Identification and Expression Analysis of Calcium-dependent Protein Kinase（CDPK）Family in Haematococcus pluvialis

doi:10.13560/j.cnki.biotech.bull.1985.2023-0719

Abstract

Abstract:

【Objective】 Calcium-dependent protein kinases（CDPK or CPK）are widely involved in plant growth and development and environmental stress response. To explore their roles involved in Haematococcus pluvialis growth, development and astaxanthin accumulation, HpCDPK gene family was identified and analyzed. 【Method】 Bioinformatics tools were used to identify the HpCDPK gene family members, and to analyze the physical and chemical properties, phylogenetic evolution, conserved motifs and functional domains of the encoded proteins, as well as the gene expression profiles under nitrogen deficiency stress. 【Result】 The results showed that seven members of HpCDPK gene family were identified, and all HpCDPK proteins contained typical EF-hand motifs and protein kinase domains. Phylogenetic analysis showed that these HpCDPKs clustered with the CrCDPKs from Chlamydomonas reinhartii and separated from the AtCDPKs of Arabidopsis thaliana, suggesting that species-specific gene replication occurred in the process of evolution. Transcriptome data analysis showed that HpCDPK gene expression was induced by a variety of stresses, among which the transcription level of HpCDPK7 gene was most significantly up-regulated under nitrogen deficiency. In addition, the analysis of the correlation between HpCDPK and the expression of genes related to carotenoid synthesis showed that HpCDPK was closely related to the expressions of genes relevant to several carotenoid synthesis in H. pluvialis, especially HpCDPK2 was positively correlated with the expression of key genes of astaxanthin synthesis（BKT and BCH）. 【Conclusion】 Seven HpCDPK genes were identified, the expression of HpCDPK7 gene was most significantly up-regulated under nitrogen deficiency and HpCDPK2 may play an active role in the synthesis and accumulation of carotenoids and astaxanthin. The results provide a reference basis for further analysis of the function and molecular mechanism of stress response and carotenoids synthesis and accumulation mediated by HpCDPK in H. pluvialis.

Key words: Haematococcus pluvialis, calcium-dependent protein kinase, sequence analysis, expression pattern, abiotic stress

LI Ya-nan, ZHANG Hao-jie, LIANG Meng-jing, LUO Tao, LI Wang-ning, ZHANG Chun-hui, JI Chun-li, LI Run-zhi, XUE Jin-ai, CUI Hong-li. Identification and Expression Analysis of Calcium-dependent Protein Kinase（CDPK）Family in Haematococcus pluvialis[J]. Biotechnology Bulletin, 2024, 40(2): 300-312.

Figures/Tables 11

Table 1 Members of CDPK gene family in H. pluvialis

基因名称 Gene name	基因编号 Gene No.	蛋白长度 Protein length/aa	分子量 MW/Da	等电点 pI	EF手型数量 EF-hand number	N-肉豆蔻酰化 N- myristoylator	N-棕榈酰化 N-palmitoylation	亚细胞定位 Subcellular localization
HpCDPK1	comp60148_c2	605	67 397.89	5.92	4	No	Yes	Cytoplasmic
HpCDPK2	comp60654_c0	545	59 849.50	6.08	4	No	Yes	Chloroplast
HpCDPK3	comp57813_c0	478	53 280.35	5.71	4	No	No	Cytoplasmic
HpCDPK4	comp60035_c0	676	73 764.11	6.03	2	No	Yes	Cytoplasmic
HpCDPK5	comp58900_c0	716	76 076.86	6.39	4	No	Yes	Nuclear
HpCDPK6	comp61638_c1	1 479	153 784.74	7.29	4	No	Yes	Nuclear
HpCDPK7	comp60433_c0	633	70 805.48	5.79	4	Yes	Yes	Mitochondrial

Fig. 1 Prediction of tertiary structures of CDPK proteins in H. pluvialis

Fig. 2 Phylogenetic tree of CDPK genes in H. pluvialis, C. reinhardtii and A. thaliana

Fig. 3 Conserved motif and functional domain of HpCDPK family members of H. pluvialis Different motif is represent by specific color. Purple hollow box indictes the Serine/ Threonine protein kinases domain（SM000220）, and green hollow box indicted the EF-hand（SM000054）

Fig. 4 Conserved motifs of HpCDPKs in H. pluvialis predicted by MEME The abscissa indicates amino acid position and the ordinate indicates the proportion of amino acid at a site

Fig. 5 Multiple sequence alignments of conserved PDK domains of HpCDPK proteins in H. pluvialis

Fig. 6 Multiple sequence alignments of conserved EF motifs of HpCDPK proteins in H. pluvialis

Fig. 7 Conserved domain and important site analysis of HpCDPKs in H. pluvialis

Fig. 8 Expression patterns of HpCDPK genes in H. pluvialis in response to abiotic stresses A: Normal light（N）, blue light（B）, and high white light（W）. B: Normal light（L）, normal light and UV-B 200 W（LU200）, normal light and UV-B 400 W（LU400）, dark（D）, dark and UV-B 200 W（DU200）, dark and UV-B 400 W（DU400）. C: High light（CK）, high light and α-ketoglutaric acid（OG）, high light and nitrogen deficiency（ND）, high light, nitrogen deficiency and α-ketoglutaric acid（ND-OG）

Fig. 9 Correlation between HpCDPKs and gene expressions for astaxanthin synthesis and accumulation

Fig. 10 Correlation between HpCDPK2, HpCDPK3, HpCDPK5 genes and gene expressions for the biosynthesis and accumulation of carotenoid and astaxanthin

References 46

[1]	Ranty B, Aldon D, Cotelle V, et al. Calcium sensors as key hubs in plant responses to biotic and abiotic stresses[J]. Front Plant Sci, 2016, 7: 327. doi: 10.3389/fpls.2016.00327 pmid: 27014336
[2]	Hepler PK. Calcium: a central regulator of plant growth and development[J]. Plant Cell, 2005, 17(8): 2142-2155. doi: 10.1105/tpc.105.032508 pmid: 16061961
[3]	Lecourieux D, Ranjeva R, Pugin A. Calcium in plant defence-signalling pathways[J]. New Phytol, 2006, 171(2): 249-269. doi: 10.1111/j.1469-8137.2006.01777.x pmid: 16866934
[4]	Sanders D, Brownlee C, Harper JF. Communicating with calcium[J]. Plant Cell, 1999, 11(4): 691-706. doi: 10.1105/tpc.11.4.691 URL
[5]	Hamel LP, Sheen J, Séguin A. Ancient signals: comparative genomics of green plant CDPKs[J]. Trends Plant Sci, 2014, 19(2): 79-89. doi: 10.1016/j.tplants.2013.10.009 URL
[6]	Cheng SH, Willmann MR, Chen HC, et al. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family[J]. Plant Physiol, 2002, 129(2): 469-485.
[7]	Boudsocq M, Sheen J. CDPKs in immune and stress signaling[J]. Trends Plant Sci, 2013, 18(1): 30-40. doi: 10.1016/j.tplants.2012.08.008 pmid: 22974587
[8]	Ludwig AA, Romeis T, Jones JDG. CDPK-mediated signalling pathways: specificity and cross-talk[J]. J Exp Bot, 2004, 55(395): 181-188. doi: 10.1093/jxb/erh008 pmid: 14623901
[9]	Harmon AC, Gribskov M, Gubrium E, et al. The CDPK superfamily of protein kinases[J]. New Phytol, 2001, 151(1): 175-183. doi: 10.1046/j.1469-8137.2001.00171.x pmid: 33873379
[10]	Yip Delormel T, Boudsocq M. Properties and functions of calcium-dependent protein kinases and their relatives in Arabidopsis thaliana[J]. New Phytol, 2019, 224(2): 585-604. doi: 10.1111/nph.16088 pmid: 31369160
[11]	Simeunovic A, Mair A, Wurzinger B, et al. Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases[J]. J Exp Bot, 2016, 67(13): 3855-3872. doi: 10.1093/jxb/erw157 pmid: 27117335
[12]	Hrabak EM, Chan CWM, Gribskov M, et al. The Arabidopsis CDPK-SnRK superfamily of protein kinases[J]. Plant Physiol, 2003, 132(2): 666-680. doi: 10.1104/pp.102.011999 URL
[13]	Li YJ, Fei XW, Dai HF, et al. Genome-wide identification of calcium-dependent protein kinases in Chlamydomonas reinhardtii and functional analyses in nitrogen deficiency-induced oil accumulation[J]. Front Plant Sci, 2019, 10: 1147. doi: 10.3389/fpls.2019.01147 URL
[14]	Romeis T, Ludwig AA, Martin R, et al. Calcium-dependent protein kinases play an essential role in a plant defence response[J]. EMBO J, 2001, 20(20): 5556-5567. doi: 10.1093/emboj/20.20.5556 pmid: 11597999
[15]	Asano T, Tanaka N, Yang GX, et al. Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice[J]. Plant Cell Physiol, 2005, 46(2): 356-366. doi: 10.1093/pcp/pci035 URL
[16]	Rutschmann F, Stalder U, Piotrowski M, et al. LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization[J]. Plant Physiol, 2002, 129(1): 156-168. doi: 10.1104/pp.000869 pmid: 12011347
[17]	Kong XP, Lv W, Jiang SS, et al. Genome-wide identification and expression analysis of calcium-dependent protein kinase in maize[J]. BMC Genomics, 2013, 14: 433. doi: 10.1186/1471-2164-14-433 pmid: 23815483
[18]	Huang K, Peng L, Liu YY, et al. Arabidopsis calcium-dependent protein kinase AtCPK1 plays a positive role in salt/drought-stress response[J]. Biochem Biophys Res Commun, 2018, 498(1): 92-98. doi: 10.1016/j.bbrc.2017.11.175 URL
[19]	Urao T, Katagiri T, Mizoguchi T, et al. Two genes that encode Ca(2+)-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana[J]. Mol Gen Genet, 1994, 244(4): 331-340. doi: 10.1007/BF00286684 URL
[20]	Boudsocq M, Willmann MR, McCormack M, et al. Differential innate immune signalling via Ca²⁺ sensor protein kinases[J]. Nature, 2010, 464(7287): 418-422. doi: 10.1038/nature08794
[21]	Zhang CH, Zhang LT, Liu JG. Exogenous sodium acetate enhances astaxanthin accumulation and photoprotection in Haematococcus pluvialis at the non-motile stage[J]. J Appl Phycol, 2019, 31(2): 1001-1008. doi: 10.1007/s10811-018-1622-z
[22]	Wang HM D, Li XC, Lee DJ, et al. Potential biomedical applications of marine algae[J]. Bioresour Technol, 2017, 244(Pt 2): 1407-1415. doi: 10.1016/j.biortech.2017.05.198 URL
[23]	Ren YY, Deng JQ, Huang JC, et al. Using green alga Haematococcus pluvialis for astaxanthin and lipid co-production: advances and outlook[J]. Bioresour Technol, 2021, 340: 125736. doi: 10.1016/j.biortech.2021.125736 URL
[24]	滕长英, 张立, 缪静, 等. 雨生红球藻虾青素积累机制的研究进展[J]. 海洋科学, 2006, 30(12): 77-81.
	Teng CY, Zhang L, Miao J, et al. Review of astaxanthin accumulating mechanism in Haematococcus pluvialis[J]. Mar Sci, 2006, 30(12): 77-81.
[25]	Lamers J, van der Meer T, Testerink C. How plants sense and respond to stressful environments[J]. Plant Physiol, 2020, 182(4): 1624-1635. doi: 10.1104/pp.19.01464 pmid: 32132112
[26]	崔红利, 许文鑫, 崔玉琳, 等. 光诱导雨生红球藻虾青素积累的信号通路转录组分析[J]. 生物工程学报, 2021, 37(4): 1260-1276.
	Cui HL, Xu WX, Cui YL, et al. Transcriptome analysis of signal transduction pathway involved in light inducing astaxanthin accumulation in Haematococcus pluvialis[J]. Chin J Biotechnol, 2021, 37(4): 1260-1276.
[27]	许文鑫, 朱琴, 朱梅, 等. UV-B对雨生红球藻生长及虾青素积累的影响[J]. 水生生物学报, 2021, 45(6): 1281-1290.
	Xu WX, Zhu Q, Zhu M, et al. Ultraviolet-b radiation enhances the growth and astaxanthin production in Haematococcus pluvialis[J]. Acta Hydrobiol Sin, 2021, 45(6): 1281-1290.
[28]	Johnson DR, Bhatnagar RS, Knoll LJ, et al. Genetic and biochemical studies of protein N-myristoylation[J]. Annu Rev Biochem, 1994, 63: 869-914. pmid: 7979256
[29]	Farazi TA, Waksman G, Gordon JI. The biology and enzymology of protein N-myristoylation[J]. J Biol Chem, 2001, 276(43): 39501-39504. doi: 10.1074/jbc.R100042200 pmid: 11527981
[30]	Yuan M, Song ZH, Ying MD, et al. N-myristoylation: from cell biology to translational medicine[J]. Acta Pharmacol Sin, 2020, 41(8): 1005-1015. doi: 10.1038/s41401-020-0388-4 pmid: 32203082
[31]	Zou JJ, Wei FJ, Wang C, et al. Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca²⁺-mediated stomatal regulation in response to drought stress[J]. Plant Physiol, 2010, 154(3): 1232-1243. doi: 10.1104/pp.110.157545 URL
[32]	Li AL, Zhu YF, Tan XM, et al. Evolutionary and functional study of the CDPK gene family in wheat(Triticum aestivum L.)[J]. Plant Mol Biol, 2008, 66(4): 429-443. doi: 10.1007/s11103-007-9281-5 URL
[33]	Wang CT, Shao JM. Characterization of the ZmCK1 gene encoding a calcium-dependent protein kinase responsive to multiple abiotic stresses in maize[J]. Plant Mol Biol Rep, 2013, 31(1): 222-230. doi: 10.1007/s11105-012-0496-5 URL
[34]	Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains[J]. Science, 1988, 241(4861): 42-52. doi: 10.1126/science.3291115 pmid: 3291115
[35]	Wen F, Ye F, Xiao ZL, et al. Genome-wide survey and expression analysis of calcium-dependent protein kinase(CDPK)in grass Brachypodium distachyon[J]. BMC Genomics, 2020, 21(1): 53. doi: 10.1186/s12864-020-6475-6
[36]	任慧, 黄烯. 紫外光B波段光信号调控植物生长发育的研究进展[J]. 厦门大学学报: 自然科学版, 2021, 60(2): 327-338.
	Ren H, Huang X. Research progress in the regulation of plant growth and development by ultraviolet-B light[J]. J Xiamen Univ Nat Sci, 2021, 60(2): 327-338.
[37]	Wargent JJ, Gegas VC, Jenkins GI, et al. UVR8 in Arabidopsis thaliana regulates multiple aspects of cellular differentiation during leaf development in response to ultraviolet B radiation[J]. New Phytol, 2009, 183(2): 315-326. doi: 10.1111/j.1469-8137.2009.02855.x pmid: 19402876
[38]	Fasano R, Gonzalez N, Tosco A, et al. Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B[J]. Mol Plant, 2014, 7(5): 773-791. doi: 10.1093/mp/ssu002 URL
[39]	Liang T, Yang Y, Liu HT. Signal transduction mediated by the plant UV-B photoreceptor UVR8[J]. New Phytol, 2019, 221(3): 1247-1252. doi: 10.1111/nph.15469 pmid: 30315741
[40]	Ramani S, Chelliah J. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures[J]. BMC Plant Biol, 2007, 7: 61. doi: 10.1186/1471-2229-7-61 URL
[41]	Frattini M, Morello L, Breviario D. Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development[J]. Plant Mol Biol, 1999, 41(6): 753-764. pmid: 10737140
[42]	Disch A, Schwender J, Müller C, et al. Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714[J]. Biochem J, 1998, 333(Pt 2)(Pt 2):381-388. doi: 10.1042/bj3330381 URL
[43]	Gilroy S, Trewavas A. Signal processing and transduction in plant cells: the end of the beginning?[J]. Nat Rev Mol Cell Biol, 2001, 2(4): 307-314.
[44]	Pecker I, Gabbay R, Cunningham FX Jr, et al. Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening[J]. Plant Mol Biol, 1996, 30(4): 807-819. doi: 10.1007/BF00019013 pmid: 8624411
[45]	Cunningham FX Jr, Gantt E. One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases[J]. Proc Natl Acad Sci USA, 2001, 98(5): 2905-2910. doi: 10.1073/pnas.051618398 pmid: 11226339
[46]	刘贯山, 陈珈. 钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用[J]. 植物学通报, 2003, 38(2): 160-167.
	Liu GS, Chen J. Roles of calcium dependent protein kinases(CDPKs)in plant calcium signal transduction[J]. Chin Bull Bot, 2003, 38(2): 160-167.