Expression profiles of genes involved in ethylene and ABA production and CK glycosylation and deglycosylation.

<p>Changes in expression were followed by qPCR in time intervals ranging from 30 minutes to 3 days in roots (<b>A</b>) and aerial part (<b>B</b>) of maize plantlets (6 to 8 days of development). <b>3MeOBAP</b> (black bars), <b>3MeOBA9THPP</b> (grey bars) and <b>3MeOBAP9G</b> (white bars) were applied in 1 μM concentration to the nutrient solution. All data are accomplished from three independent biological replicates run in at least two technical replicates. Genes for actin and elongation factor 1 were used as reference genes. Expression change due to control plants considered as statistically significant is indicated by asterisks (unpaired Student's <i>t</i> test with <i>P</i> ≤ 0.05). <b>ACS</b> – ethylene precursor synthase; <b>ACO</b> – ethylene release enzyme; <b>cZOGT</b> – cis-zeatin-<i>O</i>-glucosyltransferase; <b>bGLU</b> – <i>β</i>-glucosidase; <b>VP-14</b> – ABA biosynthetic gene. Graphs for <i>ACS</i> and <i>ACO</i> genes are arrayed from left to right side as abundance of particular gene decreases in given tissue.</p

Expression profiles of genes involved in the CK perception and signal transduction after exogenous CK treatments in maize.

<p>Changes in expression were followed by qPCR in time intervals ranging from 30 minutes to 3 days in roots (<b>A</b>) and aerial part (<b>B</b>) of maize plantlets (6 to 8 days of development). <b>3MeOBAP</b> (black bars), <b>3MeOBA9THPP</b> (grey bars) and <b>3MeOBAP9G</b> (white bars) were applied in 1 μM concentration to the nutrient solution. All data are accomplished from three independent biological replicates run in at least two technical replicates. Genes for actin and elongation factor 1 were used as reference genes. Expression change due to control plants considered as statistically significant is indicated by asterisks (unpaired Student's <i>t</i> test with <i>P</i> ≤ 0.05). <b>HK</b> – CK receptor; <b>RR</b> – CK response regulator type A. Graphs for <i>RR</i> genes are arrayed from left side of upper line to right side of the middle line as abundance of particular gene decreases in given tissue. Graphs for HK genes are ordered from left to right due to their abundance.</p

The effects of 3MeOBAP <i>N9</i>-modifications on length of the primary root (A) and the first emerged leaf (C) in 7-day-old maize seedlings and biomass production of the root system (B) and the aerial part (D).

<p>4-day-old maize plantlets were cultivated for 5 days in nutrient solution supplemented with CKs. Each value is an average ±SD of measurements of at least 120 seedlings grown in 12 independent hydroponic boxes. Solid line indicates measured parameters and dashed lines indicate SD of control plants. Asterisks indicate the significant difference (<i>P</i> ≤ 0.05) between untreated and treated plants according to Student's unpaired t-test.</p

Structure of 3MeOBAP and its novel <i>N9</i>-derivatives used in the study.

<p>Structure of 3MeOBAP and its novel <i>N9</i>-derivatives used in the study.</p

CKs content in maize roots after exogenous application of 1 μM 3MeOBAP and 3MeOBA9THPP compared to controls.

<p>CK quantitative analysis was performed by UPLC/MS 1 hour (<b>A</b>), 3 hours (<b>B</b>), 24 hours (<b>C</b>) and 72 hours (<b>D</b>) after treatment with 3MeOBAP (black bars), 3MeOBA9THPP (grey bars) and DMSO-treated plants (white bars). All values are derived from three biological replicates that were determined in at least two technical replicates. <b>3MeOBAPR</b> – 3-methoxy(-6-benzylamino)purine riboside; 3OHBAP – 3-hydroxy(-6-benzylamino)purine; <b>3OHBAPR</b> – 3-hydroxy(-6-benzylamino)purine riboside; <b>3OHBAP9G</b> – 3-hydroxy(-6-benzylamino-9-glucosyl)purine; <b>(OG)3OHBAP</b> – 3-<i>O</i>-glucosyl-6-benzylaminopurine; <b>unknown</b> – 3MeOBA9THPP with additional hydroxyl group; <b>All tZ metabolites</b> – sum of <i>trans</i>-zeatin, its <i>N9</i>-riboside, <i>N9</i>-glucoside, 5′-monophosphate nucleotide, <i>O-</i>glucoside and <i>N9</i>-riboside-<i>O</i>-glucoside; <b>All cZ and DHZ metabolites</b> – sum of above listed <i>cis</i>-zeatin and dihydrozeatin derivatives, respectively; <b>All iP metabolites</b> – sum of 6<i>-</i>isopentenylaminopurine, its <i>N9</i>-riboside, <i>N9</i>-glucoside and 5′ -monophosphate nucleotide. Absolute concentration of unknown metabolite can be misinterpreted as deuterium labeled standard is not available.</p

CKs content in maize leaves after exogenous application of 1 μM 3MeOBAP and 3MeOBA9THPP compared to controls.

<p> Samples were processed in the same way as the samples from root tissue.</p

CK receptor studies.

<p><i>β</i>-galactosidase induction in <i>E. coli</i> strain Δ<i>rcsC, cps::lacZ</i>) upon incubation with CK derivatives. Strains harboring constructs of Arabidopsis receptor AHK3 and AHK4, or maize receptor ZmHK1 and ZmHK3a were activated by the selected compounds in a dose-dependent manner. The values shown are means with ±SD (n = 3).</p

Content of CK metabolites in xylem sap of maize seedlings treated with 1 μM 3MeOBAP a 1 μM 3MeOBA9THPP.

<p>CK quantitative analysis was carried out by UPLC/MS in approximately 30 μl of collected sap 0.5 to 1 h, 2 to 3 h or 16 h after CK application. All values are derived from two biological replicates that were determined in at least two technical replicates. Concentration of all other CKs in xylem exudates were under the detection limit of the method; u.d.l. – under detection limit.</p

Primary root length and number of lateral roots of Arabidopsis seedlings grown on media supplemented with 3MeOBAP derivatives.

<p>Root length was determined in 7-day-old (<b>A</b>) and 14-day-old seedlings (<b>B</b>), and the number of emerged lateral roots in 14-day-old seedlings (<b>C</b>). Each value is an average ±SD of measurements in at least 90 plantlets grown on 9 independent vertical dishes. Solid line indicates measured parameters and dashed lines indicate SD of control plants. Asterisks indicate the significant difference (<i>P</i> ≤ 0.05) between untreated and treated plants according to Student's unpaired t-test.</p

Expression profiles of genes involved in CK biosynthesis and activation after exogenous CK treatments in maize.

<p>Changes in expression were followed by qPCR in time intervals ranging from 30 minutes to 3 days in roots (<b>A</b>) and aerial part (<b>B</b>) of maize plantlets (6 to 8 days of development). <b>3MeOBAP</b> (black bars), <b>3MeOBA9THPP</b> (grey bars) and <b>3MeOBAP9G</b> (white bars) were applied in 1 μM concentration to the nutrient solution. All data are accomplished from three independent biological replicates run in at least two technical replicates. Genes for actin and elongation factor 1 were used as reference genes. Expression change due to control plants considered as statistically significant is indicated by asterisks (unpaired Student's <i>t</i> test with <i>P</i> ≤ 0.05). <b>IPT</b> – isopentenyl transferase (IPT1, 10 – tRNA::IPT, IPT5, 6, 8 – <i>de novo</i> IPT); <b>LOG</b> – CK nucleoside 5-monophosphate phosphoribohydrolase. Graphs for both groups of genes are arrayed from left to right side as abundance of particular gene decreases in given tissue.</p