Clinorotation Affects Induction of the Heat Shock Response in Arabidopsis thaliana Seedlings

Steady growing of plants as an autotrophic link of Life Support Systems is one of the key issues for long-term manned space missions (Ferl et al., 2002; Wheeler, 2010). Their ability to cope with spaceflight environments is thus of critical importance. It has been shown that microgravity, an unusual factor for plants, does not prevent their growth and development but causes changes in structural and functional organization of cells, and is considered as a mild stressor (Medina et al., 2011; Kordyum, 2014). However, its influence on the development of adaptive reaction to other unfavorable factors is not well understood. To study effects of reduced gravity in plants in ground-based experiments, we used clinorotation. This study is focused on how clinorotation might influence a conserved reaction of cells to elevated temperatures, known as the heat shock response. A key component of this response is upregulation of heat shock proteins (HSP) (Vierling, 1991; Sørensen et al., 2003; Wang et al., 2004). Functioning as molecular chaperones, they protect and reactivate protein structures affected by stress events.

It has been found that spaceflight factors, including microgravity, activate HSP expression in plant cells (Paul et al., 2005, 2012; Zupanska et al., 2013; Hausmann et al., 2014). In our previous work, some temporary increase in the HSP70 and HSP90 levels in pea seedlings for the first hours of clinorotation was determined (Kozeko and Kordyum, 2007). In addition, seedlings grown under permanent rotation showed a perceptible increase in HSP induction in response to high temperatures, when compared to seedlings grown motionlessly (Kozeko, 2008).

Numerous investigations have confirmed the importance of HSPs in plant tolerance to heat, as well as to a range of other stressful factors (Vierling, 1991; Sørensen et al., 2003; Wang et al., 2004). Moreover, their involvement in cross-protection between different types of stress has been examined (Banti et al., 2008; Montero-Barrientos et al., 2010).

In the present work, we have studied the influence of clinorotation on gene expression of HSP70 and HSP90 during the following heat shock treatment, as well as thermotolerance in Arabidopsis thaliana seedlings. HSP70s are considered as the principal stress-induced HSP family in many species (Sørensen et al., 2003). HSP90s also respond to damaging factors (Milioni and Hatzopoulos, 1997; Wang et al., 2004). In A. thaliana, there are five homologues of HSP70s (DnaK subfamily) and four homologues of HSP90s that are localized to the cytosol. Among these AtHSP70-4, AtHSP70-5, and AtHSP90-1 are highly induced by heat (Milioni and Hatzopoulos, 1997; Lin et al., 2001; Sung et al., 2001). Refer to Table 1 for gene names and AGI codes.

In order to test whether clinorotation influences the heat shock response, seedlings grown under stationary conditions (control) or clinostat rotation for 12 d were exposed to 37°C for 0.5–2 h. Levels of mRNAs of cytosolic HSP70s and HSP90s in seedlings were quantified using RT-qPCR. We used one set of primers for AtHSP90-2, AtHSP90-3, and AtHSP90-4, since their sequences are highly homologous and we could not synthesize specific primers for each gene. In addition, these HSP90s are known to show similar pattern of gene expression at both normal and high temperatures (Milioni and Hatzopoulos, 1997). Therefore, we measured the total mRNA level of these three genes to characterize gene expression of constitutive cytosolic HSP90s.

Figure 1 shows that exposure to 37°C resulted in rapid induction of HSP genes in control and rotated seedlings, but with differences in magnitudes of their expression. The mRNA level of constitutive AtHSP70-1 steadily increased at a low rate over the duration of heat exposure, and only a tendency to its enhancement after clinorotation was observed. Induction of constitutive AtHSP70-2, AtHSP70-3, as well as AtHSP90-2, AtHSP90-3, and AtHSP90-4 (in total) reached the maximum levels at 1 h of heat treatment, while thereafter the mRNA amounts declined. At the same time, a higher expression rate for AtHSP70-2 and AtHSP70-3 and, conversely, a tendency to decline for expression of HSP90s were determined in rotated seedlings, when compared to control ones. Inducible AtHSP70-4, AtHSP70-5, and AtHSP90-1 showed strong induction by 30 min of heat treatment, its significant increase within the next 30 min and a steady high level for the second hour. After clinorotation, the induction of these genes occurred faster and reached a higher level, especially for AtHSP70-4 and AtHSP70-5.

Figure 1

As illustrated in Figure 2, comparison of the transcript abundance in rotated and control seedlings at 22 ± 1°C (0 h of heat exposure) showed an increase of 2- to 7-fold under clinorotation, with the exception of constitutive HSP90s.

Figure 2

The heat tolerance of seedlings was evaluated as a function of their survival after an exposure to 45°C for 45 min. This temperature is specified as a standard to detect alterations in A. thaliana plant thermotolerance (Silva-Correia et al., 2014). Figure 3 depicts that different survival rates were observed in heat-treated seedlings grown under clinorotation compared with stationary conditions. A portion of rotated seedlings were able to generate new leaves over the 6-d post-exposure period, whereas control seedlings showed almost complete death. Similar results were obtained with 5-d-old seedlings as illustrated in Figure 4.

Figure 3

Figure 4

HSPs/chaperones play a role in protecting cells against multiple stressful conditions (Sørensen et al., 2003; Wang et al., 2004). Their upregulation during the heat shock response provides resumption of protein homeostasis and leads to a higher level of thermotolerance (Vierling, 1991). Moreover, an increasing number of studies demonstrate that HSPs contribute to cross-protection between heat stress and other types of stress in plants (Banti et al., 2008; Montero-Barrientos et al., 2010). In this study, we applied heat treatment following clinorotation of seedlings. As previously mentioned, both factors can activate HSP synthesis. The obtained results showed that clinorotation caused the minor increase in transcript abundance of five cytosolic AtHSP70s and AtHSP90-1 under normal temperature, as well as a faster onset and enhancement of their induction during heat shock. Notably, the strongest effect was revealed for inducible AtHSP70-4, AtHSP70-5, and AtHSP90-1, which are especially important for stress tolerance (Sørensen et al., 2003). In contrast, the total expression of constitutive AtHSP90-2, AtHSP90-3, and AtHSP90-4 was not altered under clinorotation and showed less induction by heat treatment in rotated seedlings, when compared to seedlings grown under stationary conditions.

A weak increase in the expression levels of several HSPs in seedlings grown under clinorotation at normal temperature likely reflects adaptation of cells to rotation. It is noteworthy that clinorotation may have multiple impacts on organisms, including not only simulation of the microgravity effects, but also mechanical and electromagnetic influences, etc. However, in our previous work with pea seedlings, comparison of the effects of horizontal and vertical rotation showed small but statistically significant differences in HSP70 and HSP90 levels that could be attributed to simulated microgravity (Kozeko and Kordyum, 2006).

As a whole, the time course of HSP expression during heat exposure was in good agreement with the previous findings by other researchers (Milioni and Hatzopoulos, 1997; Sung et al., 2001). At that, the magnitudes of AtHSP70s and AtHSP90-1 induction were higher in rotated seedlings relative to control ones. These findings correlate with our previous Western-blot data indicating enhanced induction of HSP70 and HSP90 in pea seedlings in response to high temperatures (38–45°C) after long-term clinorotation (Kozeko, 2008). Thus, increasing the heat shock response under the influence of clinorotation was shown at both transcriptional and translational levels. However, the obtained results could indicate two opposite situations. On the one hand, the higher inducibility of several HSPs may reflect higher sensitivity of rotated seedlings to heat shock; on the other hand, it may result from preadaptation of seedlings under clinorotation. To test that, the seedling survival assay was carried out. Its results showed that seedlings grown under clinorotation withstood exposure to the near lethal temperature better than seedlings grown under stationary conditions. These data support the assumption that clinorotation may provide cross-protection of plants against fluctuations in environmental conditions, which correlates with the data on HSP expression.