Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
RESEARCH ARTICLE

Identification and functional analysis of calcium sensor calmodulins from heavy metal hyperaccumulator Noccaea caerulescens

Lu Han A B , Xiaohua Wu A B , Kailin Hou A C , Hongshan Zhang A C , Xueshuang Liang A C , Cheng Chen D , Zhijing Wang D and Chenjia Shen https://orcid.org/0000-0002-8575-0593 A C *
+ Author Affiliations
- Author Affiliations

A College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China.

B Zhejiang Key Laboratory of Organ Development and Regeneration, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China.

C Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou 310036, China.

D College of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China.

* Correspondence to: shencj@hznu.edu.cn

Handling Editor: Jian Feng Ma

Functional Plant Biology 50(4) 294-302 https://doi.org/10.1071/FP22243
Submitted: 9 October 2022  Accepted: 6 January 2023   Published: 23 January 2023

© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. is a heavy metal hyperaccumulator exhibiting extreme tolerance to various environmental stresses. To date, the functional role of Ca2+-binding protein in this plant is largely unknown. To investigate the function of calmodulins (CaMs) in N. caerulescens, CaM2, a Ca2+ sensor encoding gene, was identified and functionally characterised. Protein structure analysis showed that NcCaM2 contains four classic exchange factor (EF)-hand motifs with high sequence similarity to the CaM proteins from model plant Arabidopsis thaliana L. Tissue specific expression analysis showed that NcCaM2 is constitutively expressed in stems, leaves, and roots. Expression level of NcCaM2 was significantly upregulated under various environmental stimulus, indicating a potential involvement of NcCaM2 in the tolerance to abiotic stresses. The heterologous expression of NcCaM2 in a yeast mutant strain increased the heavy metal tolerance in yeast cells. Furthermore, the constitutive expression of NcCaM2 enhanced the heavy metal tolerance capability of transgenic tobacco (Nicotiana tabacum L.) plants. Our data suggested an important role of NcCaM2 in the responses to environmental stresses and provided a potential target gene to enhance of the ability to hyperaccumulate metals.

Keywords: abiotic stress, Ca2+ sensor, calmodulin, environmental stress, gene expression, heavy metal stress, heavy-metal tolerance, plant physiology.


References

Al-Quraan NA, Locy RD, Singh NK (2010) Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress. Plant Physiology and Biochemistry 48, 697–702.
Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress.Crossref | GoogleScholarGoogle Scholar |

Antunes G, Roque AC, Simoes de Souza FM (2016) Modelling intracellular competition for calcium: kinetic and thermodynamic control of different molecular modes of signal decoding. Scientific Reports 6, 23730
Modelling intracellular competition for calcium: kinetic and thermodynamic control of different molecular modes of signal decoding.Crossref | GoogleScholarGoogle Scholar |

Bhattacharjee S (2009) Involvement of calcium and calmodulin in oxidative and temperature stress of Amaranthus lividus L. during early germination. Journal of Environmental Biology 30, 557–562.

Boonburapong B, Buaboocha T (2007) Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins. BMC Plant Biology 7, 4
Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins.Crossref | GoogleScholarGoogle Scholar |

Cai K, Kuang L, Yue W, Xie S, Xia X, Zhang G, Wang J (2022) Calmodulin and calmodulin-like gene family in barley: identification, characterization and expression analyses. Frontiers in Plant Science 13, 964888
Calmodulin and calmodulin-like gene family in barley: identification, characterization and expression analyses.Crossref | GoogleScholarGoogle Scholar |

Cappetta E, Andolfo G, Di Matteo A, Ercolano MR (2020) Empowering crop resilience to environmental multiple stress through the modulation of key response components. Journal of Plant Physiology 246–247, 153134
Empowering crop resilience to environmental multiple stress through the modulation of key response components.Crossref | GoogleScholarGoogle Scholar |

Chinpongpanich A, Limruengroj K, Phean-o-pas S, Limpaseni T, Buaboocha T (2012) Expression analysis of calmodulin and calmodulin-like genes from rice, Oryza sativa L. BMC Research Notes 5, 625
Expression analysis of calmodulin and calmodulin-like genes from rice, Oryza sativa L.Crossref | GoogleScholarGoogle Scholar |

Craciun AR, Meyer C-L, Chen J, Roosens N, De Groodt R, Hilson P, Verbruggen N (2012) Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation. Journal of Experimental Botany 63, 4179–4189.
Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation.Crossref | GoogleScholarGoogle Scholar |

Fasani E, DalCorso G, Zorzi G, Agrimonti C, Fragni R, Visioli G, Furini A (2021) Overexpression of ZNT1 and NRAMP4 from the Ni hyperaccumulator Noccaea caerulescens population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni accumulation. International Journal of Molecular Sciences 22, 11896
Overexpression of ZNT1 and NRAMP4 from the Ni hyperaccumulator Noccaea caerulescens population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni accumulation.Crossref | GoogleScholarGoogle Scholar |

Ghorbel M, Zribi I, Missaoui K, Drira-Fakhfekh M, Azzouzi B, Brini F (2021) Differential regulation of the durum wheat Pathogenesis-related protein (PR1) by Calmodulin TdCaM1.3 protein. Molecular Biology Reports 48, 347–362.
Differential regulation of the durum wheat Pathogenesis-related protein (PR1) by Calmodulin TdCaM1.3 protein.Crossref | GoogleScholarGoogle Scholar |

Gulli M, Marchi L, Fragni R, Buschini A, Visioli G (2018) Epigenetic modifications preserve the hyperaccumulator Noccaea caerulescens from Ni geno-toxicity. Environmental and Molecular Mutagenesis 59, 464–475.
Epigenetic modifications preserve the hyperaccumulator Noccaea caerulescens from Ni geno-toxicity.Crossref | GoogleScholarGoogle Scholar |

Hashimoto K, Kudla J (2011) Calcium decoding mechanisms in plants. Biochimie 93, 2054–2059.
Calcium decoding mechanisms in plants.Crossref | GoogleScholarGoogle Scholar |

Huang Y, Chen Y, Liu Y, Mi R, Han X, Gong H, Cheng L, Chen Z (2022) Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection. Parasite Immunology 44, e12937
Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection.Crossref | GoogleScholarGoogle Scholar |

Landoni M, De Francesco A, Galbiati M, Tonelli C (2010) A loss-of-function mutation in Calmodulin2 gene affects pollen germination in Arabidopsis thaliana. Plant Molecular Biology 74, 235–247.
A loss-of-function mutation in Calmodulin2 gene affects pollen germination in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Lee H-J, Seo PJ (2021) Ca2+talyzing initial responses to environmental stresses. Trends in Plant Science 26, 849–870.
Ca2+talyzing initial responses to environmental stresses.Crossref | GoogleScholarGoogle Scholar |

Liu D, Yang Q (2020) Expression patterns of NbrgsCaM family genes in Nicotiana benthamiana and their potential roles in development and stress responses. Scientific Reports 10, 9652
Expression patterns of NbrgsCaM family genes in Nicotiana benthamiana and their potential roles in development and stress responses.Crossref | GoogleScholarGoogle Scholar |

Liu Y, Chen W, Liu L, Su Y, Li Y, Jia W, Jiao B, Wang J, Yang F, Dong F, Chai J, Zhao H, Lv M, Li Y, Zhou S (2022) Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in wheat (Triticum aestivum L.). Plant Signaling & Behavior 17, 2013646
Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in wheat (Triticum aestivum L.).Crossref | GoogleScholarGoogle Scholar |

McAinsh MR, Pittman JK (2009) Shaping the calcium signature. New Phytologist 181, 275–294.
Shaping the calcium signature.Crossref | GoogleScholarGoogle Scholar |

McCormack E, Tsai Y-C, Braam J (2005) Handling calcium signaling: Arabidopsis CaMs and CMLs. Trends in Plant Science 10, 383–389.
Handling calcium signaling: Arabidopsis CaMs and CMLs.Crossref | GoogleScholarGoogle Scholar |

Milner MJ, Mitani-Ueno N, Yamaji N, Yokosho K, Craft E, Fei Z, Ebbs S, Clemencia Zambrano M, Ma JF, Kochian LV (2014) Root and shoot transcriptome analysis of two ecotypes of Noccaea caerulescens uncovers the role of NcNramp1 in Cd hyperaccumulation. The Plant Journal 78, 398–410.
Root and shoot transcriptome analysis of two ecotypes of Noccaea caerulescens uncovers the role of NcNramp1 in Cd hyperaccumulation.Crossref | GoogleScholarGoogle Scholar |

Moyet L, Salvi D, Bouchnak I, Miras S, Perrot L, Seigneurin-Berny D, Kuntz M, Rolland N (2019) Calmodulin is involved in the dual subcellular location of two chloroplast proteins. Journal of Biological Chemistry 294, 17543–17554.
Calmodulin is involved in the dual subcellular location of two chloroplast proteins.Crossref | GoogleScholarGoogle Scholar |

Munir S, Khan MRG, Song J, Munir S, Zhang Y, Ye Z, Wang T (2016) Genome-wide identification, characterization and expression analysis of calmodulin-like (CML) proteins in tomato (Solanum lycopersicum). Plant Physiology and Biochemistry 102, 167–179.
Genome-wide identification, characterization and expression analysis of calmodulin-like (CML) proteins in tomato (Solanum lycopersicum).Crossref | GoogleScholarGoogle Scholar |

Niedbala G, Niazian M, Sabbatini P (2021) Modeling Agrobacterium-mediated gene transformation of tobacco (Nicotiana tabacum) – a model plant for gene transformation studies. Frontiers in Plant Science 12, 695110
Modeling Agrobacterium-mediated gene transformation of tobacco (Nicotiana tabacum) – a model plant for gene transformation studies.Crossref | GoogleScholarGoogle Scholar |

Nishida S, Tanikawa R, Ishida S, Yoshida J, Mizuno T, Nakanishi H, Furuta N (2020) Elevated expression of vacuolar nickel transporter gene IREG2 is associated with reduced root-to-shoot nickel translocation in Noccaea japonica. Frontiers in Plant Science 11, 610
Elevated expression of vacuolar nickel transporter gene IREG2 is associated with reduced root-to-shoot nickel translocation in Noccaea japonica.Crossref | GoogleScholarGoogle Scholar |

Ó Lochlainn S, Bowen HC, Fray RG, Hammond JP, King GJ, White PJ, Graham NS, Broadley MR (2011) Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens. PLoS ONE 6, e17814
Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens.Crossref | GoogleScholarGoogle Scholar |

Pollard AJ, Reeves RD, Baker AJM (2014) Facultative hyperaccumulation of heavy metals and metalloids. Plant Science 217-218, 8–17.
Facultative hyperaccumulation of heavy metals and metalloids.Crossref | GoogleScholarGoogle Scholar |

Raina M, Kumar A, Yadav N, Kumari S, Yusuf MA, Mustafiz A, Kumar D (2021) StCaM2, a calcium binding protein, alleviates negative effects of salinity and drought stress in tobacco. Plant Molecular Biology 106, 85–108.
StCaM2, a calcium binding protein, alleviates negative effects of salinity and drought stress in tobacco.Crossref | GoogleScholarGoogle Scholar |

Rascio N, Navari-Izzo F (2011) Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting? Plant Science 180, 169–181.
Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting?Crossref | GoogleScholarGoogle Scholar |

Rosca M, Cozma P, Minut M, Hlihor R-M, Betianu C, Diaconu M, Gavrilescu M (2021) New evidence of model crop Brassica napus L. in soil clean-up: comparison of tolerance and accumulation of lead and cadmium. Plants (Basel) 10, 2051
New evidence of model crop Brassica napus L. in soil clean-up: comparison of tolerance and accumulation of lead and cadmium.Crossref | GoogleScholarGoogle Scholar |

Sangchai P, Buaboocha T, Sirikantaramas S, Wutipraditkul N (2022) Changes in physiological responses of OsCaM1-1 overexpression in the transgenic rice under dehydration stress. Bioscience, Biotechnology and Biochemistry 86, 1211–1219.
Changes in physiological responses of OsCaM1-1 overexpression in the transgenic rice under dehydration stress.Crossref | GoogleScholarGoogle Scholar |

Shen C, Yang Y, Du L, Wang H (2015) Calmodulin-binding transcription activators and perspectives for applications in biotechnology. Applied Microbiology and Biotechnology 99, 10379–10385.
Calmodulin-binding transcription activators and perspectives for applications in biotechnology.Crossref | GoogleScholarGoogle Scholar |

Shen Q, Fu L, Su T, Ye L, Huang L, Kuang L, Wu L, Wu D, Chen Z-H, Zhang G (2020) Calmodulin HvCaM1 negatively regulates salt tolerance via modulation of HvHKT1s and HvCAMTA4. Plant Physiology 183, 1650–1662.
Calmodulin HvCaM1 negatively regulates salt tolerance via modulation of HvHKT1s and HvCAMTA4.Crossref | GoogleScholarGoogle Scholar |

Shi J, Du X (2020) Identification, characterization and expression analysis of calmodulin and calmodulin-like proteins in Solanum pennellii. Scientific Reports 10, 7474
Identification, characterization and expression analysis of calmodulin and calmodulin-like proteins in Solanum pennellii.Crossref | GoogleScholarGoogle Scholar |

Steinhorst L, Kudla J (2014) Signaling in cells and organisms – calcium holds the line. Current Opinion in Plant Biology 22, 14–21.
Signaling in cells and organisms – calcium holds the line.Crossref | GoogleScholarGoogle Scholar |

Vandelle E, Vannozzi A, Wong D, Danzi D, Digby A-M, Dal Santo S, Astegno A (2018) Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses. Plant Physiology and Biochemistry 129, 221–237.
Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses.Crossref | GoogleScholarGoogle Scholar |

Wang Y, Salt DE, Koornneef M, Aarts MGM (2022a) Construction and analysis of a Noccaea caerulescens TILLING population. BMC Plant Biology 22, 360
Construction and analysis of a Noccaea caerulescens TILLING population.Crossref | GoogleScholarGoogle Scholar |

Wang H, Feng M, Zhong X, Yu Q, Que Y, Xu L, Guo J (2022b) Identification of Saccharum CaM gene family and function characterization of ScCaM1 during cold and oxidant exposure in Pichia pastoris. Genes & Genomics 45, 103–122.
Identification of Saccharum CaM gene family and function characterization of ScCaM1 during cold and oxidant exposure in Pichia pastoris.Crossref | GoogleScholarGoogle Scholar |

Xuan Y, Zhou S, Wang L, Cheng Y, Zhao L (2010) Nitric oxide functions as a signal and acts upstream of AtCaM3 in thermotolerance in Arabidopsis seedlings. Plant Physiology 153, 1895–1906.
Nitric oxide functions as a signal and acts upstream of AtCaM3 in thermotolerance in Arabidopsis seedlings.Crossref | GoogleScholarGoogle Scholar |

Yang Y, Al-Baidhani HHJ, Harris J, Riboni M, Li Y, Mazonka I, Bazanova N, Chirkova L, Sarfraz Hussain S, Hrmova M, Haefele S, Lopato S, Kovalchuk N (2020) DREB/CBF expression in wheat and barley using the stress-inducible promoters of HD-Zip I genes: impact on plant development, stress tolerance and yield. Plant Biotechnology Journal 18, 829–844.
DREB/CBF expression in wheat and barley using the stress-inducible promoters of HD-Zip I genes: impact on plant development, stress tolerance and yield.Crossref | GoogleScholarGoogle Scholar |

Yu C, Guo H, Zhang Y, Song Y, Pi E, Yu C, Zhang L, Dong M, Zheng B, Wang H, Shen C (2017) Identification of potential genes that contributed to the variation in the taxoid contents between two Taxus species (Taxus media and Taxus mairei). Tree Physiology 37, 1659–1671.
Identification of potential genes that contributed to the variation in the taxoid contents between two Taxus species (Taxus media and Taxus mairei).Crossref | GoogleScholarGoogle Scholar |

Zeng H, Xu L, Singh A, Wang H, Du L, Poovaiah BW (2015) Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses. Frontiers in Plant Science 6, 600
Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses.Crossref | GoogleScholarGoogle Scholar |

Zhan X, Chen Z, Chen R, Shen C (2022) Environmental and genetic factors involved in plant protection-associated secondary metabolite biosynthesis pathways. Frontiers in Plant Science 13, 877304
Environmental and genetic factors involved in plant protection-associated secondary metabolite biosynthesis pathways.Crossref | GoogleScholarGoogle Scholar |

Zhang Z, Hou C, Tian W, Li L, Zhu H (2019) Electrophysiological studies revealed CaM1-mediated regulation of the Arabidopsis calcium channel CNGC12. Frontiers in Plant Science 10, 1090
Electrophysiological studies revealed CaM1-mediated regulation of the Arabidopsis calcium channel CNGC12.Crossref | GoogleScholarGoogle Scholar |

Zhang Y, Huang J, Hou Q, Liu Y, Wang J, Deng S (2021) Isolation and functional characterization of a salt-responsive calmodulin-like gene MpCML40 from semi-mangrove Millettia pinnata. International Journal of Molecular Sciences 22, 3475
Isolation and functional characterization of a salt-responsive calmodulin-like gene MpCML40 from semi-mangrove Millettia pinnata.Crossref | GoogleScholarGoogle Scholar |

Zhang X, Tian J, Li S, Liu Y, Feng T, Wang Y, Li Y, Huang X, Li D (2022) Characterization of the Calmodulin/Calmodulin-like Protein (CAM/CML) Family in Ginkgo biloba, and the influence of an ectopically expressed GbCML gene (Gb_30819) on seedling and fruit development of transgenic Arabidopsis. Plants 11, 1506
Characterization of the Calmodulin/Calmodulin-like Protein (CAM/CML) Family in Ginkgo biloba, and the influence of an ectopically expressed GbCML gene (Gb_30819) on seedling and fruit development of transgenic Arabidopsis.Crossref | GoogleScholarGoogle Scholar |