While cytokinin (CK) can delay natural leaf senescence, its effects on abiotic stress accelerated leaf senescence are less studied. Here we show N-conjugated trans-zeatin CK forms (tZ7G and tZ9G, or tZNGs) have the ability to delay salt stress senescence. Using a modified dark-induced senescence bioassay with Arabidopsis leaves, exogenous salt treatment accelerated leaf senescence as measured by lower photosystem II efficiency (Fv/Fm) and chlorophyll content. tZNGs were able to delay these parameters at concentrations as low as 10 nM similar to tZ, indicating that tZ7G and tZ9G can function in delaying salt accelerated senescence (SAS). To better understand physiological effects regulating tZNG delay of senescence, transcriptomics, proteomics, as well as CK measurements were examined. Salt treatment has strong transcriptome and proteome effects in accelerating senescence and reducing overall CK levels. Exogenous CK treatments could be quickly detected from changes seen in endogenous CK measurements, where each CK has a distinct profile contributing to transcript/protein alterations. Interestingly, transcriptomics show tZNGs are primarily responsive at later stages of salt senescence, in contrast to an immediate and continual response of tZ treatment. Known CK-regulated genes are induced by tZNGs and tZ, as corroborated by ARR:GUS reporter lines. Differences between tZNGs and tZ DEGs were revealed by WGCNA that included salt and CK specifically gene modules. In contrast, proteomic analysis revealed unique, but similar numbers of tZNG compared to tZ DAPs across senescence. GO term analysis of tZNG DEGs and DAPs showed enrichment of senescence, chloroplast, and CK signaling. Together this indicates tZNGs function as active CK forms in delaying salt accelerated leaf senescence.

Department of Molecular Biology and Radiobiology Faculty of AgriSciences Mendel University in Brno Zemědělská 1 CZ 61300 Brno Czech Republic

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany The Czech Academy of Sciences Šlechtitelů 27 CZ 77900 Olomouc Czech Republic

Rouse Life Sciences Department of Biological Sciences Auburn University Auburn AL 36849 USA

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