Stimulation of ipt overexpression as a tool to elucidate the role of cytokinins in high temperature responses of Arabidopsis thaliana

Abstract

Cytokinins (CKs) are phytohormones regulating plant growth and development as well as response to the environment. In order to evaluate their function in heat stress (HS) responses, the effect of CK elevation was determined during three types of HS - targeted to shoots, targeted to roots and applied to the whole plant. The early (30min) and longer term (3h) responses were followed at the hormonal, transcriptomic and proteomic levels in Arabidopsis transformants with dexamethasone-inducible expression of the CK biosynthetic gene isopentenyltransferase (ipt) and the corresponding wild-type (Col-0). Combination of hormonal and phenotypic analyses showed transient up-regulation of the CK/abscisic acid ratio, which controls stomatal aperture, to be more pronounced in the transformant. HS responses of the root proteome and Rubisco-immunodepleted leaf proteome were followed using 2-D gel electrophoresis and MALDI-TOF/TOF. More than 100 HS-responsive proteins were detected, most of them being modulated by CK increase. Proteome and transcriptome analyses demonstrated that CKs have longer term positive effects on the stress-related proteins and transcripts, as well as on the photosynthesis-related ones. Transient accumulation of CKs and stimulation of their signal transduction in tissue(s) not exposed to HS indicate that they are involved in plant stress responses.

Keywords: Abscisic acid; Arabidopsis thaliana; cytokinin; heat stress; isopentenyltransferase; proteome..

Fig. 1.

(A) Time course of air and soil temperature profile during a summer day in nature. (B) Transcript levels of heat stress marker gene HSA32 in leaves and roots. (C) Leaf temperature of wild-type (Col 0), non-induced, and DEX-induced ipt transformant after prolonged HS exposure. HS (40 °C) treatment was applied to the shoots (S), whole plant (SR) or roots (R). HS duration was 180min. Unstressed organs were held at 20 °C. Transcript results represent mean values±SD from three independent biological replicates with three technical replicates for each. Leaf temperature data represent means±SD (n≥50). Statistical differences are indicated by letters.

Fig. 2.

The content of active cytokinins in leaves (A) and roots (B) after exposure to different HS treatments in wild-type (Col-0) and DEX-induced trasngenic plants. Data represent means±SD from two independent biological experiments with three replicates for each bar. tZ, trans-zeatin; tZR, trans-zeatin riboside; DZ, dihydrozeatin; DZR, dihydrozeatin riboside; iP, isopentenyladenine; iPR, isopentenyladenosine; cZ, cis-zeatin. S30, 30-min HS applied to shoots; S180, 180-min HS applied to shoots; SR30, 30-min HS applied to shoots and roots; SR180, 180-min HS applied to shoots and roots; R30, 30-min HS applied to roots; R180, 180-min HS applied to roots HS.

Fig. 3.

The content of abscisic acid in leaves (A) and roots (B) after exposure to different HS in wild-type (Col-0) and DEX-induced trasngenic plants. HS specification is as described in Fig. 2. Statistical significance of the data was evaluated by ANOVA and Duncan’s multiple range test.

Fig. 4.

Effect of heat stress targeting on expression profiles of 46 selected hormone- or stress-related genes. The heat map represents fold changes in the abundance of gene transcripts in leaves (A) and roots (B) after heat stress (40 °C) targeted to shoots (S), roots (R) and whole plant (SR) compared with the control. HS duration was 30 or 180min (as indicated). The genes are grouped according to their function (cytokinin, abscisic acid, photosynthesis and stress related).

Fig. 5.

(A) The number of proteins regulated by HS in leaf and root proteome. (B) Effects of activation of ipt system (cytokinin elevation) on HS-responsible proteins under control conditions (20 °C). (C) Number of up/down-regulated protein spots in leaves of wild-type (Col-0) and DEX-induced ipt transformant after different HS-treatments compared with mock samples. Number of up/down-regulated protein spots in roots of wild-type (Col-0) and DEX-induced ipt transformant after different HS-treatments (D). HS specification is as described in Fig. 2.

Fig. 6.

(A, B) Comparison of the effect of different heat stress treatments on HS-responsive proteins in wild-type (Col-0) and DEX-induced ipt transformant leaves (A) and roots (B). In total, 146 HS-responsive protein spots were detected. HS specification is as described in Fig. 2. The heat-maps are divided into three sections: on the left side are levels of proteins regulated by HS-S (Col-0 40 °C/Col-0 21 °C), in the middle are levels of proteins regulated only by activation of the ipt system (DEX ipt/mock Col-0), on the right side are the effects of ipt expression after exposure to different HSs compared with HS-treated wild-type plants (DEX 40 °C ipt/40 °C Col-0). (C, D) PCA analysis of abundance of differentially regulated proteins in each type of HS treatment in leaves (C) and roots (D) plotted on three principle components, providing 3D plots with visualisation of similarities between specific HS treatment in wild-type (grey circles, dots and letters) and transgenic plants (red circles, dots and letters). The dimensional intersections are marked as rings (PC1×PC2), triangles (PC1×PC3) and squares PC2×PC3.

Fig. 7.

The effect of different HS treatments on the number of up- and down-regulated proteins of different biological functions in wild-type leaves (A), transformant leaves (B), wild-type roots (C) and transformant roots (D). Significant change in relative protein abundance: P<0.05. The colour code represents the functional classification acquired from the TAIR database; the biological functions are categorized according to Bevan et al. (1998). HS specification is as described in Fig. 2.