Plant hormone cytokinin at the crossroads of stress priming and control of photosynthesis

To cope with biotic and abiotic stress conditions, land plants have evolved several levels of protection, including delicate defense mechanisms to respond to changes in the environment. The benefits of inducible defense responses can be further augmented by defense priming, which allows plants to respond to a mild stimulus faster and more robustly than plants in the naïve (non-primed) state. Priming provides a low-cost protection of agriculturally important plants in a relatively safe and effective manner. Many different organic and inorganic compounds have been successfully tested to induce resistance in plants. Among the plethora of commonly used physicochemical techniques, priming by plant growth regulators (phytohormones and their derivatives) appears to be a viable approach with a wide range of applications. While several classes of plant hormones have been exploited in agriculture with promising results, much less attention has been paid to cytokinin, a major plant hormone involved in many biological processes including the regulation of photosynthesis. Cytokinins have been long known to be involved in the regulation of chlorophyll metabolism, among other functions, and are responsible for delaying the onset of senescence. A comprehensive overview of the possible mechanisms of the cytokinin-primed defense or stress-related responses, especially those related to photosynthesis, should provide better insight into some of the less understood aspects of this important group of plant growth regulators

Role of Cytokinins in Senescence, Antioxidant Defence and Photosynthesis

Cytokinins modulate a number of important developmental processes, including the last phase of leaf development, known as senescence, which is associated with chlorophyll breakdown, photosynthetic apparatus disintegration and oxidative damage. There is ample evidence that cytokinins can slow down all these senescence-accompanying changes. Here, we review relationships between the various mechanisms of action of these regulatory molecules. We highlight their connection to photosynthesis, the pivotal process that generates assimilates, however may also lead to oxidative damage. Thus, we also focus on cytokinin induction of protective responses against oxidative damage. Activation of antioxidative enzymes in senescing tissues is described as well as changes in the levels of naturally occurring antioxidative compounds, such as phenolic acids and flavonoids, in plant explants. The main goal of this review is to show how the biological activities of cytokinins may be related to their chemical structure. New links between molecular aspects of natural cytokinins and their synthetic derivatives with antisenescent properties are described. Structural motifs in cytokinin molecules that may explain why these molecules play such a significant regulatory role are outlined

Naturally Occurring and Artificial N9-Cytokinin Conjugates: From Synthesis to Biological Activity and Back

Cytokinins and their sugar or non-sugar conjugates are very active growth-promoting factors in plants, although they occur at very low concentrations. These compounds have been identified in numerous plant species. This review predominantly focuses on 9-substituted adenine-based cytokinin conjugates, both artificial and endogenous, sugar and non-sugar, and their roles in plants. Acquired information about their biological activities, interconversions, and metabolism improves understanding of their mechanisms of action and functions in planta. Although a number of 9-substituted cytokinins occur endogenously, many have also been prepared in laboratories to facilitate the clarification of their physiological roles and the determination of their biological properties. Here, we chart advances in knowledge of 9-substituted cytokinin conjugates from their discovery to current understanding and reciprocal interactions between biological properties and associated structural motifs. Current organic chemistry enables preparation of derivatives with better biological properties, such as improved anti-senescence, strong cell division stimulation, shoot forming, or more persistent stress tolerance compared to endogenous or canonical cytokinins. Many artificial cytokinin conjugates stimulate higher mass production than naturally occurring cytokinins, improve rooting, or simply have high stability or bioavailability. Thus, knowledge of the biosynthesis, metabolism, and activity of 9-substituted cytokinins in various plant species extends the scope for exploiting both natural and artificially prepared cytokinins in plant biotechnology, tissue culture, and agriculture

Arabidopsis leaves

<i>2.4. Regulation of gene expression in senescent Arabidopsis leaves</i> <p> Using BAP and DMSO treatments as references, we performed genome-wide expression profiling of senescent <i>Arabidopsis</i> leaves treated with compounds <b>3</b> and <b>6</b> in order to better understand the regulation of senescence by ArCKs at the molecular level. Compounds <b>3</b> and <b>6</b> were selected because they exhibited the highest anti-senescence activity in the detached wheat leaf bioassay (Table 2). Expression changes were monitored in detached senescent <i>Arabidopsis</i> leaves after 48 h of incubation in darkness with a 10 µM solution of one of the tested compounds or with DMSO alone; a complete list of DE genes from the three treatments (i.e. treatment with <b>3</b>, <b>6</b> and BAP) is shown in the Supplementary Table S1. Transcriptome profiling was performed using a standardized procedure developed for the detached leaf assay, which has previously been used to investigate the genetic effects of treatment with Kin derivatives that exhibit anti-senescent activity (Mik et al., 2011). While these Kin derivatives were effective anti-senescence agents under both light and constant dark conditions, their senescence-delaying activity was greatest in darkness.</p> <p> Hierarchical clustering analysis of the resulting data sets revealed that the samples treated with specific cytokinins clustered together and exhibited low variability in their responses, as did the mock-treated wild type <i>Arabidopsis</i> leaf samples (Fig. 2A). Interestingly, the BAP-treated samples formed the most distinct group and had a gene expression profile that differed significantly from those for all of the other groups. In contrast, the gene expression profiles for the groups treated with <b>3</b> and <b>6</b> were quite similar. This is readily apparent in the heat map shown in Fig. 2B, which presents data for 8659 genes whose expression changed significantly after treatment with <b>3</b>, <b>6</b> or BAP (<i>P</i> value <b>Ḉ</b> 0.01 in at least one treatment). To limit the number of target genes, we adopted more stringent statistical criteria for identifying genes whose expression had changed significantly. Specifically, the data were RMA normalized and genes were required to have a signal ratio change log 2 of <i>À</i> 0.5 or <b>Ḉ</b> <i>—</i> 0.5 in addition to a <i>P</i> -value of <0.01; see the Methods section for details. In this way we defined a group of 1128 genes whose expression changed after treatment with <b>3</b> (of which 510 were upregulated and 618 downregulated), and 944 genes whose expression changed upon treatment with <b>6</b> (548 upregulated, 396 downregulated). These two groups overlapped extensively: there were 671 genes common to both (Fig. 2C; Table S2, Supplementary data). To better understand the molecular functions of these genes, we categorized the transcripts in both groups according to their GO terms (Ashburner et al., 2000) using categories such as ‘transcription factor activity’, ‘DNA or RNA binding’, and ‘protein binding’ (Fig. 2D). This analysis indicated that the affected genes were rather evenly distributed over the defined categories.</p> <p> We then examined the genes whose expression changed significantly in the three datasets (i.e. in senescent leaves treated with <b>3</b>, <b>6</b> or BAP) in more detail. This analysis revealed that the cytokinin derivatives had distinct modes of action to those observed for the parent free bases. In keeping with previous reports (Brandstatter and Kieber, 1998; Rashotte et al., 2003), many cytokinin-related genes were upregulated in the BAP-treated leaves (Fig. 3; Table S2, Supplementary data). Importantly, these included the cytokinin response regulators <i>ARR7</i>, <i>ARR9</i>, <i>ARR5</i>, <i>ARR6</i> and <i>ARR4</i>. Exposure to high concentrations of BAP also prompted the induction of several cytokinin dehydrogenase genes including <i>CKX1</i>, <i>CKX2</i>, <i>CKX3</i> and <i>CKX4</i>. Upregulation of response regulators, several <i>CKX</i> genes, and other cytokinin response genes was also observed in senescent leaves treated with <b>3</b> or <b>6</b>.</p> <p> It is interesting to compare our results to those of a recent meta-analysis of microarray data reported by various laboratories, which identified a core list of cytokinin response genes (Brenner et al., 2012). The results of both this meta-analysis and a search of the Genevestigator database (https://genevestigator.com/gv/) conducted by ourselves indicate that tZ treatment leads to rapid reprogramming of gene expression in <i>Arabidopsis</i>. Specifically, cytokinin response regulators such as <i>ARR15</i>, <i>ARR5</i>, <i>ARR16</i>, <i>ARR7</i>, <i>ARR4</i>, <i>ARR6</i> and <i>ARR9</i> were strongly upregulated in response to tZ treatment. Other cytokinin-responsive genes identified in the meta-analysis and database search include <i>CKX4</i> and <i>CKX5</i> (which code for two cytokinin dehydrogenase isoforms), <i>AHK4</i> and <i>AHK1</i>, <i>CRF5</i> or <i>CYP735A2</i>, and <i>CYP82F1</i>. This group of core cytokinin-responsive genes clearly overlaps extensively with the list of genes whose expression was altered significantly following treatment with <b>3</b>, <b>6</b>, or BAP (Fig. 3). It is also consistent with the cytokinin bioassay results presented in the preceding section and thus confirms that the halogenated aromatic cytokinin derivatives considered in this work are indeed active cytokinins whose signaling effects partially mirror those of BAP and tZ.</p> <p> Our analysis also revealed some genes that were only affected by treatment with <b>3</b> and <b>6</b>, most of which were directly or indirectly linked to photosynthesis. Some of these genes are listed in Fig. 3, in which the most significant hits are categorized according to their function in cytokinin signaling and metabolism or photosynthesis and related categories. Importantly, genes encoding components of the photosystem II light harvesting complex (LHCII), namely At 2g 05070, At 5g 54270, At 1g 44575, At 3g 01440, At 3g 55330 and At 2g 39470, were upregulated by treatment with <b>3</b> and <b>6</b>. In contrast, BAP treatment had mostly negligible effects on these genes. In addition, our <i>in silico</i> analyses using the Genevestigator database confirmed that these genes are probably not affected by tZ treatment in <i>Arabidopsis</i> seedlings: treatments with 1 µm tZ solution for 30 min, 1 h, or 3 h had almost no measurable effects on the expression of any photosystem II-related gene. As mentioned above, the different modes of action of <b>3</b>, <b>6</b> and BAP may not be directly attributable to differential activation of cytokinin response regulators because all three of these ligands upregulated most of the <i>ARR</i> genes to a similar degree. However, <i>ARR8</i> and <i>ARR15</i> were found to be less upregulated in samples treated with BAP than those treated with <b>3</b> or <b>6</b> (Fig. 3). This may be important because <i>ARR15</i> is a negative regulator of AHK4-mediated cytokinin signal transduction whose expression is particularly strong in roots (Kiba et al., 2003). Therefore, negative regulation of the cytokinin signaling machinery may diminish some of the negative effects associated with exogenous cytokinin treatment at higher concentrations. However, we cannot exclude the possibility that the tested cytokinin derivatives may activate multiple signaling pathway(s) simultaneously, some of which may be closely related to the cytokinin pathway. One of these may be the auxin signaling pathway: we found several auxin-related genes that were differentially regulated by treatment with <b>3</b> and <b>6,</b> including <i>PIN3</i> and <i>PIN5</i> (Fig. 3). Another example is downregulation of the auxin-responsive gene At 2g 45210, which codes for senescence-associated gene 201 (SAG201), a positive regulator of senescence.</p> <p> We also found a group of genes involved in chlorophyll degradation that were downregulated in response to treatment with <b>3</b> and <b>6</b> (Fig. 3; Table S2, Supplementary data). Several of these genes were previously described as leaf anti-senescent markers that respond to auxin, cytokinin and some other molecules that regulate leaf senescence (Li et al., 2012). This group included At 4g 13250, which codes for a protein that is involved in LHCII degradation in rice, non-yellow coloring protein 1 (NYC1) (Kusaba et al., 2007). It also included genes encoding other chlorophyll/LHCII catabolic reductases such as At 4g 22920, which codes for non-yellowing protein 1 (NYE1), and At 4g 11910, which codes for non-yellowing protein 2 (NYE2). The latter gene was also downregulated in response to BAP treatment together with At 3g 44880, which codes for Pheophorbide A oxygenase/accelerated cell death 1 (PAO/ACD1).</p> <p>In conclusion, we have collected evidence that selected cytokinin derivatives have similar signaling outputs to their parent free bases in general but also exhibit selectively modulated anti-senescence activity. This modulation is primarily due to upregulation of genes coding for the subunits of LHCII and LHCI and downregulation of genes that are responsible for LHCII and chlorophyll degradation.</p>Published as part of <i>Vylíčilová, Hana, Husičková, Alexandra, Spíchal, Lukáš, Srovnal, Josef, Doležal, Karel, Plíhal, Ondřej & Plíhalová, Lucie, 2016, C 2 - substituted aromatic cytokinin sugar conjugates delay the onset of senescence by maintaining the activity of the photosynthetic apparatus, pp. 22-33 in Phytochemistry 122</i> on pages 25-28, DOI: 10.1016/j.phytochem.2015.12.001, <a href="http://zenodo.org/record/10485389">http://zenodo.org/record/10485389</a&gt

Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER. Cytokinin receptors predominantly localize to the endoplasmic reticulum. Here, Kubiasova et al. use a cytokinin fluoroprobe to show that ER-localized cytokinin receptors can enter the secretory pathway, reach the plasma membrane and undergo vesicular recycling, suggesting multiple sites of cytokinin perception

Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy

Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum

Cytokinin receptors predominantly localize to the endoplasmic reticulum. Here, Kubiasová et al. use a cytokinin fluoroprobe to show that ER-localized cytokinin receptors can enter the secretory pathway, reach the plasma membrane and undergo vesicular recycling, suggesting multiple sites of cytokinin perception