Functional analysis of tomato CHIP ubiquitin E3 ligase in heat tolerance

Abstract

Plants have evolved genetic and physiological mechanisms to mitigate the adverse effects of high temperature. CARBOXYL TERMINUS OF THE HSC70-INTERACTING PROTEINS (CHIP) is a conserved chaperone-dependent ubiquitin E3 ligase that targets misfolded proteins. Here, we report functional analysis of the SlCHIP gene from tomato (Solanum lycopersicum) in heat tolerance. SlCHIP encodes a CHIP protein with three tandem tetracopeptide repeat (TPR) motifs and a C-terminal U box domain. Phylogenetic analysis of CHIP homologs from animals, spore-bearing and seed plants revealed a tree topology similar to the evolutionary tree of the organisms. Expression of SlCHIP was induced under high temperature and was also responsive to plant stress hormones. Silencing of SlCHIP in tomato reduced heat tolerance based on increased heat stress symptoms, reduced photosynthetic activity, elevated electrolyte leakage and accumulation of insoluble protein aggregates. The accumulated protein aggregates in SlCHIP-silenced plants were still highly ubiquitinated, suggesting involvement of other E3 ligases in ubiquitination. SlCHIP restored the heat tolerance of Arabidopsis chip mutant to the wild type levels. These results indicate that tomato SlCHIP plays a critical role in heat stress responses most likely by targeting degradation of misfolded proteins that are generated during heat stress.

Figure 1

Tomato SlCHIP and SlTPR28 genes and encoded proteins. (A) PCR products of SlTPR28 (lanes 1 and 2) and SlCHIP (lanes 3 and 4) coding sequences. (B) The TPR motifs and U-box domain in predicted SlCHIP and SlTPR28 proteins. (C) Alignment of protein sequences of AtCHIP, SlCHIP and SlTPR28. The three TPR motifs were underlined and the U-box domain was highlighted by a rectangular box.

Figure 2

Phylogenetic analysis of CHIP homologs from different species. Twenty CHIP homologs with both TPR motifs and U-box domain from 16 animals and plant species were used in the construction of the phylogenetic tree. The rooted tree was generated with MEGA X v10.0.5 and FigTree v1.3.1 using Neighbor-Joining method with a p-distance model (1000 replicates). Numbers on the tree branches represent bootstrap values.

Figure 3

Expression of SlCHIP and SlTPR28 in response to heat, SA, JA and ABA. Six-week-old tomato plants were treated at 45 °C (A) or sprayed with 100 μM MeJA (B), 20 μM SA (C) and 20 μM ABA (D) for the indicated amounts of time. At least 3 leaflets of the fourth leaves from 3 individual plants for each sample were collected at 0-, 3-, 6-, and 9-h after treatment for RNA isolation and RT-qPCR analysis. Error bars indicate SE (n = 3). The experiments were repeated three times with similar results.

Figure 4

Phenotypic analysis of SlCHIP- or SlTPR28-silenced plants after heat stress. (A) SlCHIP and SlTPR28 transcript levels in SlCHIP- and SlTPR28-silenced tomato lines. The terminal leaflets in the fifth leaves of 20 individual 6-week-old plants were collected for RNA extraction and RT-qPCR analysis. Means of transcript levels were calculated from 12 plants with > 80% silencing efficiency and shown with SE (n = 12). These plants were used in the subsequent assays of heat tolerance. (B) Heat stress symptoms of SlCHIP- or SlTPR28-silenced plants. Six-weeks old mock (pTRV2), SlCHIP-silenced (pTRV2-SlCHIP) and SlTPR28-silenced (pTRV2-SlTPR28) plants were placed in a 45 °C growth chamber for 9 h. The picture was taken after 9 h heat treatment. (C) Water soaking symptom on leaflets of the fourth leaves in SlCHIP-silenced plants. Experiments were repeated three times with similar results.

Figure 5

Effects of heat stress on electrolyte leakage and CO₂ assimilation rate in SlCHIP- and SlTPR28-silenced plants. (A) Electrolyte leakage. (B) CO₂ assimilation rates. Both electrolyte leakage and CO₂ assimilation rates of the terminal leaflets of the fifth leaves were determined immediately after 9 h at 22 or 45 °C heat treatment. Means and SE were calculated from average values determined from 12 plants per experiment for each type of plants. According to Tukey's multiple comparisons test (P = 0.01), means do not differ significantly if they are indicated with the same letter. Experiments were repeated three times with similar results.

Figure 6

Accumulation of insoluble protein aggregates in SlCHIP- and SlTPR28-silenced plants under heat stress. Leaf tissues from mock (pTRV2)-, SlCHIP-silenced (pTRV2-SlCHIP) and SlTPR28-silenced (pTRV2-SlTPR28) plants were collected at indicated hours at 45 °C for extraction of total, soluble and insoluble proteins as described in Materials and Methods. The starting homogenates as total proteins and the last pellets as insoluble proteins were determined and the percentages of insoluble proteins to total proteins were calculated. Error bars indicate SE (n = 12). According to Tukey's multiple comparisons test (P = 0.01), means do not differ significantly if they are indicated with the same letter. Experiments were repeated three times with similar results.

Figure 7

Accumulation of insoluble ubiquitinated proteins in SlCHIP- and SlTPR28-silenced plants under heat stress. Soluble and insoluble proteins were prepared using low-speed centrifugation from the same amount of total proteins prepared from leaflets of the fourth leaves in SlCHIP-silenced (lane 1), SlTPR28-silenced (lane 2) and mock (lane 3) plants. The proteins were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.

Figure 8

SlCHIP rescued the heat sensitive phenotype of Arabidopsis atchip mutant. (A) Phenotypic analysis of Arabidopsis wild type Col-0, Arabidopsis atchip mutant and transgenic SlCHIP/atchip plants after 9 h heat stress. Plants were kept in 45 °C. Pictures were taken before and after 9-h heat treatment. (B) Accumulation of insoluble protein aggregates. Leaf tissues from Col-0, atchip and transgenic SlCHIP/atchip plants were collected at indicated hours at 45 °C for extraction of total, soluble and insoluble proteins as described in Materials and Methods. The starting homogenates as total proteins and the last pellets as insoluble proteins were used to calculate the percentages of insoluble proteins to total proteins. Error bars indicate SE (n = 6). According to Tukey's multiple comparisons test (P = 0.01), means do not differ significantly if they are indicated with the same letter. The experiments were repeated for three times with similar results.