A cytosolic class II small heat shock protein, PfHSP17.2, confers resistance to heat, cold, and salt stresses in transgenic <i>Arabidopsis</i>

We cloned and characterized the full-length coding sequence of a small heat shock (sHSP) gene, PfHSP17.2, from Primula forrestii leaves following heat stress treatment. Homology and phylogenetic analysis suggested that PfHSP17.2 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of transient expression of PfHSP17.2 fused with green fluorescent protein reporter. Expression analysis showed that PfHSP17.2 was highly inducible by heat stress in almost all the vegetative and generative tissues and was expressed under salt, cold, and oxidative stress conditions as well. Moreover, the expression of PfHSP17.2 in P. forrestii was detected in certain developmental growth stages. Transgenic Arabidopsis thaliana constitutively expressing PfHSP17.2 displayed increased thermotolerance and higher resistance to salt and cold compared with wild type plants. It is suggested that PfHSP17.2 plays a key role in heat and other abiotic stresses.

Keywords:
sHSPs; stress tolerance; Arabidopsis

Authorship

Lu Zhang

Department of Landscape Architecture, School of Civil Engineering and Architecture, Zhejiang Sci-Tech University, Hangzhou, Zhejiang Province, ChinaZhejiang Sci-Tech UniversityChinaHangzhou, Zhejiang Province, ChinaDepartment of Landscape Architecture, School of Civil Engineering and Architecture, Zhejiang Sci-Tech University, Hangzhou, Zhejiang Province, China

College of Landscape Architecture, Beijing Forestry University, China National Engineering Research Center for Floriculture, Beijing, ChinaBeijing Forestry UniversityChinaBeijing, ChinaCollege of Landscape Architecture, Beijing Forestry University, China National Engineering Research Center for Floriculture, Beijing, China

Weijuan Hu

Yike Gao

Huitang Pan

Qixiang Zhang Send correspondence to Qixiang Zhang. College of Landscape Architecture, Beijing Forestry University, Qinghua East Road No.35, Haidian District, 100083 Beijing, China. E-mail: zqxbifv@126.com.

Send correspondence to Qixiang Zhang. College of Landscape Architecture, Beijing Forestry University, Qinghua East Road No.35, Haidian District, 100083 Beijing, China. E-mail: zqxbifv@126.com.

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Figures | Formulas

Figures (7)
Formulas (1)

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Figure 1
Protein sequence multiple alignment of the deduced amino acid sequence of PfHSP17.2 with other plant sHSPs. The two consensus regions in sHSPs are underlined, and identical amino acid residues are highlighted. A putative nuclear localization signal is indicated by arrows and a polyproline motif at the carboxyl end of proteins is marked with asterisks. Their GenBank accession numbers are as follows: Primula forrestii (PfHSP17.2; JX025006); Arachis hypogaea (AhHSP17.6; ACF74271.1); Ammopiptanthus mongolicus (AmHSP; AGS48404); Cicer arietinum (CaHSP17.1; XP_004501443.1); Prunus salicina (PsHSP; ACV93250.1); Nelumbo nucifera (NnHSP17.5; ABK92179.1); Brachypodium distachyon (BdHSP17.8; XP_003564488.1); Medicago truncatula (MtHSP; AES73434.1); Solanum peruvianum (SpHSP17.4; AAT36481.1); Cucumis sativus (CsHSP17.9; XP_004141334.1).

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Figure 2
Phylogenetic analysis of the deduced amino acid sequence of PfHSP17.2 and other small sHSPs. The gene abbreviation and GenBank accession numbers are as follows: Primula forrestii (PfHSP17.2; JX025006), Arachis hypogaea (AhHSP17.5; ABC46712.1), Glycine max (GmHSP17.3; NP_001235293.1), Nicotiana tabacum (NtHSP; ADK36668.1), Zea mays (ZmHSP22; NP_001151139.1), Rosa chinensis (RcHSP17.8; ABK32539.1), Arabidopsis thaliana (AtHSP17.8; NP_172220.1), Glycine max (GmHSP22; HSP41_SOYBN), Medicago truncatula (MtHSP22.7; XP_003622351.1), Arabidopsis thaliana (AtHSP15.7; NP_198583.1), Triticum aestivum (TaHSP26; AAC96315.1), Oryza sativa (OsHSP26; BAA78385.1), Agrostis stolonifera (AsHSP26.8; AAN74536.1), Capsicum annuum (CaHSP; ADJ57588.1), Copaifera officinalis (CoHSP; AEX97054.1), Arabidopsis thaliana (AtHSP23.6; NP_194250.1), Arabidopsis thaliana (AtHSP23.5; NP_199957.1); Arachis hypogaea(AhHSP17.6; ACF74271.1); Ammopiptanthus mongolicus (AmHSP; AGS48404); Cicer arietinum (CaHSP17.1; XP_004501443.1); Prunus salicina (PsHSP; ACV93250.1); Nelumbo nucifera (NnHSP17.5; ABK92179.1); Brachypodium distachyon (BdHSP17.8; XP_003564488.1); Medicago truncatula (MtHSP17.6; XP_003603183.1); Solanum peruvianum (SpHSP17.4; AAT36481.1); Cucumis sativus (CsHSP17.9; XP_004141334.1), Prunus salicina (PsHSP; ACV93250.1).

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Figure 3
Subcellular localization of the PfHSP17.2:GFP fusion protein in onion epidermal cells. Cells were bombarded with DNA-coated gold particles carrying GFP or PfHSP17.2: GFP.

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Figure 4
Semi-quantitative RT-PCR of PfHSP17.2 expression in P. forrestii. (a) Heat treatment, time course induction of PfHSP17.2 by heat in P. forrestii; (b) cold treatment, time course induction of PfHSP17.2 by cold in P. forrestii; (c) Transcript levels of PfHSP17.2 in leaves under different treatments for 3 h. CK, no treatment as control (plants only irrigated with half-strength MS salt solution); (d) expression of PfHSP17.2 in different organs after incubation at 42 °C for 2 h; (e) semi-quantitative RT-PCR analysis in developing seeds and germinating seeds (f), respectively. DAP, days after pollination. HAI, hours after imbibition. YL, young leaves (5 d from emergence). OL, old leaves (two weeks from emergence). F, flower. P, petiole. R, root.

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Figure 5
PCR analysis of PfHSP17.2 transgenic plants and stress tolerance tests. (a) Semi-quantitative RT-PCR analysis of PfHSP17.2 expression in WT and PfHSP17.2 transgenic plants grown at 22°C for 4 weeks; (b) Heat treatments: aquired heat tolerance and basal heat tolerance; (c) 3-week-old plants heat treatment comparison; (d) survival rate after acquired heat treatment; (e) effect of heat stress on hypocotyl elongation of WT and PfHSP17.2-overexpressing plants; (f) MDA; (g) electrolyte leakage. For survival rate (d), hypocotyl elongation (e), MDA (f) and electrolyte leakage (g), error bars indicate SD (n=3). *p<0.05, ** p<0.01. WT, wild type; L223, L253, two transgenic Arabidopsis lines.

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Figure 6
Performance of PfHSP17.2-overexpressing transgenic plants under salt stress. (a) Germination on different salt MS (0 mm NaCl, 150 mm NaCl, 200 mm NaCl); (b) germination rate; (c) root number; (d) root length; (e) phenotypic comparison of the plants under salt stress; (f) proline content in WT and transgenic plants in response to salt stress. Error bars indicate SD (n=3). *p<0.05, ** p<0.01. WT, wild type; L223, L253, two transgenic Arabidopsis lines.

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Figure 7
Performance of PfHSP17.2-overexpressing transgenic plants under cold stress. (a) Cold tolerance of the PfHSP17.2 transformants; (b) proline content; (c) chlorophyll content; (d) POD changes. Error bars indicate SD (n=3). *p<0.05, ** p<0.01. WT, wild type; L223, L253, two transgenic Arabidopsis lines.

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Figure 1 Protein sequence multiple alignment of the deduced amino acid sequence of PfHSP17.2 with other plant sHSPs. The two consensus regions in sHSPs are underlined, and identical amino acid residues are highlighted. A putative nuclear localization signal is indicated by arrows and a polyproline motif at the carboxyl end of proteins is marked with asterisks. Their GenBank accession numbers are as follows: Primula forrestii (PfHSP17.2; JX025006); Arachis hypogaea (AhHSP17.6; ACF74271.1); Ammopiptanthus mongolicus (AmHSP; AGS48404); Cicer arietinum (CaHSP17.1; XP_004501443.1); Prunus salicina (PsHSP; ACV93250.1); Nelumbo nucifera (NnHSP17.5; ABK92179.1); Brachypodium distachyon (BdHSP17.8; XP_003564488.1); Medicago truncatula (MtHSP; AES73434.1); Solanum peruvianum (SpHSP17.4; AAT36481.1); Cucumis sativus (CsHSP17.9; XP_004141334.1).

Figure 2 Phylogenetic analysis of the deduced amino acid sequence of PfHSP17.2 and other small sHSPs. The gene abbreviation and GenBank accession numbers are as follows: Primula forrestii (PfHSP17.2; JX025006), Arachis hypogaea (AhHSP17.5; ABC46712.1), Glycine max (GmHSP17.3; NP_001235293.1), Nicotiana tabacum (NtHSP; ADK36668.1), Zea mays (ZmHSP22; NP_001151139.1), Rosa chinensis (RcHSP17.8; ABK32539.1), Arabidopsis thaliana (AtHSP17.8; NP_172220.1), Glycine max (GmHSP22; HSP41_SOYBN), Medicago truncatula (MtHSP22.7; XP_003622351.1), Arabidopsis thaliana (AtHSP15.7; NP_198583.1), Triticum aestivum (TaHSP26; AAC96315.1), Oryza sativa (OsHSP26; BAA78385.1), Agrostis stolonifera (AsHSP26.8; AAN74536.1), Capsicum annuum (CaHSP; ADJ57588.1), Copaifera officinalis (CoHSP; AEX97054.1), Arabidopsis thaliana (AtHSP23.6; NP_194250.1), Arabidopsis thaliana (AtHSP23.5; NP_199957.1); Arachis hypogaea(AhHSP17.6; ACF74271.1); Ammopiptanthus mongolicus (AmHSP; AGS48404); Cicer arietinum (CaHSP17.1; XP_004501443.1); Prunus salicina (PsHSP; ACV93250.1); Nelumbo nucifera (NnHSP17.5; ABK92179.1); Brachypodium distachyon (BdHSP17.8; XP_003564488.1); Medicago truncatula (MtHSP17.6; XP_003603183.1); Solanum peruvianum (SpHSP17.4; AAT36481.1); Cucumis sativus (CsHSP17.9; XP_004141334.1), Prunus salicina (PsHSP; ACV93250.1).

Figure 3 Subcellular localization of the PfHSP17.2:GFP fusion protein in onion epidermal cells. Cells were bombarded with DNA-coated gold particles carrying GFP or PfHSP17.2: GFP.

Figure 4 Semi-quantitative RT-PCR of PfHSP17.2 expression in P. forrestii. (a) Heat treatment, time course induction of PfHSP17.2 by heat in P. forrestii; (b) cold treatment, time course induction of PfHSP17.2 by cold in P. forrestii; (c) Transcript levels of PfHSP17.2 in leaves under different treatments for 3 h. CK, no treatment as control (plants only irrigated with half-strength MS salt solution); (d) expression of PfHSP17.2 in different organs after incubation at 42 °C for 2 h; (e) semi-quantitative RT-PCR analysis in developing seeds and germinating seeds (f), respectively. DAP, days after pollination. HAI, hours after imbibition. YL, young leaves (5 d from emergence). OL, old leaves (two weeks from emergence). F, flower. P, petiole. R, root.

Figure 5 PCR analysis of PfHSP17.2 transgenic plants and stress tolerance tests. (a) Semi-quantitative RT-PCR analysis of PfHSP17.2 expression in WT and PfHSP17.2 transgenic plants grown at 22°C for 4 weeks; (b) Heat treatments: aquired heat tolerance and basal heat tolerance; (c) 3-week-old plants heat treatment comparison; (d) survival rate after acquired heat treatment; (e) effect of heat stress on hypocotyl elongation of WT and PfHSP17.2-overexpressing plants; (f) MDA; (g) electrolyte leakage. For survival rate (d), hypocotyl elongation (e), MDA (f) and electrolyte leakage (g), error bars indicate SD (n=3). *p<0.05, ** p<0.01. WT, wild type; L223, L253, two transgenic Arabidopsis lines.

Figure 6 Performance of PfHSP17.2-overexpressing transgenic plants under salt stress. (a) Germination on different salt MS (0 mm NaCl, 150 mm NaCl, 200 mm NaCl); (b) germination rate; (c) root number; (d) root length; (e) phenotypic comparison of the plants under salt stress; (f) proline content in WT and transgenic plants in response to salt stress. Error bars indicate SD (n=3). *p<0.05, ** p<0.01. WT, wild type; L223, L253, two transgenic Arabidopsis lines.

Figure 7 Performance of PfHSP17.2-overexpressing transgenic plants under cold stress. (a) Cold tolerance of the PfHSP17.2 transformants; (b) proline content; (c) chlorophyll content; (d) POD changes. Error bars indicate SD (n=3). *p<0.05, ** p<0.01. WT, wild type; L223, L253, two transgenic Arabidopsis lines.

\begin{array}{l} C h l a = \frac{[(12.3 A 663 - 0.86 A 645) \times V]}{X \times 1000 \times W} \\ C h l b = \frac{[(19.3 A 645 - 3.6 A 663) \times V]}{X \times 1000 \times W} \\ C a r o t e n o i d s = \frac{[1000 \times A 470 - (1.82 C h a - 85.02 C h b)]}{198} \end{array}

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A cytosolic class II small heat shock protein, PfHSP17.2, confers resistance to heat, cold, and salt stresses in transgenic <i>Arabidopsis</i>

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[1] Send correspondence to Qixiang Zhang. College of Landscape Architecture, Beijing Forestry University, Qinghua East Road No.35, Haidian District, 100083 Beijing, China. E-mail: zqxbifv@126.com.