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Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana

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Abstract

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We found that embryogenic tissues of Picea balfouriana adopt different physiological pathways for long-term proliferation with 6-benzylaminopurine treatment and identified the miRNAs closely associated with proliferation.

Abstract

The long-term maintenance of somatic embryo production capacity in conifer embryogenic tissue (ET) is essential for the production of vigorous somatic seedlings. However, this ability is often lost after several months of proliferation in many conifer species including Picea balfouriana. Cytokinins are known to influence several important physiological processes during plant growth and development, including somatic embryogenesis (SE). In this study, we found that the 6-benzylaminopurine (BA) concentration influenced the yields of P. balfouriana somatic embryos and their germination response. Only ET of P. balfouriana proliferated on medium supplemented with 3.6 µM BA produced somatic embryos that germinated into normal plants. Most hormone levels increased in ET after prolonged proliferation. Moreover, antioxidant enzyme activities and polyamine contents were also significantly changed after 8 months of culture, which might be modulated by accumulated zeatin riboside (ZR). Finally, some selected microRNAs and their target genes were confirmed to be involved in the proliferation of ET of P. balfouriana and they also might be regulated by accumulated ZR. These findings may facilitate efforts to clarify basic physiological processes after the long-term proliferation stage of SE in conifers and delay the decreased production capacity of somatic embryos.

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Abbreviations

BA:

6-Benzylaminopurine

ABA:

Abscisic acid

ET:

Embryogenic tissue

FW:

Fresh weight

IAA:

Indole-3-acetic acid

PGR:

Plant growth regulator

POD:

Peroxidase

Put:

Putrescine

qRT-PCR:

Quantitative reverse transcription-polymerase chain reaction

SE:

Somatic embryogenesis

SOD:

Superoxide dismutase

Spd:

Spermidine

Spm:

Spermine

ZR:

Zeatin riboside

References

  • Abrahamsson M, Clapham D, von Arnold S (2018) Somatic embryogenesis in Scots pine (Pinus sylvestris L.). In: Jain S, Gupta P (eds) Step wise protocols for somatic embryogenesis of important woody plants, vol 84. Springer, Cham, pp 123–133 Forestry Sciences

    Chapter  Google Scholar 

  • Anil US, Rao KS (2000) Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger. Plant Physiol 123(4):1301–1312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bairu MW, Stirk WA, Dolezal K, Van Staden J (2007) Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell Tissue Organ Cult 90:15–23

    Article  CAS  Google Scholar 

  • Breton D, Harvengt L, Trontin JF, Bouvet A, Favre JM (2006) Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine. Plant Cell Tissue Organ Cult 87:95–108

    Article  Google Scholar 

  • Centeno ML, Rodríguez R, Berros B, Rodríguez A (1997) Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons. Plant Cell Rep 17:139–144

    Article  CAS  PubMed  Google Scholar 

  • Corredoira E, Ballester A, Ibarra M, Vieitez AM (2015) Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees. Tree Physiol 35:678–690

    Article  CAS  PubMed  Google Scholar 

  • Correia S, Vinhas R, Manadas B, Lourenço AS, Veríssimo P, Canhoto JM (2012) Comparative proteomic analysis of auxin-induced embryogenic and nonembryogenic tissues of the solanaceous tree Cyphomandra betacea (Tamarillo). J Proteome Res 11:1666–1675

    Article  CAS  PubMed  Google Scholar 

  • Díaz-Vivancos P, Majourhat K, Fernández JA, Hernández JA, Piqueras A (2011) Study of the antioxidant enzymatic system during shoot development from cultured intercalar meristems of saffron. Plant Growth Regul 65:119–126

    Article  CAS  Google Scholar 

  • Filipović BK, Simonović AD, Trifunovic MM, Dmitrović SS, Savić JM, Jevremović SB, Subotić AR (2015) Plant regeneration in leaf culture of Centaurium erythraea Rafn. Part 1: the role of antioxidant enzymes. Plant Cell Tissue Organ Cult 121:703–719

    Article  CAS  Google Scholar 

  • Galston AW, Flores HE (1991) Polyamines and plant morphogenesis. In: Slocum R, Flores HE (eds) The biochemistry and physiology of polyamines in plants. CRC Press, Boca Raton, pp 175–186

    Google Scholar 

  • Gemperlová L, Fischerová L, Cvikrová M, Malá J, Vondráková Z, Martincová O, Vágner M (2009) Polyamine profiles and biosynthesis in somatic embryo development and comparison of germinating somatic and zygotic embryos of Norway spruce. Tree Physiol 29:1287–1298

    Article  CAS  PubMed  Google Scholar 

  • Grieb B, Schafer F, Imani J, Mashayekhi KN, Arnholdt-Schmitt B, Neumann KH (1997) Changes in soluble proteins and phytohormone concentrations of cultured carrot petiole explants during induction of somatic embryogenesis (Daucus carota L.). J Appl Bot Food Qual 71:94–103

    CAS  Google Scholar 

  • Grzyb M, Kalandyk A, Waligórski P, Mikuła A (2017) The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern Cyathea delgadii Sternb. Plant Cell Tissue Organ Cult 129:387–397

    Article  CAS  Google Scholar 

  • Gupta PK, Holmstrom D (2005) Double staining technology for distinguishing embryogenic cultures. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, The Netherlands, pp 573–575

    Chapter  Google Scholar 

  • Häggman H, Vuosku J, Kestilä J, Suorsa M, Ruottinen M, Neubauer P, Läärä E, Sarjala T (2011) The role of spermidine and spermine in cell function and pattern formation in Scots pine embryogenesis. In: Park Y-S, Bonga JM, Park S-Y, Moon H-K (eds) Advances in somatic embryogenesis of trees and its application for the future forests and plantations. IUFRO working party 2.09.02: Somatic embryogenesis and other clonal propagation methods of forest trees, August 19–21 (Suwon, South Korea) pp 100–102

  • Hepler PK (2005) Calcium: a central regulator of plant growth and development. Plant Cell 17:2142–2155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jo L, Santos AL, Bueno CA, Barbosa HR, Floh EI (2013) Proteomic analysis and polyamines, ethylene and reactive oxygen species levels of Araucaria angustifolia (Brazilian pine) embryogenic cultures with different embryogenic potential. Tree Physiol 34:94–104

    Article  CAS  PubMed  Google Scholar 

  • Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-Walter MA (2009) Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.). Vitr Cell Dev Biol Plant 45:20–33

    Article  Google Scholar 

  • Klimaszewska K, Hargreaves C, Lelu-Walter MA, Trontin JF (2016) Chap. 7 Advances in conifer somatic embryogenesis since year 2000. In: Germanà MA, Lambardi M (eds) In Vitro embryogenesis in higher plants. Methods in molecular biology, Springer, New York, pp 131–162

    Chapter  Google Scholar 

  • Kong LS, Attree SM, Fowke LC (1997) Changes of endogenous hormone levels in developing seeds, zygotic embryos and megagametophytes in Picea glauca. Physiol Plant 101(1):23–30

    Article  CAS  Google Scholar 

  • Kvaalen H, Daehlen OG, Rognstad AT, Grønstad B, Egertsdotter U (2005) Somatic embryogenesis for plant production of Abies lasiocarpa. Can J For Res 35:1053–1060

    Article  Google Scholar 

  • Lelu-Walter MA, Pâques LE (2009) Simplified and improved somatic embryogenesis of hybrid larches (Larix × eurolepis and Larix × marschlinsii): perspectives for breeding. Ann For Sci 66:1–104

    Article  Google Scholar 

  • Li ZL, Burritt DJ (2003) Changes in endogenous polyamines during the formation of somatic embryos from isogenic lines of Dactylis glomerata L. with different regenerative capacities. Plant Growth Regul 40:65–74

    Article  CAS  Google Scholar 

  • Li WF, Zhang SG, Han SY, Wu T, Zhang JH, Qi LW (2013) Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr. Plant Cell Tissue Organ Cult 113:131–136

    Article  CAS  Google Scholar 

  • Li QF, Zhang SG, Wang JH (2014a) Transcriptome analysis of callus from P. balfouriana. BMC Genom 15:533

    Article  CAS  Google Scholar 

  • Li QF, Zhang SG, Wang JH (2014b) Transcriptomic and proteomic analyses of embryogenic tissues in P. balfouriana treated by 6-benzylaminopurine. Physiol Plant 154:95–113

    Article  CAS  PubMed  Google Scholar 

  • Li QF, Wang JH, Pulkkinen P, Kong LS (2015) Changes in the metabolome of P. balfouriana ETs that were linked to different levels of 6-BAP by gas chromatography-mass spectrometry approach. PloS One 10(10):e0141841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li QF, Deng C, Xia Y, Kong LS, Zhang HG, Zhang SG, Wang JH (2017) Identification of novel miRNAs and miRNA expression profiling in embryogenic tissues of Picea balfouriana treated by 6-benzylaminopurine. PloS One 12(5):e0176112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Litvay JD, Verma DC, Johnson MA (1985) Influence of loblolly pine (Pinus taeda L.) culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328

    Article  CAS  PubMed  Google Scholar 

  • Maruyama E, Tanaka T, Hosoi Y, Ishii K, Morohoshi N (2000) Embryogenic cell culture, protoplast regeneration, cryopreservation, biolistic gene transfer and plant regeneration in Japanese cedar (Cryptomeria japonica D. Don). Plant Biotechnol 17:281–296

    Article  CAS  Google Scholar 

  • Montalbán IA, Novák O, Rolčik J, Strnad M, Moncaleán P (2013) Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees. Physiol Plant 148:214–231

    Article  CAS  PubMed  Google Scholar 

  • Müller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453:1094–1097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Niemi K, Haggman H, Sarjala T (2002) Effects of exogenous diamines on the interaction between ectomycorrhizal fungi and adventitious root formation in Scots pine in vitro. Tree Physiol 22:373–381

    Article  CAS  PubMed  Google Scholar 

  • Ouakfaoui SE, Schnell J, Abdeen A, Colville A, Labbé H, Han SY, Baum B, Laberge S, Miki B (2010) Control of somatic embryogenesis and embryo development by AP2 transcription factors. Plant Mol Biol 74:313–326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Plačková L, Hrdlička J, Smýkalová I, Cvečková M, Novák O, Griga M, Doležal K (2015) Cytokinin profiling of long-term in vitro pea (Pisum sativum L.) shoot cultures. Plant Growth Regul 77:125–132

    Article  CAS  Google Scholar 

  • Sala K, Potocka I, Kurczynska E (2013) Spatio-temporal distribution and methyl-esterification of pectic epitopes provide evidence of developmental regulation of pectins during somatic embryogenesis in Arabidopsis thaliana. Biol Plant 57:410–416

    Article  CAS  Google Scholar 

  • Salo HM, Sarjala T, Jokela A, Häggman H, Vuosku J (2015) Moderate stress responses and specific changes in polyamine metabolism characterize Scots pine somatic embryogenesis. Tree Physiol 36:392–402

    Article  CAS  Google Scholar 

  • Santa-Catarina C, Silveira V, Balbuena TS, Viana AM, Estelita MEM, Handro W, Floh EI (2006) IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis. Plant Growth Regul 49:237–247

    Article  CAS  Google Scholar 

  • Shi J, Fu XZ, Peng T, Huang XS, Fan QJ, Liu JH (2010) Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiol 30:914–922

    Article  CAS  Google Scholar 

  • Stirk WA, Van SJ (2014) Plant growth regulators in seaweeds: occurrence, regulation and functions. In: Nathalie B (ed) Advances in botanical research, vol 71. Academic Press, Amsterdam, pp 125–159

    Google Scholar 

  • Synkova H, Semoradova S, Schnablova R, Witters E, Husak M, Valcke R (2006) Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny. Biol Plant 50:31–41

    Article  CAS  Google Scholar 

  • Tang W, Newton RJ (2005) Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos. Plant Physiol Biochem 43:760–769

    Article  CAS  PubMed  Google Scholar 

  • Tian M, Gu Q, Zhu M (2003) The involvement of hydrogen peroxide and antioxidant enzymes in the process of shoot organogenesis of strawberry callus. Plant Sci 165:701–707

    Article  CAS  Google Scholar 

  • Trontin J-F, Reymond I, Quoniou S, Canlet F, Debille S, Bruneau G, Harvengt L, Le Metté C, Vallance M, Teyssier C, Lable P, Lelu-Walter MA (2011) An overview of current achievements and shortcomings in developing Maritime pine somatic embryogenesis and enabling technologies in France. In: Park Y-S, Bonga JM, Park S-Y, Moon H-K (eds) Advances in somatic embryogenesis of trees and its application for the future forests and plantations. IUFRO working party 2.09.02: Somatic embryogenesis and other clonal propagation methods of forest trees, August 19–21 2010 (Suwon, South Korea) pp 132

  • Valero-Aracama C, Kane M, Wilson S, Philman N (2010) Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to acclimatize sea oats (Uniola paniculata L.) genotypes. Plant Growth Regul 60:43–49

    Article  CAS  Google Scholar 

  • Vanneste S, Friml J (2013) Calcium: the missing link in auxin action. Plants 2(4):650–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wickramasuriya AW, Dunwell JM (2015) Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro. BMC Genom 16:301

    Article  CAS  Google Scholar 

  • Yang Y, Han C, Liu Q, Lin B, Wang JW (2008) Effect of drought and low light on growth and enzymatic antioxidant system of Picea asperata seedlings. Acta Physiol Plant 30(4):433–440

    Article  CAS  Google Scholar 

  • Yang X, Zhang X, Yuan D, Jin F, Zhang Y, Xu J (2012) Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton. BMC Plant Biol 12:110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang ZF, Finer JJ (2016) Use of cytokinin pulse treatments and micrografting to improve sunflower (Helianthus annuus L.) plant recovery from cotyledonary tissues of mature seeds. In Vitr Cell Dev Biol Plant 52:391–399

    Article  CAS  Google Scholar 

  • Żur I, Dubas E, Krzewska M, Waligórski P, Dziurka M, Janowiak F (2015) Hormonal requirements for effective induction of microspore embryogenesis in triticale (× Triticosecale Wittm.) anther cultures. Plant Cell Rep 34:47–62

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the Forestry Station of Xianggelila Country in Yunnan Province for cone sampling in the P. balfouriana seed orchard. This study was supported by grants from the General Financial Grant from China Postdoctoral Science Foundation (2017M622717) and the Fundamental Research Funds for the Central Nonprofit Research Institution of the Chinese Academy of Forestry (TGB2013011).

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Author notes

  1. Qingfen Li and Cheng Deng contributed equally to this work.

Authors and Affiliations

  1. Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, 510642, Guangdong, China

    Qingfen Li, Cheng Deng & Xiaoyang Chen

  2. State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, China

    Tianqing Zhu, Juanjuan Ling, Shougong Zhang & Junhui Wang

  3. State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, 150040, Hei Longjiang, China

    Hanguo Zhang

  4. Department of Biology, Centre for Forest Biology, University of Victoria, Victoria, BC, V8P 5C2, Canada

    Lisheng Kong

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  1. Qingfen Li
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  2. Cheng Deng
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  3. Tianqing Zhu
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Correspondence to Xiaoyang Chen.

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Supplementary material 1 (DOC 39 KB)

Supplementary material 2 (DOC 40 KB)

468_2018_1793_MOESM3_ESM.tif

Supplementary material 3 Endogenous hormone levels during different stages of proliferation at three BA concentrations (S1 was the ETs initiated from initiation medium and then proliferated on medium with 10 μM 2,4-D and 5 μM BA for 2 weeks; S2 was the ETs proliferated on medium as above for 8 months; S3 was the ETs proliferated for 8 months and then cultured for 1 week, on medium without PGRs). a–d Changes in IAA (a), GA3 (b), ABA (c), and ZR (d) levels. The black uppercase or lowercase letters show the significance of differences among the three stages under each BA treatment (lowercase and uppercase indicate significant (P < 0.05) and very significant (P < 0.01), respectively). The pink and blue lowercase letters show the significance of differences among three BA treatments in the S2 and S3 stages, respectively. (TIF 1035 KB)

468_2018_1793_MOESM4_ESM.tif

Supplementary material 4 Ratios of different hormones among the three concentrations of BA. a–c Changes in the ratios of ABA/ZR (a), ABA/IAA (b), and IAA/ZR (c). (TIF 2090 KB)

468_2018_1793_MOESM5_ESM.tif

Supplementary material 5 Activities of antioxidant enzymes during different stages of proliferation under BA treatments. a, b Changes in the activity of SOD (a) and POD (b). (TIF 677 KB)

468_2018_1793_MOESM6_ESM.tif

Supplementary material 6 Polyamine contents at different stages of proliferation under BA treatments. a–c Changes in Put (a), Spd (b), and Spm (c). (TIF 499 KB)

468_2018_1793_MOESM7_ESM.tif

Supplementary material 7 Changes of physiological index and miRNAs levels in ETs of P. balfouriana in response to BA treatments. a Cell line 4A. b, d, f Proliferation of ETs on proliferation media containing different concentrations of BA. ETs proliferated on medium with 2.5 μM BA (b), 3.6 μM BA (d), and 5.0 μM BA (f). c, e, g Prematuration of ETs. Somatic embryos stained red (acetocarmine) and suspensors stained blue (Evan’s blue). ETs were cultured on 1/2 LM without PGRs after proliferation for 8 months on medium with 2.5 μM BA (c), 3.6 μM BA (e), and 5.0 μM BA (g). S1 was collected when the ETs proliferated on initiation medium with 10 μM 2,4-D and 5 μM BA for 2 weeks; S2 was collected when the ETs proliferated on medium containing different concentrations of BA for 8 months; S3 was collected when the ETs matured on 1/2 LM without PGRs (prematuration) for 1 week. “vs” means compared with. (TIF 155 KB)

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Li, Q., Deng, C., Zhu, T. et al. Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana. Trees 33, 469–480 (2019). https://doi.org/10.1007/s00468-018-1793-x

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