Subunits B′γ and B′ζ of protein phosphatase 2A regulate photo‐oxidative stress responses and growth in Arabidopsis thaliana

Plants survive periods of unfavourable conditions with the help of sensory mechanisms that respond to reactive oxygen species (ROS) as signalling molecules in different cellular compartments. We have previously demonstrated that protein phosphatase 2A (PP2A) impacts on organellar cross‐talk and associated pathogenesis responses in Arabidopsis thaliana. This was evidenced by drastically enhanced pathogenesis responses and cell death in cat2 pp2a‐b′γ double mutants, deficient in the main peroxisomal antioxidant enzyme CATALASE 2 and PP2A regulatory subunit B′γ (PP2A‐B′γ). In the present paper, we explored the impacts of PP2A‐B′γ and a highly similar regulatory subunit PP2A‐B′ζ in growth regulation and light stress tolerance in Arabidopsis. PP2A‐B′γ and PP2A‐B′ζ display high promoter activities in rapidly growing tissues and are required for optimal growth under favourable conditions. Upon acclimation to a combination of high light, elevated temperature and reduced availability of water, however, pp2a‐b′γζ double mutants grow similarly to the wild type and show enhanced tolerance against photo‐oxidative stress. We conclude that by controlling ROS homeostasis and signalling, PP2A‐B′γ and PP2A‐B′ζ may direct acclimation strategies upon environmental perturbations, hence acting as important determinants of defence responses and light acclimation in plants.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

The effects of extracellular adenosine 5′‐triphosphate on the tobacco proteome

Extracellular adenosine 5′‐triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca²⁺, nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non‐hydrolysable analogue as probes to identify the molecular and physiological effects of eATP‐mediated signalling in tobacco. 2‐DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence‐related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.     

Studies of the Activity of Peroxidase‐Like DNAzyme by Modifying 3′‐ or 5′‐End of Aptamers

In the presence of hemin and under appropriate conditions, some modalities of G‐quadruplexes can form a peroxidase‐like DNAzyme that has been widely used in biology. Structure? function studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G‐quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G‐quadruplex sequences by adding different lengths of oligo‐dT to the 3′‐ or 5′‐end of the aptamers. The results suggested that adding dT_n to the 5′‐end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dT_n to the 3′‐end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H₂O₂), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

Guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a ‘rare’ actinomycete

With the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) and guanosine 5′‐triphosphate 3′‐diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)‐encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild‐type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster‐specific dpgA mRNA were stabilized during the idiophase in the wild‐type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse‐labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.