Stress-induced senescence is a global agro-economic problem. Cytokinins are considered to be key plant anti-senescence hormones, but despite this practical function their use in agriculture is limited because cytokinins also inhibit root growth and development. We explored new cytokinin analogs by synthesizing a series of 1,2,3-thiadiazol-5-yl urea derivatives. The most potent compound, 1-(2-methoxy-ethyl)-3-1,2,3-thiadiazol-5-yl urea (ASES - Anti-Senescence Substance), strongly inhibited dark-induced senescence in leaves of wheat (Triticum aestivum L.) and Arabidopsis thaliana. The inhibitory effect of ASES on wheat leaf senescence was, to the best of our knowledge, the strongest of any known natural or synthetic compound. In vivo, ASES also improved the salt tolerance of young wheat plants. Interestingly, ASES did not affect root development of wheat and Arabidopsis, and molecular and classical cytokinin bioassays demonstrated that ASES exhibits very low cytokinin activity. A proteomic analysis of the ASES-treated leaves further revealed that the senescence-induced degradation of photosystem II had been very effectively blocked. Taken together, our results including data from cytokinin content analysis demonstrate that ASES delays leaf senescence by mechanism(s) different from those of cytokinins and, more effectively. No such substance has yet been described in the literature, which makes ASES an interesting tool for research of photosynthesis regulation. Its simple synthesis and high efficiency predetermine ASES to become also a potent plant stress protectant in biotechnology and agricultural industries.

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Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a. In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light–adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.