Comparative analysis of two phytochrome mutants of tomato (Micro-Tom cv.) reveals specific physiological, biochemical, and molecular responses under chilling stress

Background Phytochromes are plant photoreceptors that have long been associated with photomorphogenesis in plants; however, more recently, their crucial role in the regulation of variety of abiotic stresses has been explored. Chilling stress is one of the abiotic factors that severely affect growth, development, and productivity of crops. In the present work, we have analyzed and compared physiological, biochemical, and molecular responses in two contrasting phytochrome mutants of tomato, namely aurea (aur) and high pigment1 (hp1), along with wild-type cultivar Micro-Tom (MT) under chilling stress. In tomato, aur is phytochrome-deficient mutant while hp1 is a phytochrome-sensitive mutant. The genotype-specific physiological, biochemical, and molecular responses under chilling stress in tomato mutants strongly validated phytochrome-mediated regulation of abiotic stress. Results Here, we demonstrate that phytochrome-sensitive mutant hp1 show improved performance compared to phytochrome-deficient mutant aur and wild-type MT plants under chilling stress. Interestingly, we noticed significant increase in several photosynthetic-related parameters in hp1 under chilling stress that include photosynthetic rate, stomatal conductance, stomatal aperture, transpiration rate, chlorophyll a and carotenoids. Whereas most parameters were negatively affected in aur and MT except a slight increase in carotenoids in MT plants under chilling stress. Further, we found that PSII quantum efficiency (Fv/Fm), PSII operating efficiency (Fq′/Fm′), and non-photochemical quenching (NPQ) were all positively regulated in hp1, which demonstrate enhanced photosynthetic performance of hp1 under stress. On the other hand, Fv/Fm and Fq′/Fm′ were decreased significantly in aur and wild-type plants. In addition, NPQ was not affected in MT but declined in aur mutant after chilling stress. Noticeably, the transcript analysis show that PHY genes which were previously reported to act as molecular switches in response to several abiotic stresses were mainly induced in hp1 and repressed in aur and MT in response to stress. As expected, we also found reduced levels of malondialdehyde (MDA), enhanced activities of antioxidant enzymes, and higher accumulation of protecting osmolytes (soluble sugars, proline, glycine betaine) which further elaborate the underlying tolerance mechanism of hp1 genotype under chilling stress. Conclusion Our findings clearly demonstrate that phytochrome-sensitive and phytochrome-deficient tomato mutants respond differently under chilling stress thereby regulating physiological, biochemical, and molecular responses and thus establish a strong link between phytochromes and their role in stress tolerance.

A temperature-sensitive Mycobacterium smegmatis glgE mutation leads to a loss of GlgE enzyme activity and thermostability and the accumulation of α-maltose-1-phosphate

BackgroundThe bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis.MethodsWe reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme.ResultsThe mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 ºC. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected.ConclusionsWe have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation.General SignificanceThis study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria.

Temperature sensitive Mutant Proteome Profiling: a novel tool for the characterization of the global impact of missense mutations on the proteome

ABSTRACTTemperature sensitive (TS) mutants have been foundational in the characterization of essential genes. However, a high-throughput workflow for characterization of biophysical changes in TS mutants is lacking. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and PPIs. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. TeMPP is a precise approach to measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion.

Biofilm-associated toxin and extracellular protease cooperatively suppress competitors inBacillus subtilisbiofilms

In nature, most bacteria live in biofilms where they compete with their siblings and other species for space and nutrients. Some bacteria produce antibiotics in biofilms; however, since the diffusion of antibiotics is generally hindered in biofilms by extracellular polymeric substances, i.e., the biofilm matrix, their function remains unclear. TheBacillus subtilis yitPOMoperon is a paralog of thesdpABCoperon, which produces the secreted peptide toxin SDP. UnlikesdpABC,yitPOMis induced in biofilms by the DegS-DegU two-component regulatory system. HighyitPOMexpression leads to the production of a secreted toxin called YIT. Expression ofyitQ, which lies upstream ofyitPOM, confers resistance to the YIT toxin, suggesting that YitQ is an anti-toxin protein for the YIT toxin. The alternative sigma factor SigW also contributes to YIT toxin resistance. In a mutant lackingyitQandsigW, the YIT toxin specifically inhibits biofilm formation, and the neutral protease NprB is required for this inhibition. The requirement for NprB is eliminated by Δepsand ΔbslAmutations, either of which impairs production of biofilm matrix polymers. Overexpression of biofilm matrix polymers prevents the action of the SDP toxin but not the YIT toxin. These results indicate that, unlike the SDP toxin and conventional antibiotics, the YIT toxin can pass through layers of biofilm matrix polymers to attack cells within biofilms with assistance from NprB. When the wild-type strain and the YIT-sensitive mutant were grown together on a solid medium, the wild-type strain formed biofilms that excluded the YIT-sensitive mutant. This observation suggests that the YIT toxin protectsB. subtilisbiofilms against competitors. We propose that some bacteria have evolved specialized antibiotics that can function within biofilms.Author SummaryBiofilms are multicellular aggregates of bacteria that are formed on various living and non-living surfaces. Biofilms often cause serious problems, including food contamination and infectious diseases. Since bacteria in biofilms exhibit increased tolerance or resistance to antimicrobials, new agents and treatments for combating biofilm-related problems are required. In this study, we demonstrated thatB. subtilisproduces a secreted peptide antibiotic called the YIT toxin and its resistant protein in biofilms. A mutant lacking the resistance gene was defective in biofilm formation. This effect resulted from the ability of the YIT toxin to pass through the biofilm defense barrier and to attack biofilm cells. Thus, unlike conventional antibiotics, the YIT toxin can penetrate biofilms and suppress the growth of YIT toxin-sensitive cells within biofilms. Some bacteria produce antibiotics in biofilms, some of which can alter the bacterial composition in the biofilms. Taking these observations into consideration, our findings suggest that some bacteria produce special antibiotics that are effective against bacteria in biofilms, and these antibiotics might serve as anti-biofilm agents.