Loss of Arabidopsis 5'-3' Exoribonuclease AtXRN4 Function Enhances Heat Stress Tolerance of Plants Subjected to Severe Heat Stress.

Plant and Cell Physiology Advance Access published July 1, 2015

Running title: AtXRN4 Functions In Heat Stress Response

Corresponding author: Dr. Motoaki Seki Plant Genomic Network Research Team RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan Tel: +81-45-503-9587; Fax: +81-45-503-9584; E-mail: [email protected]

(2) environmental and stress responses (3) regulation of gene expression

Number of Table: 1 Number of black and white figures: 0 Number of color figures: 7 Number of supplemental tables: 5 Number of supplemental figures: 3

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Loss of Arabidopsis 5’-3’ exoribonuclease AtXRN4 function enhances heat stress tolerance of plants subjected to severe heat stress Running title: AtXRN4 Functions In Heat Stress Response

Anh Hai Nguyen

1,2¶

1¶

1

1

1

, Akihiro Matsui , Maho Tanaka , Kayoko Mizunashi , Kentaro Nakaminami ,

Makoto Hayashi3, Kei Iida5, Tetsuro Toyoda6, Dong Van Nguyen2 and Motoaki Seki 1,4,7* 1

Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science,

2

National Key Laboratory for Plant Cell Technology, Agricultural Genetics Institute, Hanoi, Vietnam

3

Department of Bioscience, Nagahama Institute of Bioscience and Technology, Nagahama 526-0829,

Japan 4

Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan

5

Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

6

Integrated Database Unit, Advanced Center for Computing and Communication (ACCC), RIKEN, 2-1

Hirosawa, Wako, Saitama 351-0198, Japan 7

Core Research for Evolutional Science and Technology, Japan Science and Technology, 4-1-8

Honcho, Kawaguchi, Saitama 332-0012, Japan ¶

Both authors have contributed equally to this paper

Abbreviations: AtXRN4, Arabidopsis exoribonuclease 4: GO, gene ontology; RT, reverse transcription; qPCR, quantitative polymerase chain reaction; TMHT, thermotolerance to moderately high temperature; Footnotes:


Yokohama 230-0045, Japan

Abstract mRNA degradation plays an important role in the rapid and dynamic alteration of gene expression in response to environmental stimuli. Arabidopsis 5’-3’ exoribonuclease (AtXRN4), a homolog of yeast Xrn1p, functions after a de-capping step in the degradation of uncapped RNAs. While Xrn1pdependent degradation of mRNA is the main process of mRNA decay in yeast, information pertaining to the targets of XRN4-based degradation in plants is limited. In order to better understand the biological function of AtXRN4, the current study examined the survivability of atxrn4 mutants subjected to heat stress. Results indicated that atxrn4 mutants, compared to WT plants, exhibited an increased survival rate when subjected to a short-term severe heat stress. A microarray and mRNA decay assay

(HSFA2) and ethylene response factor 1 (ERF1) mRNA. The heat-stress tolerant phenotype of atxrn4 mutants was significantly reduced or lost by mutation of HSFA2, a known key regulator of heat acclimation; thus indicating that HSFA2 is a target gene of AtXRN4-mediated mRNA degradation under both non-stress conditions and during heat acclimation.

These results demonstrate that

AtXRN4-mediated mRNA degradation is linked to the suppression of heat acclimation.

Keywords: AtXRN4, Arabidopsis, heat stress tolerance, RNA degradation


showed that loss of AtXRN4 function caused a reduction in the degradation of heat shock factor A2

Introduction Heat stress is one of the major abiotic stresses that affect plant growth and productivity. As a sessile organism, plants have to adapt to the challenges caused by significant temperature alterations in their environment (Hirayama and Shinozaki 2010). Studies in yeasts and mammals revealed that heat stress alters transcriptional initiation and repression, as well as mRNA stability (Fan et al. 2002, Grigull et al. 2004, Castells-Roca et al. 2011). Plants, as sessile organisms, can rapidly change the composition and level of global mRNA in response to heat stress (Kotak et al. 2007, von KoskullDoring et al. 2007, Larkindale and Vierling 2008, Hirayama and Shinozaki 2010). The degradation of mRNA in plants and other organisms represents a process that contributes to the regulation of both

cellular homeostasis and the regulation of gene expression in response to environmental stresses (Shim and Karin 2002, Nakaminami et al. 2012). Studies on heat stress response showed that tolerance to a severe heat stress is increased by pre-exposure of plants to a moderate temperature (Lim et al. 2006, Charng et al. 2007, Larkindale and Vierling 2008). The process of heat acclimation enhances the accumulation of many heat-shock response-related genes, including heat shock factor A2 (HSFA2); suggesting an important role for this gene during heat acclimation in Arabidopsis (Charng et al. 2007). In fact, HSFA2 has been shown to be one of the key regulators of heat-stress response (Baniwal et al. 2004, Kotak et al. 2007). HSFA2 regulates ascorbate peroxidase 2 (APX2), galactinol synthase 1 (GolS1), small heat shock proteins (sHSPs), several members of the heat shock protein 70 (HSP70) and heat shock protein 101 (HSP101) families (Nishizawa et al. 2006, Schramm et al. 2006, Ogawa et al. 2007). A study of HSFA2 overexpression indicated a correlation between the level of HSFA2 expression and heat tolerance in Arabidopsis (Ogawa et al. 2007). Recent studies have revealed that mRNA degradation pathways are common in plants and other living organisms (yeast and mammal). Several mRNA degradation pathways have been reported, such as 3’-to-5’ decay by exosomes and 5’-to-3’ decay by exoribonuclease following decapping and endonucleolytic cleavage (Chiba and Green 2009). In the 5’-to-3’ decay pathway, an exoribonuclease 4 (XRN4), which is a homolog of yeast’s Xrn1p, functions in the degradation of 5’ to 3’ uncapped mRNAs (Kastenmayer and Green 2000, Souret et al. 2004). Arabidopsis has three AtXRN enzymes (AtXRN2, AtXRN3 and AtXRN4) and only AtXRN4 exhibits activity in the cytosol (Kastenmayer and


gene expression and post-transcriptional regulation of mRNA. It is required for the maintenance of

Green 2000). AtXRN4 is also known as ethylene insensitive 5 (EIN5), since mutation of this gene produces an ethylene insensitive phenotype (Olmedo et al. 2006, Potuschak et al. 2006). Functional studies on AtXRN4 under normal (non-stress) growth conditions have indicated that more than 100 transcripts are targeted by AtXRN4 (Souret et al. 2004, Gregory et al. 2008, Estavillo et al. 2011, Rymarquis et al. 2011, Merret et al. 2013). It has also been reported that AtXRN4 is involved in the response to drought and heat stress (Estavillo et al. 2011, Merret et al. 2013), however, the biological role of AtXRN4 during stress response is not well understood. This current study focuses on the regulation of mRNA stability in response to heat stress and the biological function of AtXRN4 in this process.

as compared to WT plants. Transcriptome analysis of atxrn4 mutants revealed that the transcripts of several heat-stress-responsive genes accumulated to a higher level in mutant plants grown under non-stressed conditions. Furthermore, we found that HSFA2 is a target of AtXRN4-mediated mRNA degradation under both non-stress conditions and conditions that promote heat acclimation. We also demonstrated that AtXRN4 functions in the suppression of heat acclimation.


In our study, atxrn4 mutants exhibited increased tolerance to a short-term severe heat stress,

Results Deficiency in AtXRN4 function enhances heat stress tolerance in plants subjected to a short-term severe heat stress Results indicated that atxrn4 mutants (atxrn4-3 and atxrn4-8; Fig. 1A) exhibited greater heat tolerance, relative to WT plants, when temperature was shifted rapidly from 22 ºC to 43.5 ºC (Fig. 1B, C). Approximately 30% of WT plants survived under 3 h 30 min heat-stress, and no WT plants survived under 4 h 30 min heat-stress treatment (Fig. 1B, C). In contrast, 3 h 30 min severe (43.5 ºC) heat stress treatment of atxrn4-3 mutants caused some plant injury (bleaching of leaves) but all

º

30 min, 20% of the mutant plants survived (Fig. 1B). Importantly, the severe (43.5 C) heat stress treatment condition used in the current study was different from the moderate (35 ºC) heat-stress treatment used in a previous study (Merret et al. 2013); where it was reported that atxrn4 mutants were impaired in “thermotolerance to moderately high temperature” (TMHT: exposure to 35 ºC for 7 º

days and then returned to 22 C for 8 days). The phenotype reported in the Merret et al. (2013) study was also confirmed in the current study (Supplementary Fig. S1). The key differences between the º

º

conditions used in the two studies were the temperature (43.5 C vs 35 C) and duration (2 h 30 min 4 h 30 min vs 7 days) of the heat treatment, suggesting that AtXRN4 function is different in TMHT conditions (Merret et al. 2013) and the conditions (short-term severe heat treatment) used in the present study. The heat stress tolerance phenotype of atxrn4 mutants subjected to a short-term severe heat treatment was confirmed by using atxrn4-8, another mutant of atxrn4 (Hayashi et al. 2012) (Fig. 1A). Although this mutant exhibited a weaker heat-stress tolerant phenotype compared to the atxrn4-3 mutant, it still had a higher survival rate than WT plants. Approximately 60% and 10% of the atxrn4-8 º

mutant plants survived under a severe heat stress treatment of 43.5 C for 3 h 30 min and 4 h 30 min, respectively (Fig. 1B, C). It is plausible that the observed difference in the heat tolerance of the atxrn43 and atxrn4-8 mutants (Fig. 1B) may be due to differences in the type of mutation. Whereas, atxrn4-3 is a T-DNA insertion null mutant of the AtXRN4 gene, atxrn4-8 harbors a point mutation. Therefore, it is possible that a partial function of AtXRN4 may still remain (Hayashi et al. 2012). The atxrn4-3 mutant was used in the subsequent experiments based upon the stronger heat tolerance phenotype that it exhibits.


mutant plants survived. When the duration of the severe heat stress treatment was increased to 4 h

Loss of AtXRN4 function increases plant cooling capacity during heat stress Heat stress conditions enhance the cooling capacity and water loss in Arabidopsis leaves (Crawford et al. 2012). A difference in leaf surface temperature between the atxrn4-3 mutant and WT plants was observed in the current study. Two week-old atxrn4-3 mutant and WT seedlings were incubated at 37 ºC for 1h. Thermal images were taken to determine the surface temperature of leaves (Fig. 2A). Leaf surface temperature of atxrn4-3 mutants was lower than WT at three different ranges of air temperature (Fig. 2A). Water content was also measured in the WT and atxrn4-3 mutants in order to determine rates of water loss. The data showed a slight

increase in the rate of water loss in the atxrn4-3 mutant º

outcomes of plant responses to moderate and severe heat stresses are opposite (Larkindale et al., 2005), these observations suggest that the loss of AtXRN4 function may enhance transpiration activity, and that atxrn4-3 mutants release heat via transpiration at faster rate than WT.

Mutation of AtXRN4 alters gene expression under non-stress conditions The heat stress tolerance phenotype of atxrn4-3 mutants (Fig. 1B, C) suggests that loss of AtXRN4 function may lead to changes in gene expression. Therefore, an Arabidopsis microarray was used to compare global transcript profiles in WT and atxrn4-3 mutant plants subjected to non-stress and heat-stress conditions. Two week-old seedlings of WT and the atxrn4-3 mutant were exposed to º

º

non-stress (22 C) or heat stress (37 C) temperatures for 1 h and subsequently sampled for transcriptome analysis. Microarray data indicated that mutation of AtXRN4 greatly affected the composition of the transcriptome even under non-stressed conditions. 6144/29755 of the analyzed transcripts were significantly up- or down-regulated in the atxrn4-3 mutant, relative to WT, under nonº

stress conditions (Fig. 3A). After 1 h exposure to 37 C, the ratio was reduced to 448/29755 (Fig. 3B). (Red dots indicate transcripts whose expression level was significantly changed – q-value < 0.1 and blue dots indicate transcripts whose expression level was not significantly changed – q-value > 0.1). Upregulated transcripts, with an expression ratio of more than 1.7-fold under either non-heat stress or heat stress conditions, were examined in the atxrn4-3 mutant in order to identify target genes of AtXRN4. A total of 757 genes were identified in the atxrn4-3 mutant whose mRNA accumulation was higher, relative to the WT, under non-stress conditions (Fig. 3C, Supplementary Table S1), and


º

compared to WT at both 37 C (Fig. 2B) and 43.5 C (Fig. 2C). Although it has been reported that the

118 genes whose mRNA accumulation was higher under heat stress conditions (Fig. 3C, Supplementary Table S2). A total of 5170 genes, whose mRNA accumulation was upregulated by heat stress, were identified in WT plants (Fig. 3C, Supplementary Table S3). GO analysis of 206 genes in the atxrn4-3 mutant whose expression was upregulated in either non-stress or severe heat stress conditions, as well as upregulated in WT plants in response to heat stress, indicated that 88 of the genes were classified in the “response to stress” functional category group (Supplementary Table S4) and 14 genes were classified in the “heat response” functional category group (Table 1). These fourteen genes were upregulated in the atxrn4-3 mutant, relative to the WT, only under the non-stress condition and not under heat stress. Among the 14 heat responsive genes (Table 1), the

(MBF1C), and HSFA2) has been shown to enhance heat stress tolerance in previous studies (Nishizawa et al. 2006, Suzuki et al. 2008, Li et al. 2011). Among the 205 genes that were upregulated in the atxrn4-3 mutant (non-stress condition) and upregulated by heat in WT, the identified ERF1 and WRKY transcription factor 40 (WRKY40) (Supplementary Table S4) have been shown to positively regulate heat tolerance in Arabidopsis and tobacco, respectively (Cheng et al. 2013, Dang et al. 2013). Heat stress-inducible expression of these five genes was examined by RT-qPCR (Fig. 4). Results demonstrated that the expression of these 5 genes is higher in the atxrn4-3 mutant as compared to WT under the non-stress condition. However, after a 1-h heat stress treatment, the differences in expression between WT and the atxrn4-3 mutant were greatly reduced or nearly identical (Fig. 4). A higher amount of transcript for these genes was also confirmed by RT-qPCR in the atxrn4-8 mutant under the non-stress condition (Supplementary Fig. S2). The higher level of expression of heat response genes in the atxrn4 mutant under the non-stress condition suggests that AtXRN4 plays a role, through the 5’-to-3’ RNA decay pathway, in fine-tuning a balance between the transcription and degradation of several heat response-related genes. Loss of AtXRN4 function may affect this balance, resulting in a higher accumulation of the examined ‘heat response’ transcripts. Enhanced accumulation of these heat-shock response genes may be responsible for the heat-stress tolerance phenotype of atxrn4 mutants. As described above, loss of AtXRN4 function also enhances transpiration when plants are subjected to a moderate heat stress (37ºC) (Fig. 2). These results suggest that AtXRN4 may be involved in the regulation of stomatal development- and/or stomatal movement-related genes.


overexpression of 3 genes (WRKY transcription factor 33 (WRKY33), multiprotein bridging factor 1C

Microarray data indicated that the expression of MYB domain protein 61 (MYB61), which encodes a positive regulator of stomatal closure in Arabidopsis (R2R3-MYB transcription factor) (Liang et al. 2005); and IQ-motif protein 1 (AtIQM1), a positive regulator of stomatal aperture (Zhou et al. 2012); was reduced more than 1.7-fold and induced 2.4-fold, respectively, in the atxrn4-3 mutant under the non-stress condition (Supplementary Table S5). Stomatal aperture and water content were reduced in the atiqm1 mutant (Zhou et al. 2012). These results suggest that AtXRN4 might also function in the regulation of transpiration-response-related genes, however, the mechanisms associated with this regulation are still unclear.

At the present time, the role of AtXRN4 for the regulation of abiotic-stress-related genes is still not entirely understood. Therefore, a RNA decay rate assay of HSFA2, ERF1 and At5g42190 (as a reference gene) was conducted using cordycepin, a transcriptional inhibitor (Fig. 5). Results indicated that the degradation rate of HSFA2 and ERF1 mRNA is faster in WT relative to atxrn4-3 mutant plants, whereas the RNA degradation rate of At5g42190, a stable transcript (half-life is more than 24 h) (Narsai et al. 2007), was similar in both the WT and atxrn4-3 mutant plants subjected to a 2 h cordycepin treatment (Fig. 5). These data indicate that HSFA2 and ERF1 mRNA are targets of AtXRN4-mediated degradation under non-stressed conditions.

AtXRN4 functions in suppressing heat acclimation Our results indicated that atxrn4-3 mutant plants have a higher level of heat tolerance than WT after both types of plants have been subjected to a heat acclimation treatment (Fig. 6A). Charng et al. (2007) previously reported that the expression of HSFA2 was induced by a heat acclimation treatment and that the increased level was maintained to some extent when plants were returned to a non-stress temperature (Charng et al. 2007). In the current study, RT-qPCR analysis of HSFA2 and its º

regulated target (HSP70) revealed that after 1 h of heat acclimation at 37 C, the expression level of these two genes was rapidly induced and no significant difference in expressions was observed º

between the WT and atxrn4-3 mutant. After returning plants to 22 C, expression levels of both HSFA2 and HSP70, however, decreased more slowly in the atxrn4-3 mutant as compared to WT (Fig. 6B). These data suggest that AtXRN4 not only functions in the degradation of transcripts of heat-stress


HSFA2 and ERF1 are targets of AtXRN4-mediated mRNA degradation

response-related genes under non-stress conditions but that it also plays a role in decreasing the expression of HSFA2 and HSP70 transcripts when plants are returned to non-stress temperatures. Thus, AtXRN4 functions in lowering heat acclimation by reducing heat-stress-related gene transcripts during the recovery process.

The atxrn4-3 hsfa2 double mutant exhibits greater heat-stress sensitivity Our results indicate that AtXRN4 targets HSFA2 for degradation and that the suppression of HSFA2 in atxrn4 mutants decreases the heat tolerance in the atxrn4 mutants subjected to a short-term severe heat stress treatment (Fig. 7A). To test this, heat stress tolerance experiments with and without

an atxrn4-3hsfa2 double mutant. After a heat stress treatment (44 ºC) for 3 h 30 min, 18%, 100%, 40%, and 18% survival rates were observed in WT, atxrn4-3 mutant, atxrn4-3 hsfa2 double mutant, and hsfa2 mutant plants, respectively. After a heat acclimation pre-treatment followed by a 4-h of a heat º

stress treatment (44 C), 66%, 100%, 15%, and 4% rates of survival were observed in WT, atxrn4-3 mutant, atxrn4-3 hsfa2 double mutant, and hsfa2 mutant plants, respectively (Fig. 7B). These results demonstrated that increased accumulation and maintenance of HSFA2 transcripts, resulting from the loss of AtXRN4 function, is essential for the heat stress tolerance phenotype of atxrn4 mutant plants. These results also reinforce our hypothesis that HSFA2 mRNA is directly targeted by AtXRN4 for degradation.


a heat acclimation pre-treatment were performed using WT, an atxrn4-3 mutant, an hsfa2 mutant, and

Discussion The heat-stress tolerant phenotype of atxrn4 mutants subjected to short-term severe heat stress (Fig.1) suggests that AtXRN4 may be involved in controlling transcript accumulation of heatstress-response genes. In our study, heat-stress tolerance-related genes, such as HSFA2 and HSPs, were identified whose transcripts were highly accumulated in the atxrn4 mutants subjected to nonstress conditions (Table 1, Fig. 4, 6B, Supplementary Fig. S2). Stomatal-related genes, such as MYB61 and AtiQM1/EDA39, were also found to be downregulated and upregulated, respectively, in the atxrn4-3 mutant (Supplementary Table S5). Overexpression of several genes (HSFA2, ERF1,

previously reported to increase heat stress tolerance (Nishizawa et al. 2006, Suzuki et al. 2008, Li et al. 2011, Cheng et al. 2013, Dang et al. 2013). It is possible that the increased accumulation of these genes in atxrn4 mutants may contribute to the observed heat-stress tolerant phenotype. The microarray and RNA decay analyses indicate that HSFA2, a key regulator of the process of heat acclimation, is a direct target of AtXRN4 (Fig. 4, 5). The heat stress tolerance phenotype of atxrn4 mutants was significantly reduced or lost by mutation of HSFA2 (Fig. 7). These results support the premise that HSFA2 mRNA is a target of AtXRN4-mediated degradation in both non-stress and heat acclimation temperature conditions.

In addition, these data indicate that the AtXRN4-mediated

degradation of HSFA2 mRNA is a key process controlling the level of heat stress tolerance in Arabidopsis in response to a short-term severe heat stress. At normal (non-stress) temperature conditions, the transcriptional level of HSFA2 is very low. No differences were observed in the survival rate of WT and hsfa2 mutant plants subjected to a severe heat-stress treatment (Fig. 7A). Loss of AtXRN4 function in atxrn4 mutant plants resulted in an increased level of HSFA2 mRNA (2.2-fold) (Table 1) in non-stressed plants. However, this transcript level was still far below the level of induction that was stimulated by heat stress (more than 3,000-fold) (Table 1, Fig. 4). The fact that the atxrn4 mutants exhibited a greater level of heat stress tolerance, relative to WT plants, in response to a short-term severe heat stress treatment; and that the heat stress tolerant phenotype was lost by the inclusion of a second hsfa2 mutation, suggests that the small increase in the level of HSFA2 mRNA in the atxrn4 mutant plays an important role in the increased heat tolerance to a short-term severe heat stress. Furthermore, these findings suggest that


WRKY33, WRKY40 and MBF1C) that were found to be upregulated in the atxrn4-3 mutant have been

the balance between the accumulation and degradation of HSFA2 transcripts in WT plant is regulated by AtXRN4. The results obtained by expression profiling, mRNA decay analysis, and RT-qPCR under nonstress conditions indicates that AtXRN4 directly targets HSFA2 transcripts for degradation (Table 1, Fig. 4, 5). Our data also indicates that AtXRN4 regulates the mRNA expression of HSFA2 downstream genes such as HSP101 and HSP70. This conclusion was supported by the observation that the expression of HSP101 and HSP70 genes was 1.4 and 2.7 fold greater, respectively, in the atxrn4-3 mutant as compared to WT (Table 1, Supplementary Table S1). We also found that AtXRN4 functions in the degradation of HSFA2 transcripts after plants have been heat acclimated and then returned to

prolonging heat acclimation (Charng et al. 2007). In that study, hsfa2 mutant plants were found to lose their ability to acclimate to heat stress. A moderate heat stress resulted in a rapid accumulation of HSFA2 transcripts and a gradual decrease in HSFA2 level when plants were returned to normal growth temperatures; suggesting that a mRNA decay mechanism might be involved (Charng et al. 2007). In our study, transcript levels of both HSFA2 and HSP70 (a gene regulated by HSFA2) also increased substantially in both WT and atxrn4-3 mutants after plants were exposed to heat acclimation conditions. However, after plants were returned to normal growth temperatures, transcript levels of these genes decreased more slowly in the atxrn4-3 mutants relative to WT plants (Fig. 6B). Collectively, our results suggest that AtXRN4 degrades HSFA2 transcripts under non-stress conditions and when plants are returned to normal temperatures after being exposed to heat-acclimation temperatures. Additionally, these support the hypothesis that AtXRN4 plays a role in controlling the balance between transcription and the degradation of HSFA2 mRNA. The 2.2-fold increase in the accumulation of HSFA2 mRNA under non-stress conditions may be important for heat acclimation in Arabidopsis. Our data indicated that in addition to HSFA2, ERF1 is also a target of AtXRN4 and plays a role in the upregulation of other heat-shock-response genes (Table 1, Fig. 4, Supplementary Fig. S2) in atxrn4 mutants. As a result, it is plausible that these other genes may also be potential targets of AtXRN4 for degradation. Precisely how AtXRN4 regulates the transcript accumulation of these genes and how the increased transcript abundance of these genes contributes to the observed heat stress tolerant phenotype of atxrn4 mutants is still unclear. A heat tolerance assay (44 ºC with and without pre-exposure to heat acclimation) revealed that the atxrn4-3 hsfa2 double mutant is as or


normal growth temperatures (Fig. 6B). HSFA2 has been previously reported to be required for

more heat stress-sensitive than WT (survival rate of the atxrn4-3 hsfa2 double mutant and hsfa2 mutant are almost similar). These data suggest that the heat stress tolerant phenotype of atxrn4 mutants is mainly due to increased accumulation of HSFA2 transcripts. Although atxrn4 mutants exhibited an increased survival rate when subjected to a short-term severe heat stress (Fig. 1), the atxrn4 mutants were impaired in TMHT. The expression level of the HSFA2 transcript was not significantly different between atxrn4-3 and WT in the TMHT condition (35ºC for 5 days; Supplementary Fig. S3), which is similar to the heat stress treatment (37ºC for 1 h; Fig. 4). These results suggest that the heat stress-sensitive phenotype of AtXRN4 mutants in the TMHT condition might be due to the changes in an alternative target gene of AtXRN4.

the balance between the and degradation of HSFA2 mRNA; a key regulator of heat stress response. AtXRN4 regulates the expression level of HSFA2 transcripts at non-stress temperatures at a low level and also plays a role in decreasing heat tolerance after plants have been heat acclimated and then returned

to

non-stress

temperatures.


Collectively, our data indicate that an important biological function of AtXRN4 is to maintain

Materials and Methods Plant material and growth conditions To identify the function of AtXRN4, we used two atxrn4 mutant lines; atxrn4-3 (SALK_014209) and atxrn4-8, (Hayashi et al. 2012). Arabidopsis thaliana (L.) Heynh. Ecotype (Col-0) was used as the wild-type control in all of the comparisons of heat stress tolerance and gene expression. The hsfa2 (SALK_008979) line was used to confirm the heat response phenotype of the hsfa2 mutant Arabidopsis plants were grown in Kord/Valmark petri dishes (100 x 25 mm) containing Murashige and Skoog (MS, Nihon Seiyaku, Tokyo, Japan) medium, 0.7%(w:v) agar (Nacalai Tesque,

-2

-1

conditions consisted of a 16-h-light/8-h-dark, at 40-80 µmol photons m sec . The light period was supplied from 5:00 to 21:00. All seeds were maintained at 4 ºC for three days before sowing onto MS plates. Plants were grown for 2 - 3 weeks prior to any treatment. Heat treatments º

Two-week-old plants of both WT and atxrn4 mutants were exposed to a 43.5 C heat stress treatment (Start time: 10:30, that is, 5.5 h after starting of light condition) for a designated time period (2 h to 4.5 h) (Sanyo incubator MIR-153). Petri plates containing the plants were on elevated trays prior to placing them into the incubator in order to ensure that the heat stress was derived from the air temperature and not due to contact of the plate with the incubator surface. This process was used for all of heat treatments. Petri dishes (samples) were removed every 30 min and kept at normal growth conditions for one week. Survival rate was based on the percentage of surviving plants. Pre-exposure of plants to heat acclimation conditions was accomplished by placing two-weekº

old plants of WT, atxrn4-3, hsfa2 mutants and atxrn4-3 hsfa2 double mutant at 37 C for 1 h, and then transferring them to 22 ºC for 2 days. Plants were then subjected to 44 ºC for 3 h to 5 h 30 min. After exposure to the heat treatment, they were transferred to normal growth temperatures for 1 week. Survival rate was based on the percentage of the surviving plants. Thermal imaging and water loss assay Thermal imaging - Two week-old plants of WT and atxrn4-3 mutant were exposed to a º

moderate heat stress (37 C) for 1 h and leaf temperature was monitored using a thermal imaging camera (Avio: Neo Thermo TVS-700) at three different temperature ranges: 30.7 - 35.8 ºC, 31.6 - 36.6


inc., Kyoto, Japan), and 1% (w:v) sucrose, at pH 5.7, as described previously (Oono et al. 2003). Light

º

C, and 37 - 41.9 ºC.

Water loss assay - WT and atxrn4-3 mutant plants were grown for two weeks on MS medium followed by one week in soil conditions. Leaf samples were removed and exposed to heat stress (37 º

C and 43.5 ºC; 27.5% RH was monitored using a Highest I hygrometer, Sato Keiryoky MFG. Co., Ltd).

Fresh weight was measured every 5 min for a total of 30 min to analyze the water content in plants. Four biological replicates were used. RNA isolation for cDNA synthesis

º

were collected, frozen in liquid nitrogen, and kept at -80 C. mRNA was isolated using Invitrogen’s ConcertTM Plant RNA reagent according to the “small scale mRNA isolation” protocol provided by the manufacturer. Total mRNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). The integrity of mRNA was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Total mRNA was treated with DNase I (Invitrogen) according to the manufacturer’s protocol in TM

order to eliminate residual DNA. First strand cDNA was synthesized using a SuperScript

III Reverse

Transcriptase kit with a Random hexamer (Takara) and 1 µg mRNA of each sample. Primer design and RT-qPCR Real-time

PCR

primers

were

designed

(http://biotools.umassmed.edu/bioapps/primer3_www.cgi).

using Primer

the

Primer3

sequence

online

website

specificities

were

confirmed for each gene by using the TAIR resource (http://arabidopsis.org). Real-time PCR was ®

performed on a StepOnePlus Real-time PCR system with Fast SYBR Green Master Mix (Applied Biosystems, Foster, USA). For a 10 µl reaction mixture, 0.4 µl of each primer (10 pmole µl-1) and 1 µl (25 ng) total mRNA were added to 5 µl of SYBR master mix. After the addition of these components, 3.2 µl of nuclease free-deionized water was added to the reaction mixture. The following gene-specific primers

were

used:

ERF1,

5’-AGGATGGTTGTTCTCCGGTT-3’

and

5’-

AGACCCCAAAAGCTCCTCAA-3’;

WRKY40;

5’-TCCCAAGAAACGCAAATCCC-3’

and

5’-

ACGAGGGTAGTGTCAGAAGC-3’,

WRKY33,

5’-GTGGTGGAAGCAAGACAGTG-3’

and

5’-

ACTGCTCTCATGTCGTGTGA-3’;

MBF1C,

5’-ACCCAGGAGCAGTAACACAA-3’

and

5’-

CTCACCTCTGCTTTCACACG-3’; HSFA2, 5’-AGCTCAATACTTATGGATTCAGAAAGA-3’ and 5’-


Two week-old WT and atxrn4-3 plants were incubated at 37 ºC for 1 h. Samples (whole plants)

ATTCTGCAAACCCATGTTCC-3’;

18SrRNA,

5’-

CGTTAACGAACGAGACCTCA-3’

and

5’-

CCCAGAACATCTAAGGGCAT-3’. A standard curve using copy number was used for the quantification of Real-time PCR analysis and At18SrRNA was used as a reference gene for determining relative expression levels. At least three biological replicates were used in assaying the transcript levels of each gene. Microarray analysis Two week-old WT and atxrn4-3 mutant plants were subjected to a moderate heat stress (37 º

º

C) and non-stress (22 C) for 1 h. Individual Agilent Arabidopsis custom microarrays (GEO array

protocol. In the custom microarray, the probes were designed based on expressed genes and transcripts of the TAIR 10 genome using previous tiling-array and RNA-seq analyses (Matsui et al. 2008, Okamoto et al. 2010, Kawaguchi et al. 2012). cRNA labeling was performed using a Low Input Quick Amp Labeling kit. The microarrays were scanned with an Agilent DNA Microarray Scanner (G2539A ver.C). Raw signals less than 0.01 were adjusted to 0.01 and a 75 percentile normalization was performed for each chip according to Agilent’s data analysis protocol. The microarray data are available on the GEO web site (GEO ID: GSE66369). Genes with an expression ratio higher than 1.7 fold (t-test analysis; q-value: 0.1) were identified as upregulated genes. Analysis of mRNA decay profiles Two week-old atxrn4-3 mutant and WT plants were transferred from MS medium to water and º

kept for one day at 22 C to avoid the effect of water stress on transcription levels. Plants were then transferred to a solution containing 0.6 mM cordycepin and kept for 0 - 2 h at room temperature. For the HSFA2 decay assay, WT and mutant plants were transferred to water containing 0.01 % Triton X-100, kept for 1 day at 22 ºC and then transferred to a solution containing 0.6 mM º

cordycepin for 0 h - 2 h at 22 C. Samples were collected every 30 min, frozen in liquid nitrogen, and kept at -80 ºC for RNA isolation. Decay rates were calculated based on the log2 value of the relative expression of target transcripts. Three biological used were used for each determination.


platform: GPL19830) were used for three biological replicates, according to Agilent’s preferred

Funding information This project was supported by grants from RIKEN and the Japan Science and Technology Agency (JST), Core Research for Evolutionary Science and Technology (CREST) to M.S

Disclosures: The authors report no conflicts of interest.

Acknowledgments

mutant seeds (SALK_014209). We also thank Dr. Yukako Chiba for providing valuable advice during the course of this study.


We would like to thank The Arabidopsis Biological Resource Center for providing Arabidopsis

References Baniwal, S.K., Bharti, K., Chan, K.Y., Fauth, M., Ganguli, A., Kotak, S., et al. (2004) Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors. J. Biosci. 29: 471-487. Castells-Roca, L., Garcia-Martinez, J., Moreno, J., Herrero, E., Belli, G. and Perez-Ortin, J.E. (2011) Heat shock response in yeast involves changes in both transcription rates and mRNA stabilities. PLoS One 6: e17272. Charng, Y.Y., Liu, H.C., Liu, N.Y., Chi, W.T., Wang, C.N., Chang, S.H., et al. (2007) A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis.

Cheng, M.C., Liao, P.M., Kuo, W.W. and Lin, T.P. (2013) The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals. Plant Physiol. 162: 1566-1582. Chiba, Y. and Green, P.J. (2009) mRNA Degradation Machinery in Plants. J. Plant Biol. 52: 114-124. Crawford, A.J., McLachlan, D.H., Hetherington, A.M. and Franklin, K.A. (2012) High temperature exposure increases plant cooling capacity. Curr. Biol. 22: R396-397. Dang, F.F., Wang, Y.N., Yu, L., Eulgem, T., Lai, Y., Liu, Z.Q., et al. (2013) CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant Cell Environ. 36: 757-774. Estavillo, G.M., Crisp, P.A., Pornsiriwong, W., Wirtz, M., Collinge, D., Carrie, C., et al. (2011) Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis. Plant Cell 23: 3992-4012. Fan, J., Yang, X., Wang, W., Wood, W.H., 3rd, Becker, K.G. and Gorospe, M. (2002) Global analysis of stress-regulated mRNA turnover by using cDNA arrays. Proc. Natl Acad. Sci. USA 99: 1061110616. Gregory, B.D., O'Malley, R.C., Lister, R., Urich, M.A., Tonti-Filippini, J., Chen, H., et al. (2008) A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14: 854-866. Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M.D. and Hughes, T.R. (2004) Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol. Cell. Biol. 24: 5534-5547.


Plant Physiol. 143: 251-262.

Hayashi, M., Nanba, C., Saito, M., Kondo, M., Takeda, A., Watanabe, Y., et al. (2012) Loss of XRN4 Function Can Trigger Cosuppression in a Sequence-Dependent Manner. Plant Cell Physiol. 53: 1310-1321. Hirayama, T. and Shinozaki, K. (2010) Research on plant abiotic stress responses in the post-genome era: past, present and future. Plant J. 61: 1041-1052. Kastenmayer, J.P. and Green, P.J. (2000) Novel features of the XRN-family in Arabidopsis: evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm. Proc. Natl Acad. Sci. USA 97: 13985-13990. Kawaguchi, S., Iida, K., Harada, E., Hanada, K., Matsui, A., Okamoto, M., et al. (2012) Positional

noisy array signals or short reads. Bioinformatics 28: 929-937. Kotak, S., Larkindale, J., Lee, U., von Koskull-Doring, P., Vierling, E. and Scharf, K.D. (2007) Complexity of the heat stress response in plants. Curr. Opin. Plant Biol. 10: 310-316. Larkindale J., Hall J. D., Knight M. R., and Vierling, E. (2005) Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol. 38: 882-897. Larkindale, J. and Vierling, E. (2008) Core genome responses involved in acclimation to high temperature. Plant Physiol. 146: 748-761. Li, S., Fu, Q., Chen, L., Huang, W. and Yu, D. (2011) Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta 233: 1237-1252. Liang, Y.K., Dubos, C., Dodd, I.C., Holroyd, G.H., Hetherington, A.M. and Campbell, M.M. (2005) AtMYB61, an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana. Curr. Biol. 15: 1201-1206. Lim, C.J., Yang, K.A., Hong, J.K., Choi, J.S., Yun, D.J., Hong, J.C., et al. (2006) Gene expression profiles during heat acclimation in Arabidopsis thaliana suspension-culture cells. J. Plant Res. 119: 373-383. Matsui, A., Ishida, J., Morosawa, T., Mochizuki, Y., Kaminuma, E., Endo, T.A., et al. (2008) Arabidopsis transcriptome analysis under drought, cold, high-salinity and ABA treatment conditions using a tiling array. Plant Cell Physiol. 49: 1135-1149.


correlation analysis improves reconstruction of full-length transcripts and alternative isoforms from

Merret, R., Descombin, J., Juan, Y.T., Favory, J.J., Carpentier, M.C., Chaparro, C., et al. (2013) XRN4 and LARP1 are required for a heat-triggered mRNA decay pathway involved in plant acclimation and survival during thermal stress. Cell Rep. 5: 1279-1293. Nakaminami, K., Matsui, A., Shinozaki, K. and Seki, M. (2012) RNA regulation in plant abiotic stress responses. Biochim. Biophys. Acta 1819: 149-153. Narsai, R., Howell, K.A., Millar, A.H., O'Toole, N., Small, I. and Whelan, J. (2007) Genome-wide analysis of mRNA decay rates and their determinants in Arabidopsis thaliana. Plant Cell 19: 34183436. Nishizawa, A., Yabuta, Y., Yoshida, E., Maruta, T., Yoshimura, K. and Shigeoka, S. (2006)

environmental stress. Plant J. 48: 535-547. Ogawa, D., Yamaguchi, K. and Nishiuchi, T. (2007) High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth. J. Exp. Bot. 58: 3373-3383. Okamoto, M., Tatematsu, K., Matsui, A., Morosawa, T., Ishida, J., Tanaka, M., et al. (2010) Genomewide analysis of endogenous abscisic acid-mediated transcription in dry and imbibed seeds of Arabidopsis using tiling arrays. Plant J. 62: 39-51. Olmedo, G., Guo, H., Gregory, B.D., Nourizadeh, S.D., Aguilar-Henonin, L., Li, H., et al. (2006) ETHYLENE-INSENSITIVE5 encodes a 5'-->3' exoribonuclease required for regulation of the EIN3targeting F-box proteins EBF1/2. Proc. Natl Acad. Sci. USA 103: 13286-13293. Oono, Y., Seki, M., Nanjo, T., Narusaka, M., Fujita, M., Satoh, R., et al. (2003) Monitoring expression profiles of Arabidopsis gene expression during rehydration process after dehydration using ca 7000 full-length cDNA microarray. Plant J. 34: 868-887. Potuschak, T., Vansiri, A., Binder, B.M., Lechner, E., Vierstra, R.D. and Genschik, P. (2006) The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis. Plant Cell 18: 3047-3057. Rymarquis, L.A., Souret, F.F. and Green, P.J. (2011) Evidence that XRN4, an Arabidopsis homolog of exoribonuclease XRN1, preferentially impacts transcripts with certain sequences or in particular functional categories. RNA 17: 501-511.


Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of

Schramm, F., Ganguli, A., Kiehlmann, E., Englich, G., Walch, D. and von Koskull-Doring, P. (2006) The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis. Plant Mol. Biol. 60: 759-772. Shim, J. and Karin, M. (2002) The control of mRNA stability in response to extracellular stimuli. Mol. Cells 14: 323-331. Souret, F.F., Kastenmayer, J.P. and Green, P.J. (2004) AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. Mol. Cell 15: 173-183. Suzuki, N., Bajad, S., Shuman, J., Shulaev, V. and Mittler, R. (2008) The transcriptional co-activator MBF1c is a key regulator of thermotolerance in Arabidopsis thaliana. J. Biol. Chem. 283: 9269-

von Koskull-Doring, P., Scharf, K.D. and Nover, L. (2007) The diversity of plant heat stress transcription factors. Trends Plant Sci. 12: 452-457. Zhou, Y.P., Duan, J., Fujibe, T., Yamamoto, K.T. and Tian, C.E. (2012) AtIQM1, a novel calmodulinbinding protein, is involved in stomatal movement in Arabidopsis. Plant Mol. Biol. 79: 333-346.


9275.

Tables

Table 1. List of heat response genes (GO: 0009408), that were up-regulated in atxrn4-3 mutant subjected to non-stress conditions Non-stress Gene name

AGI code

WT

Heat-stress

WT

atxrn4-3

atxrn4-3

Averagea)

SD

Averagea)

SD

Averagea)

SD

Averagea)

SD

Nonstress ratiob)

Heatstress ratioc)

HSP20like

AT5G51440

0.6

0.1

1.9

0.4

273.1

28.7

226.9

21.0

3.0

0.8

AT5G64510

0.3

0.1

0.8

0.1

63.5

3.8

59.8

5.1

3.0

0.9

AT3G61190

0.2

0.0

0.5

0.2

0.3

0.1

0.4

0.1

3.0

1.4

HSP70

AT3G12580

3.5

0.1

9.3

1.4

971.6

60.0

738.1

9.9

2.7

0.8

HSP83/ HSP90.1

AT5G52640

1.9

0.2

5.0

0.7

1295.0

81.9

977.8

58.5

2.7

0.8

ELIP1

AT3G22840

0.2

0.0

0.6

0.1

3.4

0.5

3.8

0.6

2.7

1.1

ZAT12

AT5G59820

1.3

0.2

3.3

1.0

81.7

6.7

56.0

10.3

2.6

0.7

HSP70-3

AT3G09440

10.1

0.4

25.6

5.9

219.4

11.2

212.7

21.7

2.5

1.0

HSFA2

AT2G26150

0.1

0.0

0.3

0.1

524.0

44.0

408.6

24.8

2.2

0.8 0.8

ELIP2

AT4G14690

0.7

0.1

1.5

0.5

1.8

0.2

1.4

0.3

2.2

WRKY33

AT2G38470

0.8

0.0

1.8

0.3

2.8

0.2

3.4

0.5

2.1

1.2

EGY3

AT1G17870

0.4

0.0

0.8

0.1

185.2

23.0

139.9

15.3

2.0

0.8

MBF1C

AT3G24500

1.8

0.1

3.5

0.1

496.8

40.3

447.4

8.6

1.9

0.9

Zinc finger AT1G14200

4.4

0.4

7.8

0.6

210.0

11.0

217.4

16.6

1.8

1.0

a)

Mean ± sd of 3 biological replicates.

b)

Fold change was defined as the ratio of normalized signal value in atxrn4-3 versus WT in the non-stress condition

c)

Fold change was defined as the ratio of normalized signal value in atxrn4-3 versus WT in the heat stress condition


TIN1 BAP1

Figure Legends Fig. 1. Mutation of AtXRN4 enhances the heat tolerance of plants subjected to a short-term severe heat stress. WT (Col-0), atxrn4-3, and atxrn4-8 mutants plants were grown on MS medium for 2 weeks. Shortterm severe heat stress (43.5 ºC for 2 h to 4 h 30 min) was applied. Survival rate was calculated at 7 º

days after returning plants to 22 C. (A) The location of the T-DNA insertion in atxrn4-3 and the point mutation in atxrn4-8. (B) The survival rate of two lines of the atxrn4 mutant and WT plants exposed to a short-term severe heat stress. Triangle indicates the time point at which the survival rate was calculated. Data points represent the mean ± sd of 3 biological replicates. * indicates significantly

atxrn4 mutants (atxrn4-3 and atxrn4-8) and WT plants. The plants were subjected to a heat stress of º

º

43.5 C for 3 h 30 min and then kept at 22 C for 7 days.

Fig. 2. Transpiration rates are higher in the atxrn4-3 mutant than in WT plants subjected to heat stress. (A) Temperature of the leaf surface of atxrn4-3 mutant plants is lower than in WT plants. WT and atxrn4-3 mutant plants were grown on MS medium for 2 weeks. After a 1 h incubation at 37 ºC, leaf temperature was recorded using a thermal imaging camera (Avio: Neo Thermo TVS-700). Three biological replicates were performed by three different temperature ranges (30.7 - 35.8 ºC, 31.6 - 36.6 º

º

C, and 37 - 41.9 C). Triangle indicates the time at which photos were taken. (B) Loss of AtXRN4

function leads to an increased rate of water loss under moderate heat stress. Three-week-old WT and º

atxrn4-3 mutant plants were grown on MS medium. All leaves were cut and incubated at 37 C and 27.5% RH. The fresh weight of leaves was measured every 5 min for 30 min. Data points represent the mean ± sd of 4 biological replicates. Triangle indicates the time at which the assay was terminated. (C) Loss of AtXRN4 function leads to an increased rate of water loss under severe heat stress. Threeweek-old WT and atxrn4-3 mutant plants were grown on MS medium. All leaves were cut and incubated at 43.5 ºC and 27.5% RH. The fresh weight of leaves was measured every 5 min for 30 min. Data points represent the mean ± sd of 4 biological replicates. Triangle indicates the time at which the assay was terminated.


different means (p < 0.05) as determined with a t-test. (C) Plant survival of the heat tolerant phenotype

Fig. 3. Transcriptome analysis of atxrn4-3 mutant and WT under non-stress and heat stress conditions. º

Two week-old plants of WT and the atxrn4-3 mutant were exposed to non-stress (22 C) and heatstress (37 ºC) conditions for 1 h. Plant samples were then collected, frozen in liquid nitrogen and º

stored at -80 C. mRNA was isolated and used in the microarray analysis. (A) Correlation between the expression level of all genes in atxrn4-3 and WT plants. Data was analyzed based on a log2 value of the expression signal. Red dots indicate a gene whose mRNA accumulation is significantly different in the two plant types (q-value < 0.1). (B) The correlation between the expression level of all genes in atxrn4-3 and WT subjected to the heat stress condition. (C) Venn diagram representation of 118 genes whose expression is higher in atxrn4-3 plants subjected to heat stress, 757 genes whose

upregulated genes in WT plants subjected to heat-stress conditions.

Fig. 4. RT-qPCR analysis of heat stress-inducible genes whose mRNA accumulation is higher in the atxrn4-3 mutant than in WT plants subjected to non-stress conditions. The same mRNA samples were used for the microarray analysis and for RT-qPCR. Genes selected for RT-qPCR analysis were reported to function in heat stress response. At least three biological replicates were used for the analysis of each gene. At18SrRNA was used as a reference gene for determining relative expression. Data points represent the mean ± sd of at least 3 biological replicates. * indicates significantly different means (p < 0.05) as determined with a t-test.

Fig. 5. HSFA2 and ERF1 mRNA are potential targets of AtXRN4-mediated degradation under nonstress conditions. Two-week-old atxrn4-3 mutant and WT plants were incubated in water at 22 ºC for 1 day and then transferred to a solution containing 0.6 mM cordycepin for up to 2 h. mRNA was isolated from each collected sample and used for RT-qPCR analysis. At5g42190 was chosen as a reference gene as it is a stable transcript under the test conditions. At18SrRNA was used as a reference gene for determining relative expression. Data points represent the mean ± sd of 3 biological replicates.

Fig. 6. AtXRN4 functions in reducing heat tolerance in heat-acclimated Arabidopsis plants returned to non-stress temperatures.


expression is higher in atxrn4-3 plants subjected to non-stress conditions, and 5170 heat-stress

(A) WT (Col-0) and atxrn4 mutants were grown on MS medium for 2 weeks. Plants were incubated at º

º

37 C for 1 h, returned to normal growth conditions (22 C) for 2 days and then subjected to a heat stress (44 ºC) for 3 h to 5 h 30 min. The plants were then returned to 22 ºC and the rate of survival was calculated after 7 days. Data points represent the mean ± sd of 3 biological replicates. The triangle indicates the time point at which the survival rate was calculated. (B) Expression level of HSFA2 and º

HSP70 in WT and atxrn4-3 mutants after a heat acclimation treatment (37 C for 1 h). RT-qPCR analyses were conducted using mRNA samples isolated directly from non-stress plants. (C) 1 h after the heat acclimation treatment (1h-heat), 1 day (1 DAH), 2 days (2 DAH), and 3 days (3 DAH) after the heat acclimation treatment. At18SrRNA was used as a reference gene for determining relative

points at which mRNA samples were collected.

Fig. 7. Heat stress tolerance of atxrn4 mutants is significantly reduced or lost by an additional hsfa2 mutation. Twelve- and fourteen-day-old WT, atxrn4-3, atxrn4-3 hsfa2, and hsfa2 plants were subjected to º

various heat stress treatments and then returned to a normal (non-stress) growth temperature (22 C). Survival rate was evaluated at 7 days after returning plants to 22 ºC. (A) Survival rate of WT, atxrn4-3, º

atxrn4-3 hsfa2, and hsfa2 plants subjected to a severe heat treatment (44 C for 3.5 h). Data points represent the mean ± sd of 3 biological replicates. The triangle indicates the time point at which survival rate was calculated. (B) Survival rate of WT, atxrn4-3, atxrn4-3 hsfa2, and hsfa2 plants exposed to a heat acclimation treatment (37 ºC for 1 h), maintained at 22 ºC for 2 days, and then º

subjected to a severe heat stress (44 C for 4 h). Data points represent the mean ± sd of 3 biological replicates. The triangle indicates the time point at which survival rate was calculated.


expression. Data points represent the mean ± sd of 3 biological replicates. Triangles indicate the time

Supporting Information

Supplementary Fig. S1. Heat stress-sensitive phenotype of atxrn4-3 mutants after being subjected to a long-term moderate heat stress treatment. WT (Col-0), and atxrn4-3 mutant plants were grown on MS medium for 5 days, subjected to a longº

º

term moderate heat stress (35 C for 7 days) and then kept at 22 C for 8 days;at which time the survival rate was calculated. Data points represent the mean ± sd of 3 biological replicates.

Supplementary Fig. S2. RT-qPCR analysis of heat stress-inducible genes whose mRNA

Two week-old plants of WT (Col-0) and atxrn4-8 mutant grown under non-stress (22 ºC) conditions were used for mRNA isolation. At18SrRNA was used as a reference gene for determining relative expression. Data points represent the mean ± sd of 3 biological replicates.

Supplementary Fig. S3. RT-qPCR analysis of HSFA2 mRNA in WT and atxrn4-3 mutant during TMHT condition. WT (Col-0) and atxrn4-3 mutants were applied to TMHT condition (35 ºC) and mRNA samples were collected at 5 days for RT-qPCR analysis. At18SrRNA was used as a reference gene for determining the relative expression. Data represent the mean ± sd of 3 biological replicates.

Supplementary Table S1. List of 757 genes whose mRNA accumulation is higher in the atxrn4-3 mutant than in WT plants under non-stress conditions.

Supplementary Table S2. List of 118 genes whose mRNA accumulation is higher in the atxrn4-3 mutant than in WT plants subjected to heat stress conditions.

Supplementary Table S3. List of 5,170 genes whose expression is upregulated by heat stress in WT plants.


accumulation is higher in atxrn4-8 than in WT plants under non-stress conditions.

Supplementary Table S4. List of 88 “response to stress” category genes whose mRNA accumulation is higher in the atxrn4-3 mutant than in WT plants under either non-stress or heat stress or induced by heat stress in WT plants.

Supplementary Table S5. Expression profile of the stomata-related genes, MYB61 and AtIQM1, in the atxrn4-3 mutant and WT plants under non-stress conditions.


144x129mm (300 x 300 DPI)


Fig. 1. Mutation of AtXRN4 enhances the heat tolerance of plants subjected to a short-term severe heat stress. WT (Col-0), atxrn4-3, and atxrn4-8 mutants plants were grown on MS medium for 2 weeks. Short-term severe heat stress (43.5 ºC for 2 h to 4 h 30 min) was applied. Survival rate was calculated at 7 days after returning plants to 22 ºC. (A) Diagram indicates the location of the T-DNA insertion in atxrn4-3 and the point mutation in atxrn4-8. (B) The survival rate of two lines of the atxrn4 mutant and WT plants exposed to a short-term severe heat stress. Triangle indicates the time point at which the survival rate was calculated. Data points represent the mean ± sd of 3 biological replicates. * indicates significantly different means (p < 0.05) as determined with a t-test. (C) Plant survival of the heat tolerant phenotype atxrn4 mutants (atxrn43 and atxrn4-8) and WT plants. The plants were subjected to a heat stress of 43.5 ºC for 3 h 30 min and then kept at 22 ºC for 7 days.


80x203mm (300 x 300 DPI)


Fig. 3. Transcriptome analysis of atxrn4-3 mutant and WT under non-stress and heat stress conditions. Two week-old plants of WT and the atxrn4-3 mutant were exposed to non-stress (22 ºC) and heat-stress (37 ºC) conditions for 1 h. Plant samples were then collected, frozen in liquid nitrogen and stored at -80 ºC. mRNA was isolated and used in the microarray analysis. (A) Correlation between the expression level of all genes in atxrn4-3 and WT plants. Data was analyzed based on log2 value of the expression signal. Red dots indicate a gene whose mRNA accumulation is significantly different in the two plant types (q-value < 0.1). (B) The correlation between the expression level of all genes in atxrn4-3 and WT subjected to the heat stress condition. (C) Venn diagram representation of 118 genes whose expression is higher in atxrn4-3 plants subjected to heat stress, 757 genes whose expression is higher in atxrn4-3 plants subjected to nonstress conditions, and 5170 heat-stress upregulated genes in WT plants subjected to heat-stress conditions. 83x221mm (300 x 300 DPI)

170x95mm (300 x 300 DPI)


Fig. 4. RT-qPCR analysis of heat stress-inducible genes whose mRNA accumulation is higher in the atxrn4-3 mutant than in WT plants subjected to non-stress conditions. The same mRNA samples were used for the microarray analysis and for RT-qPCR. Genes selected for RTqPCR analysis were reported to function in heat stress response. At least three biological replicates were used for the analysis of each gene. At18SrRNA was used as a reference gene for determining relative expression. Data points represent the mean ± sd of at least 3 biological replicates. * indicates significantly different means (p < 0.05) as determined with a t-test.


Fig. 5. HSFA2 and ERF1 mRNA are potential targets of AtXRN4-mediated degradation under non-stress conditions. Two-week-old atxrn4-3 mutant and WT plants were incubated in water at 22 ºC for 1 day, and then transferred to a solution containing 0.6 mM cordycepin for up to 2 h. mRNA was isolated from each collected sample and used for RT-qPCR analysis. At5g42190 was chosen as a reference gene as it is a stable transcript under the test conditions. At18SrRNA was used as a reference gene for determining relative expression. Data points represent the mean ± sd of 3 biological replicates. 70x166mm (300 x 300 DPI)

170x128mm (300 x 300 DPI)


Fig. 6. AtXRN4 functions in reducing heat tolerance in heat-acclimated Arabidopsis plants returned to nonstress temperatures. (A) WT (Col-0) and atxrn4 mutants were grown on MS medium for 2 weeks. Plants were incubated at 37 ºC for 1 h, returned to normal growth condition (22 ºC) for 2 days and then subjected to a heat stress (44 ºC) for 3 h to 5 h 30 min. The plants were then returned to 22 ºC and the rate of survival was calculated after 7 days. Data points represent the mean ± sd of 3 biological replicates. The triangle indicates the time point at which the survival rate was calculated. (B) Expression level of HSFA2 and HSP70 in WT and atxrn4-3 mutants after a heat acclimation treatment (37 ºC for 1 h). RT-qPCR analyses were conducted using mRNA samples isolated directly from non-stress plants (C), 1 h after the heat acclimation treatment (1h-heat), 1 day (1 DAH), 2 days (2 DAH), and 3 days (3 DAH) after the heat acclimation treatment. At18SrRNA was used as a reference gene for determining relative expression. Data points represent the mean ± sd of 3 biological replicates. Triangles indicate the time points at which mRNA samples were collected.

134x141mm (300 x 300 DPI)


Fig. 7. Heat stress tolerance of atxrn4 mutants is significantly reduced or lost by an additional hsfa2 mutation. Twelve- and fourteen-day-old WT, atxrn4-3, atxrn4-3 hsfa2, and hsfa2 plants were subjected to various heat stress treatments, and then returned to a normal (non-stress) growth temperature (22 ºC). Survival rate was evaluated at 7 days after returning plants to 22 ºC. (A) Survival rate of WT, atxrn4-3, atxrn4-3 hsfa2, and hsfa2 plants subjected to a severe heat treatment (44 ºC for 3.5 h). Data points represent the mean ± sd of 3 biological replicates. The triangle indicates the time point at which survival rate was calculated. (B) Survival rate of WT, atxrn4-3, atxrn4-3 hsfa2, and hsfa2 plants exposed to a heat acclimation treatment (37 ºC for 1 h), maintained at 22 ºC for 2 days, and then subjected to a severe heat stress (44 ºC for 4 h). Data points represent the mean ± sd of 3 biological replicates. The triangle indicates the time point at which survival rate was calculated.

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A chrysanthemum heat shock protein confers tolerance to abiotic stress.

Pushing the limits of blood pressure control under severe heat stress. Focus on "Active and passive heat stress similarly compromise tolerance to a simulated hemorrhagic challenge".

Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

Overexpression of ZmMAPK1 enhances drought and heat stress in transgenic Arabidopsis thaliana.

Overexpression of Arabidopsis P3B increases heat and low temperature stress tolerance in transgenic sweetpotato.

Chloroplast Retrograde Regulation of Heat Stress Responses in Plants.

Enhancement of reproductive heat tolerance in plants.

Sm-Like Protein-Mediated RNA Metabolism Is Required for Heat Stress Tolerance in Arabidopsis.

Drought stress had a predominant effect over heat stress on three tomato cultivars subjected to combined stress.

Overexpression of small heat shock protein LimHSP16.45 in Arabidopsis enhances tolerance to abiotic stresses.

Deciphering the dynamics of changing proteins of tolerant and intolerant wheat seedlings subjected to heat stress.

Arabidopsis DPB3-1, a DREB2A interactor, specifically enhances heat stress-induced gene expression by forming a heat stress-specific transcriptional complex with NF-Y subunits.

HSFA2 orchestrates transcriptional dynamics after heat stress in Arabidopsis thaliana.

Plant Heat Adaptation: priming in response to heat stress.

Heat stress induces ferroptosis-like cell death in plants.

Can intradermal administration of angiotensin II influence human heat loss responses during whole body heat stress?

tasiRNA-ARF pathway moderates floral architecture in Arabidopsis plants subjected to drought stress.

Respiratory evaporative heat loss regulation in shorn and unshorn sheep during milk heat stress.