a 1.7-fold increase in fresh weight of calli is noted. The 20-day-old calli transfer to RI media shows 80% regeneration frequency and 6-7 plantlets/callus, which are twofold higher as compared with 30-day-old calli. The somatic embryogenesis and its regeneration on synthetic media provide an alternative for biotechnological intervention for yield improvement, in turn cost reduction for Navara rice.

Erratum: Author Correction: Optimization of in vitro culture media for improvement in yield of Navara ancient Indian medicinal rice.

[This corrects the article DOI: 10.1007/s13205-019-1797-2.].

Gamma irradiation of medicinally important plants and the enhancement of secondary metabolite production.

PURPOSE: The profitable production of some important plant-based secondary metabolites (ginsenosides, saponins, camptothecin, shikonins etc.) in vitro by gamma irradiation is a current area of interest. We reviewed different types of secondary metabolites, their mode of synthesis and effect of γ-radiation on their yield for different plants, organs and in vitro cultures (callus, suspension, hairy root). Special effort has been made to review the biochemical mechanisms underlying the increase in secondary metabolites. A comparison of yield improvement with biotic and abiotic stresses was made. RESULTS: Phenolic compounds increase with γ-irradiation in whole plants/plant parts; psoralen content in the common herb babchi (Psoralea corylifolia) was increased as high as 32-fold with γ-irradiation of seeds at 20 kGy. The capsaicinoids, a phenolic compound increased about 10% with 10 kGy in paprika (Capsicum annum L.). The in vitro studies show all the three types of secondary metabolites are reported to increase with γ-irradiation. Stevioside, total phenolic and flavonoids content were slightly increased in 15 Gy-treated callus cultures of stevia (Stevia rebaudiana Bert.). In terpenoids, total saponin and ginsenosides content were increased 1.4- and 1.8-fold, respectively, with 100 Gy for wild ginseng (Panax ginseng Meyer) hairy root cultures. In alkaloids, camptothecin yield increased as high as 20-fold with 20 Gy in callus cultures of ghanera (Nothapodytes foetida). Shikonins increased up to 4-fold with 16 Gy in suspension cultures of purple gromwell (Lithospermum erythrorhizon S.). The enzymes associated with secondary metabolite production were increased with γ-irradiation of 20 Gy; namely, phenylalanine ammonia-lyase (PAL) for phenolics, chalcone synthase (CHS) for flavonoids, squalene synthase (SS), squalene epoxidase (SE) and oxidosqualene cyclases (OSC) for ginsenosides and PHB (p-hydroxylbenzoic acid) geranyl transferase for shikonins. CONCLUSIONS: An increase in secondary metabolites in response to various biotic and abiotic stresses is compared with ionizing radiation. A ∼5- to 20-fold increase is noted with ∼20 Gy irradiation dose. It increases the yield of secondary metabolites by enhancing the activity of certain key biosynthetic enzymes. Identification of the optimum dose is the important step in the large-scale production of secondary metabolites at industrial level.

Position Based Nucleotide Analysis of miR168 Family in Higher Plants and its Targets in Mammalian Transcripts.

BACKGROUND: miRNA are the post transcriptional regulator of the genes. The conserved miR168 family is evaluated for position based nucleotide preference in higher plants. Low density lipoprotein receptor adaptor protein 1 (LDLRAP1) target validated for miR168a obtained from rice origin is reported. METHODS: The mature miRNA sequences include miR168-5p and miR168-3p, were obtained from miRBase (v21, June 2014) for 15 families (28 plants). The preferred position based nucleotide sequences were obtained using Data Analysis in Molecular Biology and Evolution software. The miR168-5p was subjected to cross kingdom analysis using psRNATarget. Target expression and functional annotation was analyzed by using Human Protein Atlas database WEB-based Gene SeTAnaLysis Toolkit. RESULTS: miR168-5p shows same nucleotides at positions 1-6, 8-9, 11-12, 15-17 and 19. Also, miR168-3p is present in 3 families (10 plants) shows the same nucleotide at position 1-11, 13-15 and 17-21. The 123 targets in human transcriptome were identified showing 58% cleavage and 41% translation repression. Low density lipoprotein receptor adaptor protein 1 (LDLRAP1) target validated for miR168a obtained from rice origin, could also be targeted from miR168 from any other plant sources. The randomly selected 10 targets include some important genes likeRPL34, ATXN1, AKAPI3 and ALS2 and is involved in transcription, cell trafficking, cell metabolism and neurodegenerative disorder. CONCLUSION: Our work suggests that miR168 family has conserved sequence in higher plants. The seed region position 2-8 shows 70-95% pairing with human targets. Cleavage site at position 10-14 and these were analysed for the base preference with the targets showed 80-96% Watson Crick pairing.

Formation of 8-oxo-7,8-dihydroguanine-radicals in gamma-irradiated DNA by multiple one-electron oxidations.

Electron spin resonance (ESR) studies of radicals formed by radiation-induced multiple one-electron oxidations of guanine moieties in DNA are reported in this work. Annealing of gamma-irradiated DNA from 77 to 235 K results in the hydration of one electron oxidized guanine (G*+) to form the 8-hydroxy-7,8-dihydroguanin-7-yl-radical (*GOH) having one beta-proton coupling of 17-28 G and an anisotropic nitrogen coupling, A(parallel), of approximately 20 G, A(perpendicular) = 0 with g(parallel) = 2.0026 and g(perpendicular) = 2.0037. Further annealing to 258 K results in the formation of a sharp singlet at g = 2.0048 with line-width of 5.3 G that is identified as the 8-oxo-7,8-dihydroguanine one-electron-oxidized radical (8-oxo-G*+). This species is formed via two one-electron oxidations of *GOH. These two one-electron oxidation steps leading to the formation of 8-oxo-G*+ from *GOH in DNA, are in accordance with the expected ease of oxidation of *GOH and 8-oxo-G. The incorporation of oxygen from water in G*+ leading to *GOH and to 8-oxo-G*+ is verified by ESR studies employing 17O isotopically enriched water, which provide unambiguous evidence for the formation of both radicals. ESR analysis of irradiated-DNA in the presence of the electron scavenger, Tl3+, demonstrates that the cationic pathway leads to the formation of the 8-oxo-G*+. In irradiated DNA-Tl3+ samples, Tl3+ captures electrons. Tl2+ thus produced is a strong oxidant (2.2 V), which is metastable at 77 K and is observed to increase the formation of G*+ and subsequently of 8-oxo-G*+ upon annealing. We find that in the absence of the electron scavenger the yield of 8-oxo-G*+ is substantially reduced as a result of electron recombinations with G*+ and possible reaction with *GOH.

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging.

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.

The formation of DNA sugar radicals from photoexcitation of guanine cation radicals.

In this investigation of radical formation and reaction in gamma- irradiated DNA and model compounds, we report the conversion of the guanine cation radical (one-electron oxidized guanine, G(.+)) to the C1' sugar radical and another sugar radical at the C3' or C4' position (designated C3'(.)/C4'(.)) by visible and UV photolysis. Electron spin resonance (ESR) spectroscopic investigations were performed on salmon testes DNA as well as 5'-dGMP, 3'-dGMP, 2'-deoxyguanosine and other nucleosides/nucleotides as model systems. DNA samples (25- 150 mg/ml D(2)O) were prepared with Tl(3+) or Fe(CN)(3-)(6) as electron scavengers. Upon gamma irradiation of such samples at 77 K, the electron-gain path in the DNA is strongly suppressed and predominantly G(.+) is found; after UV or visible photolysis, the fraction of the C1' sugar radical increases with a concomitant reduction in the fraction of G(.+). In model systems, 3'- dGMP(+.) and 5'-dGMP(+.) were produced by attack of Cl(.-)(2) on the parent nucleotide in 7 M LiCl glass. Subsequent visible photolysis of the 3'-dGMP(+.) (77 K) results predominantly in formation of C1'(.) whereas photolysis of 5'-dGMP(+.) results predominantly in formation of C3'(.)/C4'(.). We propose that sugar radical formation is a result of delocalization of the hole in the electronically excited base cation radical into the sugar ring, followed by deprotonation at specific sites on the sugar.

Efficient production of recombinant human transcription factor IIE.

Transcription factor IIE (TFIIE) is a general initiation and promoter escape factor for RNA polymerase II composed of p56 (TFIIE-alpha) and p34 (TFIIE-beta) subunits. Our laboratories experienced difficulty producing adequate quantities of recombinant human TFIIE-alpha for in vitro studies using available clones. We therefore re-engineered the TFIIE subunit production vectors and tested various Escherichia coli host strains to optimize expression. We report a much-improved system for production of pure, soluble, and active TFIIE complex for in vitro studies.