Site-specific regulation of transcriptional responses to cadmium stress in the hyperaccumulator, Sedum alfredii: based on stem parenchymal and vascular cells

  • Yan Hu
  • Lingling Xu
  • Shengke Tian
  • Lingli LuEmail author
  • Xianyong Lin


Key message

We compared the transcriptomes of parenchymal and vascular cells of Sedum alfredii stem under Cd stress to reveal gene regulatory networks underlying Cd hyperaccumulation.


Cadmium (Cd) hyperaccumulation in plants is a complex biological process controlled by gene regulatory networks. Efficient transport through vascular systems and storage by parenchymal cells are vital for Cd hyperaccumulation in the Cd hyperaccumulator Sedum alfredii, but the genes involved are poorly understood. We investigated the spatial gene expression profiles of transport and storage sites in S. alfredii stem using laser-capture microdissection coupled with RNA sequencing. Gene expression patterns in response to Cd were distinct in vascular and parenchymal cells, indicating functional divisions that corresponded to Cd transportation and storage, respectively. In vascular cells, plasma membrane-related terms enriched a large number of differentially-expressed genes (DEGs) for foundational roles in Cd transportation. Parenchymal cells contained considerable DEGs specifically concentrated on vacuole-related terms associated with Cd sequestration and detoxification. In both cell types, DEGs were classified into different metabolic pathways in a similar way, indicating the role of Cd in activating a systemic stress signalling network where ATP-binding cassette transporters and Ca2+ signal pathways were probably involved. This study identified site-specific regulation of transcriptional responses to Cd stress in S. alfredii and analysed a collection of genes that possibly function in Cd transportation and detoxification, thus providing systemic information and direction for further investigation of Cd hyperaccumulation molecular mechanisms.


Cadmium Laser-capture microdissection RNA-seq Transporter Hyperaccumulation Signalling 



The investigation of Cd distribution imaging by µ-XRF was conducted at the Advanced Photon Source, USA (Proposal No.: 23899) and we thank all the staff there. We also sincerely thank Vazyme Biotech (Nanjing, China) for their RNA sequencing service.

Author contributions

LL supervised and designed the experiments; YH and LX performed most of the experiments and analysed data with contributions from ST; YH and LL conceived the project and wrote the manuscript with contributions from XL and ST.


This work was supported by National Natural Science Foundation of China projects (31672235, 31471939), and the project from National Key Research and Development Program of China (2016YFD0800401).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

11103_2019_821_MOESM1_ESM.docx (276 kb)
Supplementary material 1 (DOCX 275 KB)


  1. Ali H, Khan E, Sajad MA (2013) Phytoremediation of heavy metals—Concepts and applications. Chemosphere 91:869–881CrossRefGoogle Scholar
  2. Andolfo G, Ruocco M, Di Donato A, Frusciante L, Lorito M, Scala F, Ercolano MR (2015) Genetic variability and evolutionary diversification of membrane ABC transporters in plants. BMC Plant Biol 15:51CrossRefGoogle Scholar
  3. Arazi T, Kaplan B, Sunkar R, Fromm H (2000) Cyclic-nucleotide- and Ca2+/calmodulin-regulated channels in plants: targets for manipulating heavy-metal tolerance, and possible physiological roles. Biochem Soc Trans 28(4):471–475CrossRefGoogle Scholar
  4. Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G (2004) GO::TermFinder—open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20:3710–3715CrossRefGoogle Scholar
  5. Carrio-Segui A, Garcia-Molina A, Sanz A, Penarrubia L (2015) Defective copper transport in the copt5 mutant affects cadmium tolerance. Plant Cell Physiol 56:442–454CrossRefGoogle Scholar
  6. Chen YK, Zhi JK, Zhang H, Li J, Zhao QH, Xu JC (2017) Transcriptome analysis of Phytolacca americana L. in response to cadmium stress. PLoS ONE 12:e0184681CrossRefGoogle Scholar
  7. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676CrossRefGoogle Scholar
  8. Cosio C, DeSantis L, Frey B, Diallo S, Keller C (2005) Distribution of cadmium in leaves of Thiaspi caerulescens. J Exp Bot 56:765–775CrossRefGoogle Scholar
  9. Craciun AR, Meyer CL, Chen J, Roosens N, Groodt RD, 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. J Exp Bot 63(11):4179–4189CrossRefGoogle Scholar
  10. Curie C, Alonso JM, Jean ML, Ecker JR, Briat JF (2000) Involvement of NRAMP1 from Arabidopsis thaliana in iron transport. Biochem J 347(3):749–755CrossRefGoogle Scholar
  11. Ebbs SD, Zambrano MC, Spiller SM, Newville M (2009) Cadmium sorption, influx, and efflux at the mesophyll layer of leaves from ecotypes of the Zn/Cd hyperaccumulator Thlaspi caerulescens. New Phytol 181:626–636CrossRefGoogle Scholar
  12. Feng Y, Wu YJ, Zhang J, Meng Q, Wang Q, Ma LY, Ma XX, Yang XE (2018) Ectopic expression of SaNRAMP3 from Sedum alfredii enhanced cadmium root-to-shoot transport in Brassica juncea. Ecotoxicol Environ Saf 156:279–286CrossRefGoogle Scholar
  13. Gallagher RI, Steven RB, Liotta LA, Espina V (2012) Laser capture microdissection: ArcturusXT infrared capture and UV cutting methods. Methods Mol Biol 23:157–178CrossRefGoogle Scholar
  14. Gao J, Sun L, Yang XE, Liu JX (2013) Transcriptomic analysis of cadmium stress response in the heavy metal hyperaccumulator Sedum alfredii Hance. PLoS ONE 8:e64643CrossRefGoogle Scholar
  15. González E, Joly S (2013) Impact of RNA-seq attributes on false positive rates in differential expression analysis of de novo assembled transcriptomes. BMC Res Notes 6:503CrossRefGoogle Scholar
  16. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Xian A, Fan L, Raychowdhury R, Zeng Q (2011) Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat Biotechnol 29:644–652CrossRefGoogle Scholar
  17. Hou D, Kai W, Liu T, Wang H, Zhi L, Jie Q, Lu L, Tian S (2017) Unique Rhizosphere Micro-characteristics Facilitate Phytoextraction of Multiple Metals in Soil by the Hyperaccumulating Plant Sedum alfredii. Environ Sci Technol 51(10):5675CrossRefGoogle Scholar
  18. Hu Y, Tian S, Foyer HC, Hou D, Wang H, Zhou W, Liu T, Ge J, Lu L, Lin X (2019) Efficient phloem transport significantly remobilizes cadmium from old to young organs in a hyperaccumulator Sedum alfredii. J Hazard Mater 365:421–429CrossRefGoogle Scholar
  19. Isaure MP, Huguet S, Meyer CL, Castillo-Michel H, Testemale D, Vantelon D, Saumitou-Laprade P, Verbruggen N, Sarret G (2015) Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the non-accumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques. J Exp Bot 66:3201–3214CrossRefGoogle Scholar
  20. Ishimaru Y, Takahashi R, Bashir K, Shimo H, Senoura T, Sugimoto K, Ono K, Yano M, Ishikawa S, Arao T, Nakanishi H, Nishizawa NK (2012) Characterizing the role of rice NRAMP5 in manganese, iron and cadmium transport. Sci Rep 2:286CrossRefGoogle Scholar
  21. Kim DY, Bovet L, Maeshima M, Martinoia E, Lee Y (2007) The ABC transporter AtPDR8 is a cadmium extrusion pump conferring heavy metal resistance. Plant J 50:207–218CrossRefGoogle Scholar
  22. Klaassen CD, Liu J, Choudhuri S (1999) Metallothionein: An intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39:267–294CrossRefGoogle Scholar
  23. Koren Š, Arčon I, Kump P, Nečemer M, Vogel-mikuš K (2013) Influence of CdCl2 and CdSO4 supplementation on Cd distribution and ligand environment in leaves of the Cd hyperaccumulator Noccaea (Thlaspi) praecox. Plant Soil 370:125–148CrossRefGoogle Scholar
  24. Korenkov V, Park S, Cheng NH, Sreevidya C, Lachmansingh J, Morris J, Hirschi K, Wagner GJ (2007) Enhanced Cd2+ selective root-tonoplast-transport in tobaccos expressing Arabidopsis cation exchangers. Planta 225:403–411CrossRefGoogle Scholar
  25. Kramer U (2010) Metal hyperaccumulation in plants. Annu Rev Plant Biol 61:517–534CrossRefGoogle Scholar
  26. Küpper H, Kochian LV (2010) Transcriptional regulation of metal transport genes and mineral nutrition during acclimatization to cadmium and zinc in the Cd/Zn hyperaccumulator, Thlaspi caerulescens (Ganges population). New Phytol 185:114–129CrossRefGoogle Scholar
  27. Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212:75–84CrossRefGoogle Scholar
  28. Lanquar V, Ramos MSl, Lelièvre F, Barbier-Brygoo H, Krieger-Liszkay A, Krämer U, Thomine S (2010) Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deficiency. Plant Physiol 152(4):1986–1999CrossRefGoogle Scholar
  29. Laurent C, Lekeux G, Ukuwela AA, Xiao Z, Charlier JB, Bosman B et al (2016) Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis. Plant Mol Biol 90:453–466CrossRefGoogle Scholar
  30. Lecourieux D, Raneva R, Pugin A (2006) Calcium in plant defence-signalling pathways. New Phytol 171:249–269CrossRefGoogle Scholar
  31. Liu H, Zhao H, Wu L, Liu A, Zhao FJ, Xu W (2017) Heavy metal ATPase 3 (HMA3) confers cadmium hypertolerance on the cadmium/zinc hyperaccumulator Sedum plumbizincicola. New Phytol 215(2):687CrossRefGoogle Scholar
  32. Lu LL, Tian SK, Yang XE, Wang XC, Brown P, Li TQ, He ZL (2008) Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii. J Exp Bot 59(11):3203–3213CrossRefGoogle Scholar
  33. Mendoza-Cozatl DG, Xie QQ, Akmakjian GZ, Jobe TO, Patel A, Stacey MG, Song LH, Demoin DW, Jurisson SS, Stacey G et al (2014) OPT3 is a component of the iron-signaling network between leaves and roots and misregulation of OPT3 leads to an over-accumulation of cadmium in seeds. Mol Plant 7:1455–1469CrossRefGoogle Scholar
  34. Migocka M, Kosieradzka A, Papierniak A, Maciaszczyk-Dziubinska E, Posyniak E, Garbiec A, Filleur S (2014) Two metal-tolerance proteins, MTP1 and MTP4, are involved in Zn homeostasis and Cd sequestration in cucumber cells. J Exp Bot 66:1001–1015CrossRefGoogle Scholar
  35. Milner MJ, Mitani-Ueno N, Yamaji N, Yokosho K, Craft E, Fei ZJ, Ebbs S, Zambrano MC, Ma JF, Kochian LV (2014) Root and shoot transcriptome analysis of two ecotypes of Noccaea caerulescens uncovers the role of NcNramp1 in Cd hyperaccumulation. Plant J 78:398–410CrossRefGoogle Scholar
  36. Miyadate H, Adachi S, Hiraizumi A, Tezuka K, Nakazawa N, Kawamoto T, Katou K, Kodama I, Sakurai K, Takahashi H (2011) OsHMA3, a P1B-type of ATPase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles. New Phytol 189(1):190–199CrossRefGoogle Scholar
  37. Morel M, Crouzet J, Gravot A, Auroy P, Leonhardt N, Vavasseur A, Richaud P (2009) AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis. Plant Physiol 149(2):894–904CrossRefGoogle Scholar
  38. Nakazono M, Qiu F, Borsuk LA, Schnable PS (2003) Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15:583–596CrossRefGoogle Scholar
  39. Navariizzo F (2011) Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting? Plant Sci 180:169–181CrossRefGoogle Scholar
  40. Nouet C, Charlier JB, Carnol M, Bosman B, Farnir F, Motte P, Hanikenne M (2015) Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri. J Exp Bot 66(19):5783–5795CrossRefGoogle Scholar
  41. Ogo Y, Kakei Y, Itai RN, Kobayashi T, Nakanishi H, Takahashi H, Nakazono M, Nishizawa NK (2014) Spatial transcriptomes of iron-deficient and cadmium-stressed rice. New Phytol 201:781–794CrossRefGoogle Scholar
  42. Park J, Song WY, Ko D, Eom Y, Hansen TH, Schiller M, Lee TG, Martinoia E, Lee Y (2012) The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury. Plant J 69:278–288CrossRefGoogle Scholar
  43. Peng J, Ding G, Meng S, Yi H, Gong J (2017) Enhanced metal tolerance correlates with heterotypic variation in SpMTL, a metallothionein-like protein from the hyperaccumulator Sedum Plumbizincicola. Plant Cell Environ 40(8):1368-1378CrossRefGoogle Scholar
  44. Peralta-Videa JR, Lopez ML, Narayan M, Saupe G, Gardea-Torresdey J (2009) The biochemistry of environmental heavy metal uptake by plants: Implications for the food chain. Int J Biochem Cell B 41:1665–1677CrossRefGoogle Scholar
  45. Perochon A, Aldon D, Galaud JP, Ranty B (2011) Calmodulin and calmodulin-like proteins in plant calcium signaling. Biochimie 93:2048–2053CrossRefGoogle Scholar
  46. Rodríguez-Serrano M, Romero-Puertas MC, Zabalza AN, Corpas FJ, Gomez M, Del Rio LA, Sandalio LM (2006) Cadmium effect on the oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of ROS and NO accumulation in vivo. Plant Cell Environ 29:1532–1544CrossRefGoogle Scholar
  47. Sancenón V, Puig S, Mateu-Andrés I, Dorcey E, Thiele DJ, Peñarrubia L (2004) The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development. J Biol Chem 279:15348–15355CrossRefGoogle Scholar
  48. Sasaki A, Yamaji N, Yokosho K, Ma JF (2012) Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice. Plant Cell 24(5):2155–2167CrossRefGoogle Scholar
  49. Shi X, Sun HJ, Chen YT, Pan HW, Wang SF (2016) Transcriptome sequencing and expression analysis of cadmium (Cd) transport and detoxification related genes in Cd-accumulating Salix integra. Front Plant Sci 7:1577Google Scholar
  50. Shigaki T, Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cation/H+ exchangers in plants. Plant Biol 8:419–429CrossRefGoogle Scholar
  51. Tian SK, Lu LL, Labavitch J, Yang XE, He ZL, Hu HN, Sarangi R, Newville M, Commisso J, Brown P (2011a) Cellular sequestration of cadmium in the hyperaccumulator plant species Sedum alfredii. Plant Physiol 157:1914–1925CrossRefGoogle Scholar
  52. Tian SK, Lu LL, Zhang J, Wang K, Brown P, He ZL, Liang J, Yang XE (2011b) Calcium protects roots of Sedum alfredii H. against cadmium-induced oxidative stress. Chemosphere 84(1):63–69CrossRefGoogle Scholar
  53. Tian S, Xie R, Wang H, Hu Y, Ge J, Liao X, Gao X, Brown PH, Lin X, Lu L (2016) Calcium deficiency triggers phloem remobilization of cadmium in a hyper–accumulating species. Plant Physiol 172:2300–2313CrossRefGoogle Scholar
  54. Tian SK, Xie RH, Wang HX, Hu Y, Hou DD, Liao XC, Brown PH, Yang HX, Lin XY, Labavitch JM et al (2017) Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii. J Exp Bot 68:2387–2398CrossRefGoogle Scholar
  55. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578CrossRefGoogle Scholar
  56. Ueno D, Yamaji N, Kono I, Huang CF, Ando T, Yano M, Ma JF (2010) Gene limiting cadmium accumulation in rice. Proc Natl Acad Sci USA 107(38):16500–16505CrossRefGoogle Scholar
  57. Ueno D, Milner MJ, Yamaji N, Yokosho K, Koyama E, Zambrano MC, Kaskie M, Ebbs S, Kochian LV, Ma JF (2011) Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens. Plant J 66(5):852–862CrossRefGoogle Scholar
  58. Verbruggen N, Hermans C, Schat H (2009) Molecular mechanism of metal hyperaccumulation in plants. New Phytol 181:759–776CrossRefGoogle Scholar
  59. Vogel-Mikuš K, Simčič J, Pelicon P, Budnar M, Kump P, Nečemer M, Mesjasz-Przybyłowicz J, Przybyłowicz WJ, Regvar M (2008) Comparison of essential and non-essential element distribution in leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as revealed by micro-PIXE. Plant Cell Environ 31:1484–1496CrossRefGoogle Scholar
  60. Weber M, Harada E, Vess C, Roepenack-Lahaye EV, Clemens S (2004) Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J 37:269–281CrossRefGoogle Scholar
  61. White PJ (2006) A comparison of the Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes. New Phytol 170:239–260CrossRefGoogle Scholar
  62. White PJ, Broadley MR (2003) Calcium in plants. Ann Bot 92:487–511CrossRefGoogle Scholar
  63. Wojcik M, Vangronsveld J, D’Haen J, Tukiendorf A (2005) Cadmium tolerance in Thlaspi caerulescens-II. Localization of cadmium in Thlaspi caerulescens. Environ Exp Bot 53:163–171Google Scholar
  64. Wu Q, Shigaki T, Williams KA, Han JS, Kim CK, Hirschi KD, Park S (2011) Expression of an Arabidopsis Ca2+/H+ antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation. J Plant Physiol 168:167–173CrossRefGoogle Scholar
  65. Xu J, Sun J, Du L, Liu X (2012) Comparative transcriptome analysis of cadmium responses in Solanum nigrum and Solanum torvum. New Phytol 196:110–124CrossRefGoogle Scholar
  66. Yamaguchi H, Fukuoka H, Arao T, Ohyama A, Nunome T, Miyatake K, Negoro S (2010) Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum. J Exp Bot 61:423–437CrossRefGoogle Scholar
  67. Yang XE, Stoffella PJ (2004) Cadmium tolerance and hyperaccumulation in a new zn-hyperaccumulating plant species (Sedum Alfredii Hance). Plant Soil 259:181–189CrossRefGoogle Scholar
  68. Zhang Z, Yu Q, Du H, Ai W, Yao X, Mendoza-Cózatl DG, Qiu B (2016) Enhanced cadmium efflux and root-to-shoot translocation are conserved in the hyperaccumulator Sedum alfredii (Crassulaceae family). FEBS Lett 590(12):1757CrossRefGoogle Scholar
  69. Zheng LQ, Yamaji N, Yokosho K, Ma JF (2012) YSL16 is a phloem-localized transporter of the copper-nicotianamine complex that is responsible for copper distribution in rice. Plant Cell 24:3767–3782CrossRefGoogle Scholar
  70. Zhou Q, Guo JJ, He CT, Shen C, Huang YY, Chen JX, Guo JH, Yuan JG, Yang ZY (2016) Comparative transcriptome analysis between low- and high-cadmium-accumulating genotypes of pakchoi (Brassica chinensis L.) in response to cadmium stress. Environ Sci Technol 50:6485–6494CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Zhejiang Provincial Key Laboratory of Subtropic Soil and Plant Nutrition, College of Environmental & Resource ScienceZhejiang UniversityHangzhouChina
  2. 2.Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource ScienceZhejiang UniversityHangzhouChina

Personalised recommendations