AtCHIP, a U-box-containing E3 ubiquitin ligase, plays a critical role in temperature stress tolerance in Arabidopsis

Abstract

The Arabidopsis gene AtCHIP encodes a protein with three tetratricopeptide repeats and a U-box domain, which is structurally similar to the animal CHIP proteins, a new class of E3 ubiquitin ligases. Like animal CHIP proteins, AtCHIP has E3 ubiquitin ligase activity in vitro. AtCHIP is a single-copy gene, and its transcript is up-regulated by several stress conditions such as low and high temperatures. However, increased AtCHIP expression alone was not correlated with increased stress tolerance; in fact, overexpression of AtCHIP in Arabidopsis rendered plants more sensitive to both low- and high-temperature treatments. Higher electrolyte leakage was observed in leaves of AtCHIP overexpression plants after chilling temperature treatment, suggesting that membrane function is likely impaired in these plants under such a condition. These results indicate that AtCHIP plays an important role in plant cellular metabolism under temperature stress conditions.

Figure 1.

Sequence comparison of AtCHIP with animal CHIP proteins. The sequences of fruitfly CHIP (dCHIP), human CHIP (hCHIP), and mouse CHIP (mCHIP) were from Ballinger et al. (1999). The three lines with arrows underline TPRs, and the fourth line underlines the U-box domain.

Figure 2.

In vitro experiment demonstrating that AtCHIP is an E3 ligase. Ub, Ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme.

Figure 3.

DNA-blot analysis of AtCHIP in Arabidopsis. B, BamHI; E, EcoRI; H, HindIII; X, XhoI. The HindIII digestion of λDNA was used as length markers (at left in kilobases).

Figure 4.

RNA-blot analysis on AtCHIP transcript. The genes used as probes are listed on the right side: GST6, encoding glutathione S-transferase 6; UBQ3, a polyubiquitin gene used as an RNA-loading control.

Figure 5.

RNA-blot analysis of wild-type plants and AtCHIP transgenic plants. The 18S rRNA gene was used as an RNA-loading control. WT, Wild type; lanes 1 to 15, 15 independent transgenic lines.

Figure 6.

Western-blot analysis of wild-type and AtCHIP transgenic plants. A, Wild-type plant; B to E, four independent AtCHIP high-expression lines (nos. 1, 2, 8, and 12 on the RNA blot shown in Fig. 5); F, AtCHIP low-expression line (no. 3 on the RNA blot). GapC, Cytosolic glyceraldehyde-3-phosphate-dehydrogenase as a loading control. The intensities of AtCHIP bands are normalized with the GapC band in each lane in densitometry analysis.

Figure 7.

Phenotypes of wild-type and AtCHIP transgenic plants after chilling temperature treatment. Eighteen-day-old Arabidopsis plants were treated with chilling temperature (12°C) for 42 d. Wild-type plants (B) continued to grow and flower, whereas the growth of AtCHIP high-expression plants (A and C, two independent transgenic lines) was greatly inhibited.

Figure 8.

Phenotypes of wild-type plants, unrelated transgenic plants, and AtCHIP transgenic plants after chilling temperature treatment. Eighteen-day-old Arabidopsis plants were treated with chilling temperature (7°C) for 32 d. Wild-type plants (A), AtCHIP low-expression plants (E), and unrelated transgenic plants (F) continued to grow, whereas AtCHIP high-expression plants (B–D, three independent lines) grew very little during this time.

Figure 9.

Phenotypes of wild-type and AtCHIP high-expression plants after high-temperature treatment. Eighteen-day-old Arabidopsis plants were moved to a growth chamber with a temperature cycle set at 34°C for 2 h and 24°C for 22 h each day for 24 d. The wild-type plant (left) continued to grow and produced normal amount of seeds, whereas the AtCHIP high-expression plant (right) grew but produced few seeds.

Figure 10.

Analysis of membrane damage of wild-type and AtCHIP transgenic plants after cold (7°C) treatment. WT, Wild type; A and B, AtCHIP high-expression lines; C, low-expression line. Data represent mean ± sd, n = 6.