Эффект неполного нокаутирования гена пластидной крахмалфосфорилазы NtPHO1-L1 на метаболизм углеводов и каротиноидов в листьях Nicotiana tabacum L.

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Метаболизм крахмала регулируется сложной каталитической сетью, одним из ключевых ферментов которой является пластидная крахмалфосфорилаза PHO1. В нашем исследовании с использованием системы CRISPR-Cas9 были получены растения табака (Nicotiana tabacum L.) с неполным нокаутом гена NtPHO1-L1 за счет делеционных вариантов каталитического домена белка NtPHO1-L1, приводящих к формированию нефункциональных форм фермента. Редактированные линии отличались от растений дикого типа повышенным накоплением крахмала и пониженным содержанием сахаров, хлорофиллов и каротиноидов в ткани листа. Показано, что в сравнении с контролем редактированные растения характеризовались дифференциальной экспрессией генов метаболизма крахмала (NtPHO1-L1, NtGWD, NtBAM1, NtBAM9, NtAI) и каротиноидов (NtPSY2, NtPDS, NtZDS, NtCRTISO, NtVDE), а также генов, кодирующих MADS-доменные транскрипционные факторы (NtFUL1, NtSEP1, NtSEP2, NtSEP3), которые предположительно участвуют в регуляции транскрипции исследуемых генов метаболизма. Предположено, что неполный нокаут NtPHO1-L1 приводит к изменению функциональной активности крахмалфосфорилазы табака. Это, в свою очередь, может влиять на скоординированную работу ферментов катаболизма крахмала, а также синтеза хлорофиллов и каротиноидов, возможно, за счет дифференциальной экспрессии MADS-box генов. Наши результаты подчеркивают критическую регуляторную роль пластидной крахмалфосфорилазы в метаболизме транзиторного крахмала, а также в стимулирующем влиянии на фотосинтез растения.

Full Text

Restricted Access

About the authors

А. В. Нежданова

Институт биоинженерии им. К.Г. Скрябина Федерального исследовательского центра “Фундаментальные основы биотехнологии” Российской академии наук

Email: kulakova_97@mail.ru
Russian Federation, Москва

А. В. Кулакова

Институт биоинженерии им. К.Г. Скрябина Федерального исследовательского центра “Фундаментальные основы биотехнологии” Российской академии наук

Author for correspondence.
Email: kulakova_97@mail.ru
Russian Federation, Москва

М. А. Слугина

Институт биоинженерии им. К.Г. Скрябина Федерального исследовательского центра “Фундаментальные основы биотехнологии” Российской академии наук

Email: kulakova_97@mail.ru
Russian Federation, Москва

А. М. Камионская

Институт биоинженерии им. К.Г. Скрябина Федерального исследовательского центра “Фундаментальные основы биотехнологии” Российской академии наук

Email: kulakova_97@mail.ru
Russian Federation, Москва

Е. З. Кочиева

Институт биоинженерии им. К.Г. Скрябина Федерального исследовательского центра “Фундаментальные основы биотехнологии” Российской академии наук

Email: kulakova_97@mail.ru
Russian Federation, Москва

А. В. Щенникова

Институт биоинженерии им. К.Г. Скрябина Федерального исследовательского центра “Фундаментальные основы биотехнологии” Российской академии наук

Email: kulakova_97@mail.ru
Russian Federation, Москва

References

  1. Zeeman S.C., Smith S.M., Smith A.M. The diurnal metabolism of leaf starch // Biochem. J. 2007. V. 401. P. 13. https:doi.org/10.1042/BJ20061393
  2. Lloyd J.R., Kossmann J. Starch trek: the search for yield // Front. Plant Sci. 2019. V. 9: 1930. https:doi.org/10.3389/fpls.2018.01930
  3. Thalmann M., Santelia D. Starch as a determinant of plant fitness under abiotic stress // New Phytol. 2017. V. 214. P. 943. https:doi.org/10.1111/nph.14491
  4. Shoaib N., Liu L., Ali A., Mughal N., Yu G., Huang Y. Molecular functions and pathways of plastidial starch phosphorylase (PHO1) in starch metabolism: current and future perspectives // Int. J. Mol. Sci. 2021. V. 22: 10450. https:doi.org/10.3390/ijms221910450
  5. Lütken H., Lloyd J.R., Glaring M.A., Baunsgaard L., Laursen K.H., Haldrup A., Kossmann J., Blennow A. Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato // Planta. 2010. V. 232. P. 1127. https:doi.org/10.1007/s00425-010-1245-3
  6. Rathore R.S., Garg N., Garg S., Kumar A. Starch phosphorylase: role in starch metabolism and biotechnological applications // Crit. Rev. Biotechnol. 2009. V. 29. P. 214. https:doi.org/10.1080/07388550902926063
  7. Cuesta-Seijo J.A., Ruzanski C., Krucewicz K., Meier S., Hägglund P., Svensson B., Palcic M.M. Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development // PLoS One. 2017. V. 12: e0175488. https:doi.org/10.1371/journal.pone.0175488
  8. Flores-Castellanos J., Fettke J. The plastidial glucan phosphorylase affects the maltooligosaccharide metabolism in parenchyma cells of potato (Solanum tuberosum L.) tuber discs // Plant Cell Physiol. 2023. V. 64. P. 422. https:doi.org/10.1093/pcp/pcac174
  9. Sonnewald U., Basner A., Greve B., Steup M. A second L-type isozyme of potato glucan phosphorylase: cloning, antisense inhibition and expression analysis // Plant Mol. Biol. 1995. V. 27. P. 567. https:doi.org/10.1007/BF00019322
  10. Chen H.M., Chang S.C., Wu C.C., Cuo T.S., Wu J.S., Juang R.H. Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis // Physiol. Plant. 2002. V. 114. P. 506. https:doi.org/10.1034/j.1399-3054.2002.1140402.x
  11. Camirand A., St-Pierre B., Marineau C., Brisson N. Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene // Mol. Gen. Genet. 1990. V. 224. P. 33. https:doi.org/10.1007/BF00259448
  12. Fettke J., Poeste S., Eckermann N., Tiessen A., Pauly M., Geigenberger P., Steup M. Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme // Plant Cell Physiol. 2005. V. 46. P. 1987. https:doi.org/10.1093/pcp/pci214
  13. Schopper S., Mühlenbock P., Sörensson C., Hellborg L., Lenman M., Widell S., Fettke J., Andreasson E. Arabidopsis cytosolic alpha-glycan phosphorylase, PHS2, is important during carbohydrate imbalanced conditions // Plant Biol.. 2015. V. 17. P. 74. https:doi.org/10.1111/plb.12190
  14. Zeeman S.C., Thorneycroft D., Schupp N., Chapple A., Weck M., Dunstan H., Haldimann P., Bechtold N., Smith A.M., Smith S.M. The role of plastidial α-glucan phosphorylase in starch degradation and tolerance of abiotic stress in Arabidopsis leaves // Plant Physiol. 2004. V. 135. P. 849. https:doi.org/10.1104/pp.103.032631
  15. Satoh H., Shibahara K., Tokunaga T., Nishi A., Tasaki M., Hwang S.-K., Okita T., Kaneko N., Fujita N., Yoshida M., Hosaka Y., Sato A., Utsumi Y., Ohdan T., Nakamura Y. Mutation of the plastidial α-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm // Plant Cell. 2008. V. 20. P. 1833. https:doi.org/10.1105/tpc.107.054007
  16. Yu Y., Mu H.H., Wasserman B.P., Carman G.M. Identification of the maize amyloplast stromal 112-kd protein as a plastidic starch phosphorylase // Plant Physiol. 2001. V. 125. P. 351. https:doi.org/10.1104/pp.125.1.351
  17. Mizuno S., Kamiyoshihara Y., Shiba H., Shinmachi F., Watanabe K., Tateishi A. Plastidial starch phosphorylase is highly associated with starch accumulation process in developing squash (Cucurbita sp.) fruit // Physiol. Plant. 2019. V. 167. P. 264. https:doi.org/10.1111/ppl.12886
  18. Hwang S.K., Singh S., Cakir B., Satoh H., Okita T.W. The plastidial starch phosphorylase from rice endosperm: catalytic properties at low temperature // Planta. 2016. V. 243. P. 999. https:doi.org/10.1007/s00425-015-2461-7
  19. Fettke J., Leifels L., Brust H., Herbst K., Steup M. Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature // J. Exp. Bot. 2012. V. 63. P. 3011. https:doi.org/10.1093/jxb/ers014
  20. Orawetz T., Malinova I., Orzechowski S., Fettke J. Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature // Plant Physiol. Biochem. 2016. V. 100. P. 141. https:doi.org/10.1016/j.plaphy.2016.01.013
  21. Higgins J.E., Kosar‐Hashemi B., Li Z., Howitt C.A., Larroque O., Flanagan B., Morell M.K., Rahman S. Characterization of starch phosphorylases in barley grains // J. Sci. Food Agric. 2013. V. 93. P. 2137. https:doi.org/10.1002/jsfa.6019
  22. Schreiber L., Nader-Nieto A.C., Schönhals E.M., Walkemeier B., Gebhardt C. SNPs in genes functional in starch-sugar interconversion associate with natural variation of tuber starch and sugar content of potato (Solanum tuberosum L.) // G3: Genes, Genomes, Genetics. 2014. V. 4. P. 1797. https:doi.org/10.1534/g3.114.012377
  23. Albrecht T., Koch A., Lode A., Greve B., Schneider-Mergener J., Steup M. Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization // Planta. 2001. V. 213. P. 602. https:doi.org/10.1007/s004250100525
  24. Slugina M.A., Shchennikova A.V., Kochieva E.Z. The expression pattern of the Pho1a genes encoding plastidic starch phosphorylase correlates with the degradation of starch during fruit ripening in green-fruited and red-fruited tomato species // Funct. Plant Biol. 2019. V. 46. P. 1146. https:doi.org/10.1071/FP18317
  25. Slugina M.A., Meleshin A.A., Kochieva E.Z., Shchennikova A.V. The opposite effect of low temperature on the Pho1a starch phosphorylase gene expression in Solanum tuberosum L. tubers and Petota species leaves // Am. J. Potato Res. 2020. V. 97. P. 78. https:doi.org/10.1007/s12230-019-09758-z
  26. Nezhdanova A.V., Efremov G.I., Slugina M.A., Kamionskaya A.M., Kochieva E.Z., Shchennikova A.V. Effect of a radical mutation in plastidic starch phosphorylase PHO1a on potato growth and cold stress response // Horticulturae. 2022. V. 8: 730. https:doi.org/10.3390/horticulturae8080730
  27. Sharma S., Friberg M., Vogel P., Turesson H., Olsson N., Andersson M., Hofvander P. Pho1a (plastid starch phosphorylase) is duplicated and essential for normal starch granule phenotype in tubers of Solanum tuberosum L. // Front. Plant Sci. 2023. V. 14: 1220973. https:doi.org/10.3389/fpls.2023.1220973
  28. Jacobs T.B., LaFayette P.R., Schmitz R.J., Parrott W.A. Targeted genome modifications in soybean with CRISPR/Cas9 // BMC Biotechnol. 2015. V. 15: 16. https:doi.org/10.1186/s12896-015-0131-2
  29. Nezhdanova A.V., Slugina M.A., Kulakova A.V., Kamionskaya A.M., Kochieva E.Z., Shchennikova A.V. Effect of mosaic knockout of phytoene desaturase gene NtPDS on biosynthesis of carotenoids in Nicotiana tabacum L.// Russ. J. Plant Physiol. 2023. V. 70: 116. https:doi.org/10.1134/S1021443723601271
  30. Slugina M.A. Transcription factor RIPENING INHIBITOR and its homologs in regulation of fleshy fruit ripening of various plant species // Russ. J. Plant Physiol. 2021. V. 68: 783. https:doi.org/10.1134/S1021443721050186
  31. Parenicová L., de Folter S., Kieffer M., Horner D.S., Favalli C., Busscher J., Cook H.E., Ingram R.M., Kater M.M., Davies B., Angenent G.C., Colombo L. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world // Plant Cell. 2003. V. 15. P. 1538. https:doi.org/10.1105/tpc.011544
  32. AbdElgawad H., Avramova V., Baggerman G., Van Raemdonck G., Valkenborg D., Van Ostade X., Guisez Y., Prinsen E., Asard H., Van den Ende W., Beemster G.T.S. Starch biosynthesis contributes to the maintenance of photosynthesis and leaf growth under drought stress in maize // Plant Cell Environ. 2020. V. 43. P. 2254. https:doi.org/10.1111/pce.13813
  33. Hou J., Zhang H., Liu J., Reid S., Liu T., Xu S., Tian Z., Sonnewald U., Song B., Xie C. Amylases StAmy23, StBAM1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways // J. Exp. Bot. 2017. V. 68. P. 2317. https:doi.org/10.1093/jxb/erx076
  34. Zhang H., Liu J., Hou J., Yao Y., Lin Y., Ou Y., Song B., Xie C. The potato amylase inhibitor gene SbAI regulates cold-induced sweetening in potato tubers by modulating amylase activity // Plant Biotech. J. 2014. V. 12. P. 984. https:doi.org/10.1111/pbi.12221
  35. Rosas-Saavedra C., Stange C. Biosynthesis of carotenoids in plants: enzymes and color // Subcell. Biochem. 2016. V. 79. P. 35. https:doi.org/10.1007/978-3-319-39126-7_2
  36. Cordenunsi-Lysenko B.R., Nascimento J.R.O., Castro-Alves V.C., Purgatto E., Fabi J.P., Peroni-Okyta F.H.G. The starch is (not) just another brick in the wall: the primary metabolism of sugars during banana ripening // Front. Plant Sci. 2019. V. 10: 391. https:doi.org/10.3389/fpls.2019.00391

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Results of editing the NtPHO1-L1 plastid starch phosphorylase gene: (a) alignment of the target (editable) coding sequence of the NtPHO1-L1 gene (clones 1-10 of the edited Nt1 line in comparison with the unedited sequence of genes LOC107810306 and LOC107814807); (b) alignment of variants of the edited protein sequence NtPHO1-L1 in comparison with an unedited version (LOC107810306 and LOC107814807); (c) a comparative analysis of the secondary structure of the unedited enzyme NtPHO1‒L1 and protein with the KRY→N substitution introduced as a result of editing. A fragment of the sequence where the substitution occurred is shown (marked with an arrow). The simulation was carried out in the Phyre2 program based on the well-known c5lrbB_ matrix (alpha-1,4 glucan phosphorylase); 86% of both sequences were modeled with 100% confidence.

Download (656KB)
Indexing metadata
3. Fig. 2. Nicotiana tabacum transgenic lines (Nt1-11, Nt1-13 and Nt1-15) with mosaic knockout gene of plastid starch phosphorylase NtPHO1-L1 in comparison with nontransgenic control (WT): (a) ‒ photos of plants WT, Nt1-11, Nt1-13 and Nt1-15 at the stage blooms (scale 10 cm); (b) ‒ comparison of the main characteristics of the control (WT, average for 20 plants) and edited T1 lines from Nt1 (average for 20 plants Nt1-1–Nt1-20; further, the graph shows individual values for each of the analyzed plants Nt1-11, Nt1-13 and Nt1-15). *P < 0.05 is a statistically significant difference between T1 and Nt1 vs. WT.

Download (467KB)
Indexing metadata
4. 3. The content of starch, sucrose, glucose, fructose (mg/g of crude weight), the sum of carotenoids and chlorophylls a and b (mcg/g of crude weight) in the leaf tissue of plants Nt1-11, Nt1-13 and Nt1-15 in comparison with nontransgenic control WT. *P < 0.05 is a statistically significant difference between T1 and Nt1 vs. WT.

Download (310KB)
Indexing metadata
5. Fig. 4. Expression of starch metabolism genes (NtPHO1-L1, NtGWD, NtBAM1, NtBAM9, NtAI) and carotenoid biosynthesis (NtPSY2, NtPDS, NtCRTISO, NtZDS, NtVDE) in leaf tissue of plants Nt1-11, Nt1-13 and Nt1-15 in comparison with nontransgenic control of WT. *P < 0.05 is a statistically significant difference between T1 and Nt1 vs. WT.

Download (391KB)
Indexing metadata
6. Fig. 5. Expression of genes of MADS-domain TF (NtFUL1, NtSEP1, NtSEP2, NtSEP3) in leaf tissue of plants Nt1-11, Nt1-13 and Nt1-15 in comparison with nontransgenic control of WT. *P < 0.05 is a statistically significant difference between T1 and Nt1 vs. WT.

Download (183KB)
Indexing metadata
7. Supplement
Download (18KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences