Arabidopsis thaliana Phosphoinositide-Specific ... - Oxford Journals

Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 3 (AtPLC3) and AtPLC9 have an Additive Effect on Thermotolerance 1

Hebei Key Laboratory of Molecular and Cellular Biology, Hebei 050024, China Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Science, Hebei Normal University, Hebei 050024, China 3 Hebei Collaboration Innovation Center for Cell Signaling, Shijiazhuang, Hebei 050024, China 4 Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, China 5 These authors contributed equally to this work. 2

*Corresponding author: E-mail, [email protected]; Tel, 86-311-80787512; Fax, 86-311-80787549. (Received April 25, 2014; Accepted August 19, 2014)

The heat stress response is an important adaptation, enabling plants to survive challenging environmental conditions. Our previous work demonstrated that Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 9 (AtPLC9) plays an important role in thermotolerance. During prolonged heat treatment, mutants of AtPLC3 showed decreased heat resistance. We observed no obvious phenotypic differences between plc3 mutants and wild type (WT) seedlings under normal growth conditions, but after heat shock, the plc3 seedlings displayed a decline in thermotolerance compared with WT, and also showed a 40–50% decrease in survival rate and chlorophyll contents. Expression of AtPLC3 in plc3 mutants rescued the heat-sensitive phenotype; the AtPLC3-overexpressing lines also exhibited much higher heat resistance than WT and vector-only controls. The double mutants of plc3 and plc9 displayed increased sensitivity to heat stress, compared with either single mutant. In transgenic lines containing a AtPLC3:GUS promoter fusion, GUS staining showed that AtPLC3 expresses in all tissues, except anthers and young root tips. Using the Ca2+-sensitive fluorescent probe Fluo3/AM and aequorin reconstitution, we showed that plc3 mutants show a reduction in the heat-induced Ca2+ increase. The expression of HSP genes (HSP18.2, HSP25.3, HSP70-1 and HSP83) was down-regulated in plc3 mutants and up-regulated in AtPLC3-overexpressing lines after heat shock. These results indicated that AtPLC3 also plays a role in thermotolerance in Arabidopsis, and that AtPLC3 and AtPLC9 function additionally to each other. Keywords: Arabidopsis AtPLC3 Ca2+ Heat shock Signal transduction Thermotolerance.

Introduction Plants are exposed to a wide variety of abiotic and biotic environmental stresses, including high and low temperatures out of the range they can readily tolerate. In recent years, global warming has threatened agricultural production with higher

temperatures, often accompanying drought or other stresses, which have reduced global harvests (Battisti and Naylor 2009, Hockley et al. 2009, Holden 2009). Therefore, research on how to improve the heat resistance of plants has emerged as an increasingly important field of study. Plants perceive an environmental temperature above the normal optimum as heat shock (HS). The production and accumulation of heat shock proteins (HSPs) plays an essential role in the HS response of plants (Vierling 1991, Wang et al. 2004, Kotak et al. 2007). Misfolded proteins are accumulated in the plant cells when under high temperature. The HS stress triggers the expression of some up-regulated genes such as HSP70, HSP90, HSP101 and others HSPs particularly small HSPs, which play an important role in protecting plant cells during high temperature conditions (Conde et al. 2011, Hahn et al. 2011, Hu et al. 2012, Kim et al. 2012, Waters 2013, Wu et al. 2013, Zhong et al. 2013). Heat shock transcription factors (HSFs) bind to heat shock elements (HSEs) to regulate the expression of HSPs. HSFs such as HSFA1s, HSFA2, HsfB1, HsfB2b and other HSFs, which are activated and mobilized under heat stress, transduce the HS signals and affect the heat resistance in plants (Ikeda et al. 2011, Liu et al. 2011, Scharf et al. 2012, Yu et al. 2012). Previous studies on HS have mainly focused on this transcriptional regulation, and research on heat shock signal transduction remains sparse, with only a few signaling components identified so far (Kotak et al. 2007). Moreover, plants perceive and transmit HS signals by a complex pathway (Saidi et al. 2011). Based on our previous work (Li et al. 2004, Liu et al. 2003, Liu et al. 2005, Liu et al. 2006b, Liu et al. 2007), we proposed a Ca2+/ Calmodulin (CaM) pathway for HS signal transduction (Liu et al. 2008). That work showed that Ca2+ and CaM are involved in HS signal transduction in wheat, where intracellular Ca2+ starts increasing after 1 min of HS, and reaches a maximum after 3 min. Also, CaM functions upstream of HSPs in HS signal transduction (Liu et al. 2003) and AtCaM3 plays an important role in HS signal transduction as a component of the Ca2+/CaM pathway. For example, AtCaM3 knockout mutants display a clear reduction in thermotolerance and AtCaM3 overexpression lines display a clear increase in thermotolerance compared

Plant Cell Physiol. 55(11): 1873–1883 (2014) doi:10.1093/pcp/pcu116, Advance Access publication on 22 August 2014, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

Regular Paper

Kang Gao1,2,3,5, Yu-Liang Liu1,2,3,5, Bing Li1,2,3, Ren-Gang Zhou4, Da-Ye Sun1,2,3 and Shu-Zhi Zheng1,2,3,*

K. Gao et al. | AtPLC3 modulates thermotolerance in Arabidopsis

with WT. AtCAM3 also affects the binding activity of HSF to the HSE and thus alters HSP levels (Zhang et al. 2009). OsCaM1-1 is also one of the important components in the HS signal pathway in rice (Oryza sativa L.). HS induces an increase in intracellular Ca2+ and OsCaM1-1 gene expression and the OsCaM1-1 overexpression enhances the thermotolerance in the transgenic Arabidopsis (Wu et al. 2012). Downstream of CaM, the CaMbinding protein kinase AtCBK3 interacts with and phosphorylates AtHSFA1a. AtCBK3 affects the thermotolerance of Arabidopsis by regulating the binding activity of HSFs to HSE and thus affecting the accumulation of HSPs (Liu et al. 2008). It has been demonstrated that, after HS, there is a rapid and specific increase in intracellular Ca2+, through Ca2+ influxing across the plasma membrane or Ca2+ being released from the intracellular calcium stores (Gong et al. 1998, Liu et al. 2003, Wu et al. 2012). Recently, many expert works have demonstrated that ion channels in the plasma membrane involved in the intracellular Ca2+ increase after HS and affect the thermotolerance in plants (Finka et al. 2012, Mach 2012, Tunc-Ozdemir et al. 2013). Besides the ion channels, the other pathways that participate in the elevation of Ca2+ after HS remains unclear. Polyphosphoinositides may also affect the HS pathway. The phosphoinositide-specific phospholipase C (PLC) hydrolyses phosphatidylinositol 4,5-bisphosphate (PI (4,5)P2) (MuellerRoeber and Pical 2002, Pokotylo et al. 2014, Thole and Nielsen 2008) to generate two important second messengers, inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Heat stress induces a rapid and significant PIP2 increase in tobacco BY-2 cells, Arabidopsis seedlings and rice leaves, and this accumulation of PIP2 in plasma membrane and nucleus indicates that PIP2 functions in the heat response in plants (Mishkind et al. 2009, Horvath et al. 2012). Phosphatidic acid (PA) synthesis occurs through two pathways, one is catalyzed by the phosphorylation of diacyglycerol (DAG) and the other is synthesized by the action of phospholipase D (PLD) (Pokotylo et al. 2014). The rapid induction of phospholipase D (PLD) activity after HS is the source of the PA that accumulates during heat stress (Mishkind et al. 2009, Horvath et al. 2012). Some indirect evidence suggests that HS activates PLC and the PLC inhibitor U73122 blocks the HS response (Liu et al. 2006a, Liu et al. 2006c). PLCs have been implicated in multiple responses to external stimuli in a variety of plants (Munnik 2001, Xue et al. 2007, Chen et al. 2011). For example, the expression of potato (Solanum tuberosum) StPLC1, 2, 3 changes under drought and damage stress (Kopka et al. 1998). In Commelina communis, abscisic acid (ABA) stimulates PLC activity, and plants treated with the PLC inhibitor U73122, show reductions in the ABAinduced oscillations of cytosolic Ca2+ concentration (Staxen et al. 1999). In mung bean (Vigna radiata L.), abiotic stress conditions (drought and high salinity) induce the expression of Vr-PLC3, which shows no expression under normal conditions (Kim et al. 2004). Six cDNAs encoding PLC isoforms have been isolated from tomato (Solanum lycopersicum, Sl), and SlPLC4 and SlPLC6 are required for the hypersensitive response and disease resistance (Vossen et al. 2010). HS activates PLC in 1874

pea leaves and salicylic acid (SA) is involved in thermotolerance (Liu et al. 2006a, Liu et al. 2006c). In tomato, PLC3 and PLC6 gene expression is up-regulated by high temperature (Abd-ElHaliem et al. 2012). The Arabidopsis thaliana genome encodes nine PI-PLCs (Mueller-Roeber and Pical 2002, Pokotylo et al. 2014). All the AtPLC isoforms, except AtPLC2, which is constitutively expressed, show increased or decreased expression in response to varied environmental stimuli, such as cold, drought, salt, nutrients in Murashige and Skoog salts, dehydration and ABA (Hunt et al. 2004, Tasma et al. 2008, Pokotylo et al. 2014). AtPLC9 expression was detected in all parts of Arabidopsis seedlings (Zheng et al. 2012). Although their expression has been examined, the functions of AtPLCs in HS remain unclear. Our previous work showed that HS induces an increase in IP3 and caged IP3 increases expression from a GUS reporter driven by the HSP18.2 promoter, even in the absence of HS; also, U73122 inhibits this induction. This result indicates that IP3/PLC functions in the induction of HSP gene expression by HS (Liu et al. 2006b). Our recent work showed that PLC may function as an upstream component in the Ca2+/CaM HS pathway. We found that AtPLC9 (Zheng et al. 2012), along with the membrane Ca2+ channel AtCNGC6 (Gao et al. 2012), function as early, primary mechanisms for Ca2+ increase after HS. We used a reverse genetics approach to demonstrate that AtPLC9 plays an important role in thermotolerance and heat shock signal transduction. After HS treatment, the survival rates and chlorophyll contents decreased significantly in plc9 plants compared with WT. The IP3 contents were also lower in plc9 than in WT seedlings. The [Ca2+]i concentration decreased about 30% in plc9 after HS. Also, the accumulation of HSP18.2 and HSP25.3 proteins significantly decreased in plc9, by about 50% and 30%, respectively. Although the IP3 content, [Ca2+]i concentration and HSP accumulation all were reduced in plc9, they still showed some induction (Zheng et al. 2012). Also, we found that AtPLC3 may affect the heat tolerance of Arabidopsis seedlings under prolonged HS. Hence, to further analyze the function of AtPLC3 in HS and test whether AtPLC3 functions by the same mechanism as AtPLC9, here we examined T-DNA insertion mutants of AtPLC3.

Results Arabidopsis plc3 mutants show decreased heat tolerance We obtained seeds of AtPLC3 (At4g38530) mutants from the Arabidopsis Biological Resource Center (ABRC) and identified the homozygous T-DNA insertion mutants. We named the two T-DNA insertion mutants, salk_037453 and salk_054406, as plc3-1 and plc3-2, respectively (Fig. 1B). RT-PCR showed that the homozygous T-DNA insertion mutants of AtPLC3 displayed no detectable full-length transcripts (Fig. 1B). The 14-day-old plc3-1 and plc3-2 seedlings showed normal growth, similar to WT seedlings, under normal conditions (Supplementary Fig. S1). In contrast, under heat shock at 45 C for 65 min, plc3-1 and plc3-2 seedlings showed serious growth delays, while WT

Plant Cell Physiol. 55(11): 1873–1883 (2014) doi:10.1093/pcp/pcu116

displayed no growth or visible phenotype under normal conditions (Fig. 2A) and after heat treatment, expression of AtPLC3 in plc3-1 complemented the plc3 mutant defects in heat tolerance (Fig. 2B). The complemented lines exhibited higher survival rates and higher chlorophyll contents than plc3-1 after HS (Fig. 2C and D). These results demonstrated that expression of AtPLC3 in plc3-1 improves the heat tolerance and thus the plc31 mutation is responsible for the heat tolerance defect.

AtPLC3 overexpressing lines show increased heat tolerance

Fig. 1 plc3 exhibits thermosensitivity. (A) Seven-day-old Arabidopsis seedlings were HS treated at 45 C for 65 min and then returned to 22 C for another 7 d of recovery. (B) The T-DNA insertion position and identification of the AtPLC3 transcript. First row: the intron and exon organization of AtPLC3 and the insertion locations of the T-DNA alleles. Introns and exons are shown in lines and solid boxes, respectively. The positions of the T-DNA insertions are indicated by triangles. The locations of primers used for RT-PCR are indicated by arrows. The second row: RT-PCR analysis of the AtPLC3 transcript in WT and mutant seedlings. The Actin expression levels were used as a control in the RT-PCR analysis. (C) The survival rate of WT, plc3-1 and plc3-2 after HS at 45 C for 65 min. Each value is the mean ± SE, n = 3 (30 seedlings in each experiment). (D) The chlorophyll content of WT, plc3-1 and plc3-2 after HS. Each value is the mean ± SE of three biological replicates (n = 3). A total of 0.1 g of fresh tissue was used in each experiment for chlorophyll extraction.

To further confirm the role of AtPLC3 in the heat tolerance of Arabidopsis seedlings, we obtained AtPLC3-overexpressing transgenic seedlings using a pCAMBIA1300::35S::AtPLC3 construct. RT-PCR analysis of RNA isolated from seedlings showed that AtPLC3 expression in the two overexpression lines (OE1, OE2) was higher than that in WT (Supplementary Fig. S2). Under normal conditions, we observed no obvious phenotypic change in the overexpression lines, WT seedlings and control plants transformed with the empty pCAMBIA1300 vector (p1300) (Fig. 3A). When 7day-old seedlings of AtPLC3-overexpressing line, WT and vector control were exposed to 45 C for 75 min, and then returned to 22 C for another 7 d, the AtPLC3-overexpressing lines (OE1, OE2) displayed increased heat tolerance compared to the WT seedlings and vector control (Fig. 3B). After HS, the AtPLC3-overexpressing lines (OE1, OE2) showed enhanced survival rates (Fig. 3C) and chlorophyll contents (Fig. 3D) compared with WT seedlings and vector controls. These results suggested that heat tolerance improved in the AtPLC3-overexpressing lines.

The expression pattern of AtPLC3 seedlings survived and produced new, green leaves (Fig. 1A). The WT seedlings showed a survival rate of about 90% after heat shock, but plc3-1 and plc3-2 mutants showed only a 30– 40% survival rate (Fig. 1C). Also, the WT seedlings had chlorophyll contents of 4 mg/g FW fresh tissue, compared to 2.5 mg/g FW for the mutants (Fig. 1D). Thus, the survival rate and chlorophyll content after heat shock were about 40–50% lower for plc3-1 and plc3-2 mutants than for the WT plants. These results suggested that the mutations in AtPLC3 affect thermotolerance in Arabidopsis.

Genetic complementation of AtPLC3 rescues the heat sensitivity of plc3 mutants To confirm whether absence of AtPLC3 gene expression causes the decline in heat tolerance, we performed genetic complementation analysis. For this analysis, we transformed a WT AtPLC3 cDNA under the control of the 35S promoter into plc3-1 mutant plants. The plc3-1 and plc3-2 mutants show the same decrease in thermotolerance; therefore, we only tested plc3-1 in the following experiments. RT-PCR analysis showed that AtPLC3 expression reached similar levels to WT in the two homozygous complemented lines (Comp1 and Comp2) (Supplementary Fig. S2). The complemented lines

To determine the expression pattern of AtPLC3, we generated transgenic Arabidopsis plants with an AtPLC3 promoter::GUS reporter fusion construct. We observed GUS staining in the hypocotyl and cotyledon of 4-day-old seedlings, but not in the root (Fig. 4A and B). However, in 10-day-old seedlings, we observed GUS staining in all leaves and roots, especially in the vascular parts of these tissues (Fig. 4C and D). We observed GUS staining throughout the mature leaves, flowers and inflorescences, but not in siliques (Fig. 4E–I), indicating that AtPLC3 and AtPLC9 show similarly ubiquitous expression (Zheng et al. 2012). However, we did observe a few differences. First, AtPLC3 showed no expression in roots of 4-day-old plants and low expression in the roots of 10-day-old plants (Fig. 4B), whereas AtPLC9 showed high expression in roots. Second, AtPLC3 showed no expression in siliques, where AtPLC9 showed low expression. The similar expression patterns of AtPLC3 and AtPLC9 (Zheng et al. 2012) indicate that they could have similar biological functions in regulating plant development and responding to stress. To examine the expression of AtPLC3 and AtPLC9 after HS treatment, we also performed real-time PCR on wild type plants after a short heat treatment at 37 C for 0, 1, 2, 3 4, 5, 10 and 15 min. The results showed that the expression of AtPLC3 1875


Fig. 2 Expression of AtPLC3 in plc3 mutants rescues the thermosensitive phenotype after HS. (A) Seedlings grown on MS plates at 22 C for 14 d were used as a control. WT, wild type; plc3-1, the salk_037453 mutant of AtPLC3; Comp1 and Comp2 the complemented lines. (B) WT, plc3-1, and two complemented lines (Comp1 and Comp2) under 45 C HS conditions. (C) Survival rate for WT, plc3-1 and two complemented lines (Comp1 and Comp2) under normal and HS conditions (30 seedlings per plate). (D) Chlorophyll contents of WT, plc3-1 and two complemented lines (Comp1 and Comp2) under normal and HS conditions with 0.1 g of fresh tissue in each experiment. Each data point is the mean ± SE of three experiments (n = 3).

increased during HS, while AtPLC9 expression remained the same (Supplementary Fig. S3).

The plc3 plc9 double mutants show a more severe heat-sensitive phenotype than the single mutants To test whether AtPLC3 and AtPLC9 have redundant biological functions, we crossed plc3 and plc9 plants to obtain plc3 plc9 double mutants. The 14-day-old plc3 plc9 seedlings displayed similar growth phenotypes to plc3, plc9 and WT plants (Fig. 5A). When we treated the 7-day-old seedlings with HS (45 C) for 65 min, the double mutants displayed a much more severe thermosensitive phenotype (Fig. 5B), with less than 40% of the double mutant seedlings surviving after a 7-d recovery, compared with survival rates of about 80% for WT, 65% for plc3 and 45% for plc9 (Fig. 5C). Plants of the four different genotypes displayed similar chlorophyll contents before HS (Fig. 5D). However, when the seedlings were exposed to 45 C for 65 min and then to 22 C for 7 d, the chlorophyll content in the double mutants decreased 1876

seriously to just 2 mg/g FW. By contrast, the single mutant plants showed a less severe effect; plc3 had 3 mg/g FW and plc9 had 2.5 mg/g FW, compared with the 3.5 mg/g FW observed for WT (Fig. 5D). These results demonstrate that AtPLC3 and AtPLC9 play an important role in thermotolerance in Arabidopsis and the two genes function additionally to each other.

AtPLC3 affects the [Ca2+]i changes induced by HS To investigate the mechanism by which AtPLC3 affects thermotolerance, we analysed the kinetic changes of [Ca2+]i during HS. First, we used the Ca2+ sensitive fluorescent probe Fluo-3/AM to investigate the effect of AtPLC3 on [Ca2+]i changes induced by HS. We isolated protoplasts from the leaves of 4-week-old Arabidopsis seedlings and incubated them in buffer containing 10 mm Fluo-3/AM at 4 C in the dark for 2 h. Then, we tracked the fluorescence of the protoplasts by laser scanning confocal microscopy. We measured the fluorescence intensity every 30 s, up to 8 min, and obtained an average of at least six cells in three different experiments. The


Fig. 3 Overexpression of AtPLC3 improves the thermotolerance of Arabidopsis seedlings. (A) Seedlings grown on MS plates under normal conditions for 14 d were used as a control. (B) WT, p1300, and two overexpressing lines (OE1 and OE2) under 45 C HS conditions (75 min). p1300, the vector control. (C) Survival rate for WT, p1300, OE1 and OE2 under normal and HS conditions with 30 seedlings per plate. (D) Chlorophyll contents of the seedlings under normal and HS conditions with 0.1 g fresh tissue in each experiment. Each data point is the mean ± SE of three experiments (n = 3).

fluorescence in the protoplasts of WT seedlings remained constant during the experiment at 22 C (Fig. 6A). The autofluorescence of chloroplasts also remained constant over time when we heat treated protoplasts at 37 C (Fig. 6B), suggesting that HS had a negligible effect on autofluorescence. The fluorescence of Fluo-3AM in the protoplast increased during HS, suggesting a significant increase in [Ca2+]i both for WT and plc3 during heat shock (Fig. 6C, D and E). After 8 min of HS, the [Ca2+]i reached a maximum, at about 2-fold for WT and 1.5fold for plc3 (Fig. 6E). Furthermore, we generated a stable transgenic line that constitutively expressed aequorin, according to the method described by Zheng (Zheng et al. 2012). The maximum luminescence, caused by the discharge of excess Ca2+, was similar between WT-AQ and plc3-AQ. Under normal conditions, WTAQ and plc3-AQ showed nearly identical [Ca2+]i concentrations. However, after HS treatment, [Ca2+]i increased for WT-AQ and plc3-AQ (Fig. 6F). This increase in [Ca2+]i occurred within 3 min after HS, and after 15–20 min, the [Ca2+]i

concentration reached a maximum of about 1.2-fold for plc3AQ compared with 1.5-fold for WT-AQ (Fig. 6G). The reduced [Ca2+]i concentration in plc3-AQ after HS indicated that AtPLC3 affects the [Ca2+]i change during HS.

AtPLC3 affects the expression of HSPs To test whether AtPLC3 affects the expression of HSP genes, we used real-time PCR to measure the expression of AtHSP18.2, AtHSP25.3, AtHSP70-1 and AtHSP83. We heat-treated 10-dayold seedlings of WT, plc3, complemented lines (Comp1, Comp2) and overexpressing lines (OE1, OE2) at 37 C for 1 h. RNA was isolated and used as the template for real-time PCR. After HS, the expression levels of AtHSP18.2, AtHSP25.3, AtHSP70-1 and AtHSP83 were about 30% lower in plc3 than in WT (Fig. 7A–D). Expression of the four HSPs in the complemented lines (Comp1, Comp2) was higher than that in plc3, and similar to that in WT seedlings (Fig. 7A–D). In the overexpressing lines, the expression levels of the four HSPs were 1.5to 2.0-fold higher than in WT (Fig. 7A–D). The results here 1877


Fig. 4 The expression pattern of AtPLC3. Spatiotemporal expression pattern of AtPLC3 using AtPLC3Pro::GUS transgenetic seedlings via histochemical GUS staining. Bars = 1 mm. (A to D) GUS staining of 4-day-old (A) and 10-day-old (C) whole seedlings and the root tips of 4-day-old (B) and 10-day-old (D) seedlings of the transformants containing AtPLC3pro:GUS. (E to I) GUS staining in rosette leaf (E), open flower (F), stamen (G), pistil (H) and silique (I).

suggested that the thermotolerance changes in plc3 and transgenic seedlings may be due to differential expression of HSPs.

Discussion AtPLC3 and AtPLC9 are functionally additional to each other Our previous work demonstrated that deletion of AtPLC9 results in decreased thermotolerance and overexpression of AtPLC9 results in increased thermotolerance. When the Arabidopsis seedlings were heat-treated at 45 C for 60 min, only the plc9 mutants showed a thermosensitive phenotype; when the heated treatment was prolonged to 65 min, plc3 mutants also displayed a thermosensitive phenotype, but their phenotype was less severe than that of plc9 mutants (Zheng et al. 2012). In this further study of the mechanism of the HS signaling pathway in Arabidopsis, we find that, in addition to AtPLC9, AtPLC3 also affects thermotolerance. Therefore, we speculated that AtPLC3 may play a similar role to AtPLC9 in thermotolerance in Arabidopsis and that they may be functionally additional to each other. In this study, we used T-DNA insertion mutants of AtPLC3 to obtain direct evidence of the effect of this gene on 1878

thermotolerance. The plc3-1 and plc3-2 mutants show no growth defects under normal conditions (Supplementary Fig. S1), but show impaired thermotolerance after HS (Fig. 1A). Also, AtPLC3-overexpressing lines show improved thermotolerance (Fig. 3). All these results suggest that AtPLC3 also plays a role in thermotolerance in Arabidopsis. The plc3 plc9 double mutants show more severe thermosensitivity than the single mutants (Fig. 4B), indicating that AtPLC3 and AtPLC9 have redundant functions. We observed AtPLC3 expression in all tissues except the roots, an expression pattern that generally overlaps with the expression pattern of AtPLC9 (Fig. 5). The similar expression pattern also suggested that AtPLC3 and AtPLC9 may function in a similar way to each other.

AtPLC3 and AtPLC9 regulate the thermotolerance of Arabidopsis by the same mechanisms Ca2+ as a principal second messenger affects many plant responses to environmental stress (Sanders et al. 1999, Kudla et al. 2010, Monshausen 2012). For example, a rapid and transient increase of cytosolic Ca2+ occurs during HS in tobacco, as shown in plants transiently expressing aequorin (Gong et al. 1998). Our recent results indicated that AtPLC9 is involved in the Ca2+ changes after HS, but has no effect on the Ca2+ influx through plasma membrane Ca2+ channels. So, we speculated


Fig. 5 The plc3 plc9 double mutants are more thermosensitive after HS. (A) Fourteen-day-old plants grown on the MS plates at 22 C were used as a control. WT, wild type; plc3-1, salk_037453; plc9-1, salk_120782; plc3 plc9 (plc3-1 and plc9-1 double mutants). (B) Seven-day-old seedlings were HS treated at 45 C for 65 min and then returned to 22 C for another 7 d for recovery. (C) Survival rates for WT, plc3-1, plc9-1 and plc3 plc9 after HS at 45 C for 65 min. Each value is the mean ± SE for three biological replicates (n = 3), 30 seedlings in each experiment. (D) The chlorophyll content of WT, plc3-1, plc9-1 and plc3 plc9 after HS. Each value is the mean ± SE of three biological replicates. A total of 0.1 g of fresh tissue were used in each experiment for chlorophyll extraction.

that AtPLC9 may affect the intracellular Ca2+ concentration by altering the release of Ca2+ from intracellular stores (Zheng et al. 2012). However, the Ca2+ concentration still increases after HS in the plc9 mutants, suggested that there may be other mechanisms regulating the change of [Ca2+]i in the HS pathway, such as other AtPLCs or the Ca2+ channels in the plasma membrane (Zheng et al. 2012). The purpose of this study was to identify the other possible mechanism for Ca2+ increases in HS pathway. Ca2+ concentration showed less of an increase in plc3-1 mutants after HS (Fig. 6). We tested [Ca2+]i using two methods, Fluo3-AM and transgenic seedlings expressing aequorin. These methods showed slight differences in the timing of the [Ca2+]i increase and its reaching maximum levels. This discrepancy might be caused by differences in the experimental materials (protoplasts vs. seedlings) or in the method of delivering the HS (hot water bath vs. hot air heating). Nevertheless, the consistent difference between WT and plc3 in the [Ca2+]i concentration after HS suggests that AtPLC3 affects the [Ca2+]i increase in the HS pathway.

Biological organisms respond to external thermal stimulation by producing HSPs, which play an important role in the acquisition of thermotolerance in plants (Queitsch et al. 2000, Sanmiya et al. 2004, Miroshnichenko et al. 2005, Charng et al. 2006, Waters 2013). Our results here demonstrated that AtPLC3 affects the expression of HSP genes. HSP18.2, HSP25.3, HSP70-1 and HSP83 show higher expression in the lines with higher expression of AtPLC3 such as WT, complemented and overexpression lines, and lower expression in plc3 mutants after HS (Fig. 7). These observations indicate that AtPLC3 regulates the expression of HSP genes. The expression of HSPs is regulated by the binding activity of HSFs to HSEs (Wahid et al. 2007). [Ca2+]i increase enhances this binding activity, resulting in increased expression of HSP genes (Liu et al. 2003, Li et al. 2004) . Taken together, our results indicate that AtPLC3 may affect the thermotolerance of Arabidopsis by changing the Ca2+ content and the expression of HSPs, the same mechanism as AtPLC9. However, AtPLC3 and AtPLC9 may have functional differences. For example, we found that heat stress increased the expression of AtPLC3, but did not 1879


Fig. 6 AtPLC3 affects the [Ca2+]i increase after HS. The protoplasts from leaves of Arabidopsis seedlings were incubated in medium containing Fluo-3/AM with a final concentration of 5 mM at 4 C in the dark for 2 h. Then the protoplasts loaded with Fluo-3/AM were incubated at 37 C or at 22 C as a control. The fluorescence intensity of Fluo-3/AM was observed using a Zeiss LSM confocal microscope and was measured every 30 s. The final fluorescence intensity was an average value for six cells from three different experiments. plc3-AQ, plc3-1 seedlings expressing aequorin; WT-AQ, WT seedlings expressing aequorin. (A) The fluorescence of Fluo-3AM in WT at 22 C. (B) The autofluorescence of chloroplasts in the protoplast at 37 C. C and D) The fluorescence of Fluo-3/AM for WT and plc3 at 37 C, respectively. (E) The kinetics curve of [Ca2+]i in the protoplast before and after HS. The fluorescent intensity represents [Ca2+]i. (F and G) The Ca2+ concentration and relative Ca2+ value in WT-AQ and plc3-AQ seedlings. WT-AQ tissues under HS (green line), plc3-AQ tissues under HS (blue line), WT-AQ tissues under 22 C (black line) and plc3-AQ tissues under 22 C (red line). (G) Relative Ca2+ concentration before and after HS in WT-AQ and plc3-AQ. The Ca2+ concentration in WT-AQ before HS was set as 100%; all other concentrations were compared to this value. Each data point represents the mean ± SE of three biological replicates (n = 3).

affect AtPLC9 expression (Supplementary Fig. S3). Also, the plc3 and plc9 mutants showed different thermosensitivity phenotypes, with plc3 mutants displaying a reduced thermotolerance phenotype only with longer heat treatment (Zheng et al. 2012). All this suggested that the phospholipase activity of AtPLC9 protein may be inactive under normal growth conditions. When the seedlings receive a heat signal, a rapid pathway activates AtPLC9. If the heat stimulus persists, then a later pathway activates AtPLC3. AtPLC3 may thus function with AtPLC9 in the HS pathway and also affect thermotolerance in Arabidopsis. The Ca2+-permeable channel, cyclic nucleotide-gated ion channel 6 (CNGC6), also affects the Ca2+ increase after HS in Arabidopsis (Gao et al. 2012). The work by Finka et al. demonstrates that CNGCb in Physcomitrella patens and CNGC2 in Arabidopsis thaliana acted as the primary thermosensors in land plants (Finka et al. 2012, Mach 2012). Furthermore, genetic evidence identifies that CNGC16 is critical for pollen fertility under HS conditions and in drought stress response in Arabidopsis (Tunc-Ozdemir et al. 2013). Based on these results 1880

and our previous studies, we conclude that PLCs and CNGCs may be the early primary response factors affecting the Ca2+ increase after HS and may be upstream of the Ca2+/CaM HS pathway. The HS signal activates PLCs and the CNGC channels, followed by a Ca2+ increase, which then promotes the expression and accumulation of AtCaM3. The active AtCBK3 kinase then phosphorylates AtHSFA1a, causing the HSPs to accumulate and thus producing thermotolerance of Arabidopsis. However, this hypothesis model based on our previous and recent work will require additional experiments to define the upstream and downstream relationships between the components in this model.

Materials and Methods Plant materials and growth conditions Seeds of Arabidopsis thaliana (Columbia) were surface-sterilized for 30 s in 75% (v/v) ethanol, plated on MS medium containing 1.0% (w/v) sucrose and 0.3% (w/v) phytagel (Sigma), kept at 4 C for 3 d in the dark, and then grown in growth chambers (16/8 h photoperiod) at 22 C.


constructs pCAMBIA1300-35S:AtPLC3 and pCAMBIA1300AtPLC3promoter::GUS were transformed into Agrobacterium tumefaciens strain GV3101, and then introduced into plc3 and WT seedlings by the floral dip method (Clough and Bent 1998). All the transformants were selected on MS plates containing 25 mg mL–1 hygromycin. The T4 homozygous transgenic lines were used in all experiments.

Real-time quantitative RT-PCR

Fig. 7 Effect of AtPLC3 on HSP gene expression during HS. (A–D) Real-time PCR analyses of the expression of HSP genes in WT, plc3, two complemented lines (Comp1, Comp2) and two AtPLC3-overexpressing lines (OE1, OE2). The 7-day-old seedlings were heat treated at 37 C for 1 h. After HS, the RNA was isolated and used for template in real-time PCR analyses. The expression levels of AtHSP18.2 (A), AtHSP25.3 (B), AtHSP70-1 (C), AtHSP83 (D) in WT, plc3, 3-12, 11-4, 24-8 and 25-1. The expression level of HSP genes in WT samples was set to 1.

Thermotolerance analysis The seedlings of different genotypes were planted on the same MS plate. In the thermotolerance assay, the 7-day-old seedlings grown at 22 C were exposed to 45 C in a hot chamber with lights on (120 mmol m–2 s–1) for 65 or 75 min and then returned to 22 C for recovery. The growth condition for recovering was the same as before stress. After 7 d, the seedlings were photographed and their viabilities were recorded. Seedlings that were green and growing new leaves were defined as survivors. The chlorophyll content of the seedlings was determined as described by Arnon (Arnon 1949).

RT-PCR analysis Transcript abundance for AtPLC3 in WT, T-DNA insertion mutant plants and transgenic plants was determined by RT-PCR. Total RNA was isolated from 10day-old seedlings, then translated into cDNA and used as the template for the RT-PCR. The AtPLC3 coding region was amplified using the forward primer FP 50 -CGCTACCTCTTCAGCGATACC-30 and the reverse primer RP 50 -CGTACCC ACATCCACCATTC-30 ; Actin7 (At5g09810) was used as the loading control using FP 50 -AGGCACCTCTTAACCCTAAAGC-30 and RP, 50 -GGAC A ACGGA ATCTCTCAGC-30 for forward and reverse primers.

Plasmid constructs and plant transformation The AtPLC3 coding region was PCR-amplified with cDNA from Arabidopsis thaliana (Columbia-0) seedlings using FP 50 -GCTCTAGAATGTCGGAGAGTT TCAAAGTGTG-30 and RP 50 -GAGCTCTCAACGAAACGTATAAGGAGGATC30 primers. The PCR product was cloned into the binary vector pCAMBIA1300 digested with Xbal/Sacl. The AtPLC3 promoter region was amplified with genomics DNA from Columbia-0 seedlings using FP 50 -AACT GCAGCCCTCATCTCATCCGCCACA-30 and RP 50 -TATCCCGGGCTCTCCGAC ATTCTTCTTCTTC-30 primers. The PCR product (3204 bp) was cloned into the binary vector pCAMBIA1300:GUS digested with Pstl/Sma. The resulting

Primer pairs used in the real-time PCR were designed using Primer Express (Applied Biosystems). The primers used in the real-time PCR were: AtHsp18.2 (At5g59720) 50 -TCGTGATGTGGCAGCGTTTA-30 (forward) and 50 -AAGTCCG CTTTGAACACATGTG-30 (reverse); AtHsp25.3 (At4g27670) 50 -GACGTCTCTC CTTTCGGATTGT-30 (forward) and 50 -CTCCACTTCCTCCTCTGTTTCTTC-30 (reverse); AtHsp83 (At5g52640) 50 -GCTGCTAGGATTCACAGGATG-30 (forward) and 50 -TCCTCCATCTTGCTC TCTTCA-30 (reverse). AtHsp70-1 50 -TGA CATGAAATTGTGGCCATTC-30 (forward) and 50 -CCTTGTATTCGACGTAGAT CATTGG-30 (reverse); Actin8 (At1g49240) 50 -TGTGACAATGGTACTGGA ATGG-30 (forward) and 50 -TTGGATTGTGCTTCATCACC-30 (reverse). Total RNA (500 ng) was isolated from 10-day-old seedlings grown at 22 C, using the Trizol reagent (Invitrogen). The first strand cDNA synthesis was performed using PrimeScript RT Reagent Kit (TaKaRa). For real-time PCR, SYBR Premix Ex Taq (TaKaRa) was used according to the manufacturer’s instructions. The PCR program was as described by Liu et al. (Liu et al. 2008) and Zhang et al. (Zhang et al. 2009).

Measurement of [Ca2+]i Arabidopsis mesophyll protoplasts were extracted from the leaves of 4-weekold seedlings. The fresh leaves were cut into 0.5–1 mm strips with fresh razor blades without wounding, then digested in 5 mL enzyme solution (1–1.5% cellulase R10, 0.2–0.4% macerozyme R10 (Yakult Honsha, Tokyo, Japan), 0.4 M mannitol, 20 mM KCl, 20 mM MES, pH 5.7, 10 mM CaCl2, 5 mMm-mercaptoethanol (optional), 0.1% BSA). The leaf strips were incubated at 22 C in the dark for 2 h. The enzyme solution containing the mesophyll protoplasts was filtered using a 35–70-mm nylon mesh and then centrifuged at 4 C 100 g for 3 min to pellet the protoplasts. Fluo3/AM was used as the Ca2+-sensitive fluorescent probe. For measurement of [Ca2+]i, the protoplasts were loaded with the Fluo-3/AM dye and observed by confocal microscopy. The extracted protoplasts were incubated in Ca2+ buffer containing 10 mm Fluo-3/AM at 4 C in the dark for 2 h. Then the protoplasts were observed using the Zeiss LSM 510 confocal microscope. The 37 C hot buffer was added when the heat treatment was needed. Excitation (488 ± 10 nm) and emission filters (530 ± 40 nm) were used in this experiment. The autofluorescence of protoplast was collected meanwhile using the 615-nm channel. The change of fluorescence intensity in the protoplasts was recorded over time. Software algorithms used for image processing and the fluorescence intensity calculation method were according to Liu et al. (Liu et al. 2003).

Aequorin reconstitution and Ca2+ measurement The stable aequorin-expressing plc3 lines (plc3-AQ) were generated by crossing plc3-1 and a transgenic line containing the 35S::aequorin construct seedlings (WT-AQ). The Ca2+ concentration measurement and calculation were according to Zheng (Zheng et al. 2012). WT-AQ and plc3-AQ seedlings incubated with coelenterazine-h Ca2+ buffer (0.1 mM KCl, 1 mM CaCl2, 10 mM MES, pH 5.0 and 2.5 mM coelenterazine-h) were put in a luminometer (Centro LB 960) set at 37 C for HS or 22 C as control. Changes in the Ca2+ concentration in all the seedlings under HS and normal conditions were measured every 30 s and continued for 25 min.

GUS staining GUS expression staining was carried out according to the method in Jefferson (Jefferson et al. 1987). Tissues were incubated in X-Gluc reaction solution (Liu et al. 2008, Zhang et al. 2009) at 37 C for 1–12 h, and then washed with 75% ethanol three times and then with 95% ethanol until the chlorophyll was completely removed.

1881


Supplementary data Supplementary data are available at PCP online.

Funding This work was supported by Genetically Modified (GM) Special Project, “Important trait gene cloning and functional verification”, Ministry of Agriculture, China (2014ZX08009003-002).

Acknowledgments We thank Dr Jennifer Mach for English language editing.

References Abd-El-Haliem, A., Meijer, H.J., Tameling, W.I., Vossen, J.H. and Joosten, M.H. (2012) Defense activation triggers differential expression of phospholipase-C (PLC) genes and elevated temperature induces phosphatidic acid (PA) accumulation in tomato. Plant Signaling Behav 7: 1073–1078. Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 1–15. Battisti, D.S. and Naylor, R.L. (2009) Historical warnings of future food insecurity with unprecedented seasonal heat. Science 323: 240–244. Charng, Y.Y., Liu, H.C., Liu, N.Y., Hsu, F.C. and Ko, S.S. (2006) Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation. Plant Physiol. 140: 1297–1305. Chen, G., Snyder, C.L., Greer, M.S. and Weselake, R.J. (2011) Biology and biochemistry of plant phospholipases. Crit. Rev. Plant Sci. 30: 239–258. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743. Conde, A., Chaves, M.M. and Geros, H. (2011) Membrane transport, sensing and signaling in plant adaptation to environmental stress. Plant Cell Physiol. 52: 1583–1602. Finka, A., Cuendet, A.F., Maathuis, F.J., Saidi, Y. and Goloubinoff, P. (2012) Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance. Plant Cell 24: 3333–3348. Gao, F., Han, X., Wu, J., Zheng, S., Shang, Z., Sun, D. et al. (2012) A heatactivated calcium-permeable channel—Arabidopsis cyclic nucleotidegated ion channel 6—is involved in heat shock responses. Plant J. 70: 1056–1069. Gong, M., van der Luit, A.H., Knight, M.R. and Trewavas, A.J. (1998) Heatshock-induced changes in intracellular Ca2+ level in tobacco seedlings in relation to thermotolerance. Plant Physiol. 116: 429–437. Hahn, A., Bublak, D., Schleiff, E. and Scharf, K.D. (2011) Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. Plant Cell 23: 741–755. Hockley, N., Gibbons, J.M. and Edwards-Jones, G. (2009) Risks of extreme heat and unpredictability. Science 324: 177–179; author reply 177–179. Holden, C. (2009) Climate change. Higher temperatures seen reducing global harvests. Science 323: 193. Horvath, I., Glatz, A., Nakamoto, H., Mishkind, M.L., Munnik, T., Saidi, Y. et al. (2012) Heat shock response in photosynthetic organisms: membrane and lipid connections. Prog. Lipid Res. 51: 208–220. Hu, C., Lin, S.Y., Chi, W.T. and Charng, Y.Y. (2012) Recent gene duplication and subfunctionalization produced a mitochondrial GrpE, the

1882

nucleotide exchange factor of the Hsp70 complex, specialized in thermotolerance to chronic heat stress in Arabidopsis. Plant Physiol. 158: 747–758. Hunt, L., Otterhag, L., Lee, J.C., Lasheen, T., Hunt, J., Seki, M. et al. (2004) Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms. New Phytologist 162: 643–654. Ikeda, M., Mitsuda, N. and Ohme-Takagi, M. (2011) Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance. Plant Physiol. 157: 1243–1254. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907. Kim, M., Lee, U., Small, I., des Francs-Small, C.C. and Vierling, E. (2012) Mutations in an Arabidopsis mitochondrial transcription termination factor-related protein enhance thermotolerance in the absence of the major molecular chaperone HSP101. Plant Cell 24: 3349–3365. Kim, Y.J., Kim, J.E., Lee, J.H., Lee, M.H., Jung, H.W., Bahk, Y.Y. et al. (2004) The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.). FEBS Lett. 556: 127–136. Kopka, J., Pical, C., Gray, J.E. and Muller-Rober, B. (1998) Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato. Plant Physiol. 116: 239–250. 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. Kudla, J., Batistic, O. and Hashimoto, K. (2010) Calcium signals: the lead currency of plant information processing. Plant Cell 22: 541–563. Li, B., Liu, H.T., Sun, D.Y. and Zhou, R.G. (2004) Ca(2+) and calmodulin modulate DNA-binding activity of maize heat shock transcription factor in vitro. Plant Cell Physiol. 45: 627–634. Liu, H.-T., Liu, Y.-Y., Pan, Q.-H., Yang, H.-R., Zhan, J.-C. and Huang, W.-D. (2006a) Novel interrelationship between salicylic acid, abscisic acid, and PIP2-specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves. J. Exp. Bot. 57: 3337–3347. Liu, H.-T., Sun, D.-Y. and Zhou, R.-G. (2005) Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis. Plant Cell Environ. 28: 1276–1284. Liu, H.C., Liao, H.T. and Charng, Y.Y. (2011) The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses in Arabidopsis. Plant Cell Environ. 34: 738–751. Liu, H.T., Gao, F., Cui, S.J., Han, J.L., Sun, D.Y. and Zhou, R.G. (2006b) Primary evidence for involvement of IP3 in heat-shock signal transduction in Arabidopsis. Cell Res. 16: 394–400. Liu, H.T., Gao, F., Li, G.L., Han, J.L., Liu, D.L., Sun, D.Y. et al. (2008) The calmodulin-binding protein kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana. Plant J. 55: 760–773. Liu, H.T., Huang, W.D., Pan, Q.H., Weng, F.H., Zhan, J.C., Liu, Y. et al. (2006c) Contributions of PIP(2)-specific-phospholipase C and free salicylic acid to heat acclimation-induced thermotolerance in pea leaves. J. Plant Physiol. 163: 405–416. Liu, H.T., Li, B., Shang, Z.L., Li, X.Z., Mu, R.L., Sun, D.Y. et al. (2003) Calmodulin is involved in heat shock signal transduction in wheat. Plant Physiol. 132: 1186–1195. Liu, H.T., Li, G.L., Chang, H., Sun, D.Y., Zhou, R.G. and Li, B. (2007) Calmodulin-binding protein phosphatase PP7 is involved in thermotolerance in Arabidopsis. Plant Cell Environ. 30: 156–164. Mach, J. (2012) Calcium channels and acquired thermotolerance: here comes the sun and it’s all right. Plant Cell 24: 3167. Miroshnichenko, S., Tripp, J., Nieden, U., Neumann, D., Conrad, U. and Manteuffel, R. (2005) Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures. Plant J. 41: 269–281.


Mishkind, M., Vermeer, J.E., Darwish, E. and Munnik, T. (2009) Heat stress activates phospholipase D and triggers PIP accumulation at the plasma membrane and nucleus. Plant J. 60: 10–21. Monshausen, G.B. (2012) Visualizing Ca(2+) signatures in plants. Curr. Opin. Plant Biol 15: 677–682. Mueller-Roeber, B. and Pical, C. (2002) Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C. Plant Physiol. 130: 22–46. Munnik, T. (2001) Phosphatidic acid: an emerging plant lipid second messenger. Trends Plant Sci. 6: 227–233. Pokotylo, I., Kolesnikov, Y., Kravets, V., Zachowski, A. and Ruelland, E. (2014) Plant phosphoinositide-dependent phospholipases C: variations around a canonical theme. Biochimie 96: 144–157. Queitsch, C., Hong, S.W., Vierling, E. and Lindquist, S. (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12: 479–492. Saidi, Y., Finka, A. and Goloubinoff, P. (2011) Heat perception and signalling in plants: a tortuous path to thermotolerance. New Phytol. 190: 556–565. Sanders, D., Brownlee, C. and Harper, J.F. (1999) Communicating with calcium. Plant Cell 11: 691–706. Sanmiya, K., Suzuki, K., Egawa, Y. and Shono, M. (2004) Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants. FEBS Lett. 557: 265–268. Scharf, K.D., Berberich, T., Ebersberger, I. and Nover, L. (2012) The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim. Biophys. Acta 1819: 104–119. Staxen, I., Pical, C., Montgomery, L.T., Gray, J.E., Hetherington, A.M. and McAinsh, M.R. (1999) Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C. Proc. Natl Acad. Sci. USA 96: 1779–1784. Tasma, I.M., Brendel, V., Whitham, S.A. and Bhattacharyya, M.K. (2008) Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana. Plant Physiol. Biochem. 46: 627–637. Thole, J.M. and Nielsen, E. (2008) Phosphoinositides in plants: novel functions in membrane trafficking. Curr. Opin. Plant Biol. 11: 620–631. Tunc-Ozdemir, M., Tang, C., Ishka, M.R., Brown, E., Groves, N.R., Myers, C.T. et al. (2013) A cyclic nucleotide-gated channel (CNGC16) in pollen is

critical for stress tolerance in pollen reproductive development. Plant Physiol 161: 1010–1020. Vierling, E. (1991) The roles of heat shock proteins in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 579–620. Vossen, J.H., Abd-El-Haliem, A., Fradin, E.F., van den Berg, G.C., Ekengren, S.K., Meijer, H.J. et al. (2010) Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance. Plant J. 62: 224–239. Wahid, A., Gelani, S., Ashraf, M. and Foolad, M.R. (2007) Heat tolerance in plants: an overview. Environ. Exp. Bot. 61: 199–223. Wang, W., Vinocur, B., Shoseyov, O. and Altman, A. (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9: 244–252. Waters, E.R. (2013) The evolution, function, structure, and expression of the plant sHSPs. J. Exp. Bot. 64: 391–403. Wu, H.C., Luo, D.L., Vignols, F. and Jinn, T.L. (2012) Heat shock-induced biphasic Ca(2+) signature and OsCaM1-1 nuclear localization mediate downstream signalling in acquisition of thermotolerance in rice (Oryza sativa L.). Plant Cell Environ. 35: 1543–1557. Wu, T.Y., Juan, Y.T., Hsu, Y.H., Wu, S.H., Liao, H.T., Fung, R.W. et al. (2013) Interplay between heat shock proteins HSP101 and HSA32 prolongs heat acclimation memory posttranscriptionally in Arabidopsis. Plant Physiol. 161: 2075–2084. Xue, H., Chen, X. and Li, G. (2007) Involvement of phospholipid signaling in plant growth and hormone effects. Curr. Opin. Plant Biol. 10: 483–489. Yu, H.D., Yang, X.F., Chen, S.T., Wang, Y.T., Li, J.K., Shen, Q. et al. (2012) Downregulation of chloroplast RPS1 negatively modulates nuclear heat-responsive expression of HsfA2 and its target genes in Arabidopsis. PLoS gen. 8: e1002669. Zhang, W., Zhou, R.G., Gao, Y.J., Zheng, S.Z., Xu, P., Zhang, S.Q. et al. (2009) Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis. Plant Physiol. 149: 1773–1784. Zheng, S.Z., Liu, Y.L., Li, B., Shang, Z.L., Zhou, R.G. and Sun, D.Y. (2012) Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis. Plant J. 69: 689–700. Zhong, L., Zhou, W., Wang, H., Ding, S., Lu, Q., Wen, X. et al. (2013) Chloroplast small heat shock protein HSP21 interacts with plastid nucleoid protein pTAC5 and is essential for chloroplast development in Arabidopsis under heat stress. Plant Cell 25: 2925–2943.

1883