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. 2016 Apr 12;12(10):e1162368. doi: 10.1080/15592324.2016.1162368

The role of AtPLC3 and AtPLC9 in thermotolerance in Arabidopsis

Huimin Ren 1,*, Kang Gao 1,*, Yuliang Liu 1, Daye Sun 1, Shuzhi Zheng 1,✉
PMCID: PMC5647968  PMID: 27070271

ABSTRACT

Plants respond and adapt to temperature changes by many ways. In this article, we provide a supplement to previous work about AtPLC3. The subcellular localization showed that AtPLC3 was located in the plasma membrane and nucleus which differed from AtPLC9 localization. Furthermore, we measured the contents of IP3 before and after HS in AtPLC3 mutant, complemented and overexpressing lines. The results showed that the increase in IP3 after HS was partially dependent on AtPLC3 activity. To sum up, the similar expression patterns and phenotypes suggested that AtPLC3 and AtPLC9 may regulate the thermotolerance of Arabidopsis by the same mechanisms.

KEYWORDS: Arabidopsis, AtPLC3, AtPLC9, Ca2+, heat shock, IP3, thermotolerance

As sessile organisms, plants experience many kinds of abiotic stresses, including high temperature. Plants respond and adapt to temperature changes by thermotolerance pathways that cause accumulation of heat shock proteins (HSPs), increase membrane fluidity, and activate Ca2+ channels.1 The recent acceleration of global climate change has depressed agricultural production,2,3 with high temperature strongly affecting crop yields. To mitigate these changes, research on heat shock (HS) aims to understand organisms’ response to high temperature. Since 1962, when Ritosa identified the HS response in Drosophila,4 continuing research on the HS response has examined how organisms respond to heat.5

The cell membrane plays an extremely important role in identification and conversion of signals in signal transduction. Our work aims to understand how plants sense heat and transmit HS signals from the external environment to the interior, with a particular interest in membrane proteins. Until now, our work and studies by Saidi et al. showed that cyclic nucleotide-gated ion channel (CNGC) and phosphoinositide-specific phospholipase C (PI-PLCs) proteins play important roles in thermotolerance in moss and Arabidopsis.6-8 In animals, PtdIns-PLCs mainly localize in the cell membrane. Our previous work also showed that the Ca2+/calmodulin (CaM) signal transduction pathway has a significant role in the HS response in Arabidopsis. We first used genetic studies to prove that PI-PLC participates in the HS response in Arabidopsis.9-13 Our results showed that plc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (Col-0) plants after HS. AtPLC3 also has an import role in the HS signal transduction pathway. Our previous work suggested that AtPLC9 may be activated rapidly in seedlings grown at high temperature and then AtPLC3 may be activated during continuous HS treatment.7,8

The classic second messenger inositol triphosphate (IP3) is produced through hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) by membrane-bound PtdIns-PLCs. IP3 has an important role in releasing intracellular calcium stores in animal cells;14-18 high temperature causes a rapid increase in IP3 which induces the increase in intracellular Ca2+ after HS.7,10

Herein, we provide a supplement to previous work on AtPLC3, including the measurement of IP3 accumulation before and after 37°C treatment, and the subcellular localization of AtPLC3 in tobacco and Arabidopsis.

AtPLC3 localizes in the plasma membrane and nucleus

To observe the subcellular location of AtPLC3, we constructed a vector with the AtPLC3 promoter (pAtPLC3) driving the expression of the AtPLC3 coding sequence fused to Green Fluorescent Protein (GFP) in the pAtPLC3:AtPLC3-GFP construct. The pAtPLC3:AtPLC3-GFP construct was transiently transformed into tobacco and also expressed in transgenic Arabidopsis. Localization of AtPLC3 was analyzed by a Zeiss LCM 710confocal microscopy using the 488 nm fluorescence and bright-field channels and the results showed that AtPLC3 localizes in the plasma membrane and nucleus in tobacco (Fig. 1A, 1B) and transgenic Arabidopsis thaliana (Fig. 1E, 1F). We also made a vector expressing GFP under the control of the CaMV 35S promoter (35S:GFP) and used it as a negative control (Fig. 1C, 1D). These results revealed that AtPLC3 is located in the nucleus in addition to the cytomembrane. The subcellular location of AtPLC3 differed from that AtPLC9, which just localized in the membrane,7,8 which may reflect their different functions.

Figure 1.

Figure 1.

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Subcellular localization of AtPLC3 expressed under the AtPLC3 promoter and changes in IP3 contents before or after HS. A & B: Localization of AtPLC3-GFP expressed from the pAtPLC3:AtPLC3-GFP construct in mesophyll cells of transgenic tobacco was analyzed using 488 nm fluorescence and bright-field channels. The red arrows indicate the nucleus and the white arrows indicate the cell membrane. C & D: Localization of GFP expressed from the 35S:GFP construct in mesophyll cells of transgenic tobacco was analyzed using the 488 nm fluorescence and bright-field channels. E & F: Localization of AtPLC3-GFP expressed from the pAtPLC3:AtPLC3-GFP construct in root cells of transgenic Arabidopsis thaliana was analyzed using the 488 nm fluorescence and bright-field channels. The red arrows indicate the nucleus and the white arrows indicate the cell membrane. G: Time course of the increase in IP3 contents induced by heat shock (HS) at 37℃ for 0, 2, and 4 min. The IP3 content at 0 min (no HS treatment) was set as 100%. IP3 was extracted from 10-day-old seedlings and assayed by Radio receptor Assay kit (Perkin Elmer http://www.perkinelmer.com). Each point is the mean±SE of 3 biological replicates. H: The level of IP3 in wild type (WT), plc3 mutants, 2 complemented lines (COMP1 and COMP2) and 2 overexpression lines (OE1 and OE2) before (22℃) and after (37℃, 2 min) HS treatment. *P < 0.05.

AtPLC3 mediates a heat-induced increase in IP3

To examine the function of AtPLC3, we measured the contents of IP3 before and after HS. To this end, 10-day-old seedlings (Col-0) were exposed to HS at 37°C for 0, 2, and 4 minutes. Within 2 minutes of HS, IP3 increased to 1.5-fold compared to normal levels (Fig. 1G). We also compared the levels of IP3 in wild type, plc3, 2 complemented lines (COMP1 and COMP2), and 2 AtPLC3 overexpressing lines (OE1 and OE2) before (22°C) and after (37°C, 2 min) HS treatment. We found the plc3 mutants had lower levels of IP3 compared with wild type after HS. Also, OE1 and OE2 had significantly higher levels of IP3 compared with wild type after HS, and COMP1 and COMP2 showed no significant differences with wild type (Fig. 1H). Thus, these results indicate that the increase in IP3 after HS is partially dependent on AtPLC3 activity.

Taking all our results together, the similar expression patterns of AtPLC3 and AtPLC9 and phenotypes of plc3 and plc9 mutants after HS suggested that these phosphoinositide-specific phospholipase C proteins may function redundantly. The similar changes in Ca2+ and IP3 in plc3 and plc9 mutants after HS suggested that AtPLC3 and AtPLC9 regulate the thermotolerance of Arabidopsis by the same mechanisms. However, AtPLC3 localizes to the nucleus and the membrane; whether AtPLC3 shifts from the nucleus to the cell membrane may be an interesting question for future studies.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by natural science research project of Hebei Province Department of Education Found QN2015254 and QN2015190.

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