Light Regulates the Cytokinin-Dependent Cold Stress Responses in Arabidopsis

Abstract

To elucidate the effect of light intensity on the cold response (5°C; 7 days) in Arabidopsis thaliana, we compared the following parameters under standard light (150 μmol m^-2 s^-1), low light (20 μmol m^-2 s^-1), and dark conditions: membrane damage, photosynthetic parameters, cytokinin oxidase/dehydrogenase (CKX) activity, phytohormone levels, and transcription of selected stress- and hormone-related genes and proteome. The impact of cytokinins (CKs), hormones directly interacting with the light signaling pathway, on cold responses was evaluated using transformants overexpressing CK biosynthetic gene isopentenyl transferase (DEX:IPT) or CK degradation gene HvCKX2 (DEX:CKX) under a dexamethasone-inducible promoter. In wild-type plants, cold treatment under light conditions caused down-regulation of CKs (in shoots) and auxin, while abscisic acid (ABA), jasmonates, and salicylic acid (SA) were up-regulated, especially under low light. Cold treatment in the dark strongly suppressed all phytohormones, except ABA. DEX:IPT plants showed enhanced stress tolerance associated with elevated CK and SA levels in shoots and auxin in apices. Contrarily, DEX:CKX plants had weaker stress tolerance accompanied by lowered levels of CKs and auxins. Nevertheless, cold substantially diminished the impact from the inserted genes. Cold stress in dark minimized differences among the genotypes. Cold treatments in light strongly up-regulated stress marker genes RD29A, especially in roots, and CBF1-3 in shoots. Under control conditions, their levels were higher in DEX:CKX plants, but after 7-day stress, DEX:IPT plants exhibited the highest transcription. Transcription of genes related to CK metabolism and signaling showed a tendency to re-establish, at least partially, CK homeostasis in both transformants. Up-regulation of strigolactone-related genes in apices and leaves indicated their role in suppressing shoot growth. The analysis of leaf proteome revealed over 20,000 peptides, representing 3,800 proteins and 2,212 protein families (data available via ProteomeXchange, identifier PXD020480). Cold stress induced proteins involved in ABA and jasmonate metabolism, antioxidant enzymes, and enzymes of flavonoid and glucosinolate biosynthesis. DEX:IPT plants up-regulated phospholipase D and MAP-kinase 4. Cold stress response at the proteome level was similar in all genotypes under optimal light intensity, differing significantly under low light. The data characterized the decisive effect of light-CK cross-talk in the regulation of cold stress responses.

Keywords: acclimation; cold stress; cytokinin; cytokinin oxidase/dehydrogenase; isopentenyl transferase; karrikin; light intensity; phytohormone.

FIGURE 1

Lipid peroxidation expressed as malondialdehyde (MDA) content in leaves of wild-type (WT) and transformants DEX:IPT and DEX:CKX. See Table 1 for the description of experimental conditions. Plants were either activated with dexamethasone (DEX; diluted in DMSO) or treated with DMSO only. Means ± SD are shown. Biological samples from three independent experiments were analyzed (n = 3). The differences between DEX and DMSO treatments within each experimental variant were evaluated by Student’s t test (significant differences at p < 0.05 are indicated with an asterisk). The comparison among all experimental variants within the DEX (lowercase letters) or DMSO (capital letters) treatments was evaluated by one-way ANOVA with Tukey’s post hoc test (p < 0.05).

FIGURE 2

The levels of (A) CK bases [trans-zeatin (tZ), cis-zeatin (cZ), dihydrozeatin (DZ), and N⁶-(Δ²-isopentenyl)adenine (iP)]; (B) CK ribosides [trans-zeatin riboside (tZR), cis-zeatin riboside (cZR), dihydrozeatin riboside (DZR), and N⁶-(Δ²-isopentenyl)adenosine (iPR)]; and (C) CK phosphates [trans-zeatin riboside monophosphate (tZRMP), cis-zeatin riboside monophosphate (cZRMP), dihydrozeatin riboside monophosphate (DZRMP), and N⁶-(Δ²-isopentenyl)adenosine monophosphate (iPRMP)] in apices, leaves, and roots of wild-type (WT) and transformants with high CK synthesis after activation by dexamethasone (DEX:IPT), and high CK degradation after activation by dexamethasone (DEX:CKX) exposed to cold treatments under different light conditions (see Table 1 for the description). The expression of the transgenes was activated by dexamethasone (DEX). The statistical analyses of the total CK bases/ribosides/phosphates content were performed among all experimental variants within each tissue by one-way ANOVA with Tukey’s post hoc test in four independent biological replicates (significant differences at p < 0.05, n = 4, are indicated by different letters). Means ± SD and detailed statistics of individual CKs are shown in Supplementary Table 2.

FIGURE 3

The relative levels of phytohormones in apices, leaves, and roots of WT, DEX:IPT, and DEX:CKX plants expressed as a log₂ change of the ratio between the genotype under individual treatments and the WT under control conditions within each tissue separately. The expression of the transgenes was activated by dexamethasone (DEX). See Table 1 for the description of experimental variants. ABA, abscisic acid; SA, salicylic acid; JA, jasmonic acid; GA19, gibberellin GA₁₉; IAA, auxin indole-3-acetic acid. The means ± SD and detailed statistics of hormones are shown in Supplementary Table 2.

FIGURE 4

The independent component analysis (ICA) of (A) hormonal and (B) transcriptomic data in apices, leaves, and roots of WT, DEX:IPT, and DEX:CKX plants exposed to cold stress under different light conditions (see Table 1 for the description of variants). All samples are shown as individual points (four biological replicates of each variant). Groups with high diversity are highlighted by circles. In the case of transcriptomic data, the inserted genes were excluded from the analysis.

FIGURE 5

The activity of cytokinin oxidase/dehydrogenase (CKX) enzyme in leaves of WT, DEX:IPT, and DEX:CKX plants exposed to cold treatments under different light conditions. Plants were activated by dexamethasone diluted in DMSO (DEX) or treated by pure DMSO. See Table 1 for the description of experimental variants and Figure 1 for statistics (n = 3).

FIGURE 6

The transcription changes of selected genes related to WT in control conditions within each tissue separately. Means ± SD and detailed statistics of gene transcription are shown in Supplementary Table 3. Gray squares indicate the transcription level to be below the detection limit. The expression of the transgenes was activated by dexamethasone (DEX).

FIGURE 7

The independent component analysis (ICA) of proteome profiles in leaves of WT, DEX:IPT, and DEX:CKX plants. (A) Cold stress response at standard light intensity (C-NL) masks the effect of altered cytokinin pool. Results represent the quantitative analysis of 2,067 proteins in four replicates. (B) The response to cold stress under low light (C-LL) and dark (C-D) conditions in comparison with plants grown under control conditions. The ICA separates light intensity and cytokinin effects in the IC1 and IC2 axes, respectively. The results represent the quantitative analysis of 1,950 proteins in four replicates.

FIGURE 8

Modulation of cold-responsive proteins in plants with an altered cytokinin pool. Interactions and functional clusters of identified cold-responsive proteins (compared to control, 20°C, 150 μmol m^–2 s^–1; p < 0.05) were highlighted by String (Szklarczyk et al., 2019). The line thickness indicates the strength of data support, and the minimum required interaction score is 0.4 (medium confidence). See proteomic data in ProteomeXchange for details.