Cytokinin oxidase/dehydrogenase (CKX) is a key enzyme responsible for the degradation of endogenous cytokinins. However, the origins and roles of CKX genes in angiosperm evolution remain unclear. Based on comprehensive bioinformatic and transgenic plant analyses, we demonstrate that the CKXs of land plants most likely originated from an ancient chlamydial endosymbiont during primary endosymbiosis. We refer to the CKXs retaining evolutionarily ancient characteristics as “ancient CKXs” and those that have expanded and functionally diverged in angiosperms as “non-ancient CKXs”. We show that the expression of some non-ancient CKXs is rapidly inducible within 15 min upon the dehydration of Arabidopsis, while the ancient CKX (AtCKX7) is not drought responsive. Tobacco plants overexpressing a non-ancient CKX display improved oxidative and drought tolerance and root growth. Previous mutant studies have shown that non-ancient CKXs regulate organ development, particularly that of flowers. Furthermore, ancient CKXs preferentially degrade cis-zeatin (cZ)-type cytokinins, while non-ancient CKXs preferentially target N⁶-(Δ²-isopentenyl) adenines (iPs) and trans-zeatins (tZs). Based on the results of this work, an accompanying study (Wang et al. 10.1038/s41438-019-0211-x) and previous studies, we hypothesize that non-ancient CKXs and their preferred substrates of iP/tZ-type cytokinins regulate angiosperm organ development and environmental stress responses, while ancient CKXs and their preferred substrates of cZs play a housekeeping role, which echoes the conclusions and hypothesis described in the accompanying report (Wang, X. et al. Evolution and roles of cytokinin genes in angiosperms 1: Doancient IPTs play housekeeping while non-ancient IPTs play regulatory roles? Hortic Res7, (2020). 10.1038/s41438-019-0211-x).

Isopentenyltransferase (IPT) genes, including those encoding ATP/ADP-IPTs and tRNA-IPTs, control the rate-limiting steps of the biosynthesis of N⁶-(Δ²-isopentenyl)adenine (iP)-type and trans-zeatin (tZ)-type cytokinins and cis-zeatin (cZ)-type cytokinins, respectively. However, the evolution and roles of these IPTs in angiosperms are not well understood. Here, we report comprehensive analyses of the origins, evolution, expression patterns, and possible roles of ATP/ADP-IPTs and tRNA-IPTs in angiosperms. We found that Class I and II tRNA-IPTs likely coexisted in the last common ancestor of eukaryotes, while ATP/ADP-IPTs likely originated from a Class II tRNA-IPT before the divergence of angiosperms. tRNA-IPTs are conservatively retained as 2–3 copies, but ATP/ADP-IPTs exhibit considerable expansion and diversification. Additionally, tRNA-IPTs are constitutively expressed throughout the plant, whereas the expression of ATP/ADP-IPTs is tissue-specific and rapidly downregulated by abiotic stresses. Furthermore, previous studies and our present study indicate that ATP/ADP-IPTs and their products, iPs/tZs, may regulate responses to environmental stresses and organ development in angiosperms. We therefore hypothesize that tRNA-IPTs and the associated cZs play a housekeeping role, whereas ATP/ADP-IPTs and the associated iP/tZ-type cytokinins play regulatory roles in organ development and stress responses in angiosperms, which echoes the conclusions and hypothesis presented in the accompanying study by Wang, X. et al Evolution and roles of cytokinin genes in angiosperms 2: Do ancient CKXs play housekeeping roles while non-ancient CKXs play regulatory roles? Hortic Res10.1038/s41438-020-0246-z.

Environmental stresses negatively affect the growth and development of plants. Several previous studies have elucidated the response mechanisms of plants to drought and heat applied separately; however, these two abiotic stresses often coincide in environmental conditions. The global climate change pattern has projected that combined drought and heat stresses will tend to increase in the near future. In this study, we down-regulated the expression of a cytokinin receptor gene SlHK2 using RNAi and investigated the role of this gene in regulating plant responses to individual drought, heat, and combined stresses (drought + heat) in tomato. Compared to the wild-type (WT), SlHK2 RNAi plants exhibited fewer stress symptoms in response to individual and combined stress treatments. The enhanced abiotic stress tolerance of SlHK2 RNAi plants can be associated with increased membrane stability, osmoprotectant accumulation, and antioxidant enzyme activities. Furthermore, photosynthesis machinery was also protected in SlHK2 RNAi plants. Collectively, our results show that down-regulation of the cytokinin receptor gene SlHK2, and consequently cytokinin signaling, can improve plant tolerance to drought, heat, and combined stress.

The development and ripening of fresh fruits is an important trait for agricultural production and fundamental research. Almost all plant hormones participate in this process. Strigolactones (SLs) are a new class of plant hormones that regulate plant organ development and stress tolerance, but little is known about their roles in fruit development. In this study, we identified SL biosynthetic and signaling genes in woodland strawberry, a typical non-climacteric fruit, and analyzed the expression patterns of these genes in different plant tissues and developing fruits. One D27, two MAX1, and one LBO gene were identified as involved in SL biosynthesis, and one D14, one D3, and two D53 genes as related to SL signaling. The proteins encoded by these genes had similar motifs as SL biosynthetic and signaling proteins in rice and Arabidopsis. The genes had different expression levels in the root, stem, leaf, and petiole of woodland strawberry. In addition, the expression of most SL biosynthetic genes was high in developing carpel, anther, and style, while that of SL signaling genes was high in carpel and style, but low in anther, suggesting active SL biosynthesis and signaling in the developing carpel and style. Notably, the expression of SL biosynthetic and signaling genes was significantly increased in the receptacle after pollination and decreased during receptacle development. Moreover, low or no expression of these genes was detected in ripening fruits. Our results suggest that SLs play a role in the early stages of woodland strawberry fruit development. Our findings provide insight into the function of SLs and will facilitate further study of the regulation by SLs of fresh fruit development.