Abstract
Phalaenopsis ‘Sogo Vivien’ is a hybrid orchid that can produce a mutant with foliar variegation. Variegation can be divided into structural-type and pigment-type variegation. Foliar variegation was mostly caused by mutations in plastid genes. Even though there are various studies on foliar variegation in other plants, studies on foliar variegation in orchids are quite limited. This study aimed to determine the variegated leaf phenotype characteristics and their cause in P. ‘Sogo Vivien’. This study observed the anatomy of variegated leaves by comparing the green and white parts of the leaf, measured the concentration of chlorophyll pigments, and detected leaf starch content between both parts. In addition, polymorphism analysis was also performed on the VAR2 gene partial sequences. Based on leaf anatomy structure, the difference between variegated zones is only in the number of chloroplasts, the diameter of parenchyma cells, and chloroplasts. The chloroplast diameter within the white part is smaller than the green part. The chloroplasts’ diameters in the green part are two times larger than the white part, with four times more in amount. The two parts were formed due to differences in the distribution pattern of functional chloroplasts in the leaves. The concentration of chlorophyll in the green part is higher than in the white part, a characteristic of the chlorophyll-type variegation. The green parts have more starch grains than the white parts. Stomata distance between variegated zones on the abaxial side is different, and it was suggested as a distinctive character between variegated zones. A thymine insertion in exon 1 contributed to nonsense and missense mutations on the VAR2 gene isolated from the white part. This mutation caused the amino acid motif to change from KAKFQ to KA—Q. This mutation is suggested to cause the variegated leaf phenomenon in P. ‘Sogo Vivien’.
This is a preview of subscription content, log in via an institution to check access.
References
Aluru MR, Yu F, Fu A, Rodermel S (2006) Arabidopsis variegation mutants: new insights into chloroplast biogenesis. J Exp Bot 57:1871–1881. https://doi.org/10.1093/jxb/erj008
Aphalo PJ, Sanchez RA (1986) Stomatal responses to light and drought stress in variegated leaves of Hedera helix. Plant Physiol 81:768–773
Chang H, Zhang L, Xie H, Liu J, Xi Z, Xu X (2021) The conservation of chloroplast genome structure and improved resolution of infrafamilial relationships of Crassulaceae. Front Plant Sci 12:1–13. https://doi.org/10.3389/fpls.2021.631884
Chater CCC, Caine RS, Fleming AJ, Gray JE (2017) Origins and evolution of stomatal development. Plant Physiol 174:624–638. https://doi.org/10.1104/pp.17.00183
Chen M, Choi Y, Do, Voytas DF, Rodermel S (2000) Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. Plant J 22:303–313. https://doi.org/10.1046/j.1365-313x.2000.00738.x
Chen MCM, Chao P, Huang M, Yang J, Zhang ZW, Lin K, Yang CM (2012) Chlorophyllase activity in green and non-green tissues of variegated plants. South Afr J Bot 81:44–49. https://doi.org/10.1016/j.sajb.2012.04.004
Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890. https://doi.org/10.1093/nar/16.22.10881
Croft H, Chen JM (2017) Leaf pigment content. Elsevier Inc, Canada
Dobrogojski J, Adamiec M, Luciński R (2020) The chloroplast genome: a review. Acta Physiol Plant 42:1–13. https://doi.org/10.1007/s11738-020-03089-x
Dogra V, Duan J, Lee KP, Chanhong K (2019) Impaired PSII proteostasis triggers a UPR-like response in the var2 mutant of Arabidopsis. J Exp Bot 70:3075–3088. https://doi.org/10.1093/jxb/erz151
Hastuti ED, Anggoro S, Pribadi R (2012) The effects of environmental factors on the dynamic growth pattern of mangrove Avicennia marina. J Coast Dev 16(1):57–61
Hong T, Lin H, He D (2018) Characteristics and correlations of leaf stomata in different Aleurites montana provenances. PLoS ONE 13:1–10. https://doi.org/10.1371/journal.pone.0208899
Hu Y, Yang L, Gao C, Liao D, Long L, Qiu J, Wei H, Deng Q, Zhou Y (2022) A comparative study on the leaf anatomical structure of Camellia Oleifera in a low-hot valley area in Guizhou Province, China. PLoS ONE 17:1–21. https://doi.org/10.1371/journal.pone.0262509
Janska H, Kwasniak M, Szczepanowska J (2013) Protein quality control in organelles: AAA/FtsH story. Biochim Biophys Acta 1833:381–387. https://doi.org/10.1016/j.bbamcr.2012.03.016
Jeran N, Rotasperti L, Frabetti G, Calabritto A, Pesaresi P, Tadini L (2021) The PUB4 E3 ubiquitin ligase is responsible for the variegated phenotype observed upon alteration of chloroplast protein homeostasis in Arabidopsis cotyledons. Genes 12:1–15. https://doi.org/10.3390/genes12091387
Kato Y, Sakamoto W (2018) FtsH protease in the thylakoid membrane: physiological functions and the regulation of protease activity. Front Plant Sci 9:1–8. https://doi.org/10.3389/fpls.2018.00855
Kodama Y (2016) Time gating of chloroplast autofluorescence allows clearer fluorescence imaging in Planta. PLoS ONE 11:1–8. https://doi.org/10.1371/journal.pone.0152484
Lawson T, Blatt MR (2014) Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency. Plant Physiol 164:1556–1570. https://doi.org/10.1104/pp.114.237107
Lehmann MM, Ghiasi S, George GM, Cormier MA, Gessler A, Saurer M, Werner RA (2019) Influence of starch deficiency on photosynthetic and post-photosynthetic carbon isotope fractionations. J Exp Bot 70:1829–1841. https://doi.org/10.1093/jxb/erz045
Li Q, Chen J, McConnell DB, Henny RJ (2007) A simple and effective method for quantifying leaf variegation. Horttechnology 17:285–288. https://doi.org/10.21273/horttech.17.3.285
Liu S, Zheng L, Jia J, Guo J, Zheng M, Zhao J, Shao J, Liu X, An L, Yu F et al (2019) Chloroplast translation elongation factor EF-Tu/SVR11 is involved in VAR2-mediated leaf variegation and leaf development in Arabidopsis. Front Plant Sci 10:1–11. https://doi.org/10.3389/fpls.2019.00295
Liu C, Li Y, Xu L, Li M, Wang J, Yan P, He N (2021) Stomatal arrangement pattern: a new direction to explore plant adaptation and evolution. Front Plant Sci 12:1–7. https://doi.org/10.3389/fpls.2021.655255
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325. https://doi.org/10.1093/nar/8.19.4321
Mursyanti E, Purwantoro A, Moeljopawiro S, Semiarti E (2015) Induction of somatic embryogenesis through overexpression of ATRKD4 genes in Phalaenopsis Sogo Vivien. Indones J Biotechnol 20:42–53. https://doi.org/10.22146/ijbiotech.15276
Mursyanti E, Purwantoro A, Moeljopawiro S, Semiarti E (2016) Micropropagation of mini orchid hybrid Phalaenopsis Sogo Vivien. J Trop Biodivers Biotechnol 1:45–53. https://doi.org/10.22146/jtbb.12933
Nishimura K, Kato Y, Sakamoto W (2016) Chloroplast proteases: updates on proteolysis within and across suborganellar compartments. Plant Physiol 171:2280–2293. https://doi.org/10.1104/pp.16.00330
Nopitasari S, Setiawati Y, Lawrie MD, Purwantoro A, Widada J, Sasongko AB, Yoshioka Y, Matsumoto S, Ninomiya K, Asano Y et al (2020) Development of an Agrobacterium-delivered CRISPR/Cas9 for Phalaenopsis amabilis (L.) Blume genome editing system. In: AIP Conf Proc, vol 2260. https://doi.org/10.1063/5.0015868
Nugroho LH, Purnomo I (2012) Struktur and perkembangan tumbuhan. Penebar Swadaya, Jakarta
Pao SH, Liu JW, Yang JY, Chesson P, Sheue CR (2020) Uncovering the mechanisms of novel foliar variegation patterns caused by structures and pigments. Taiwania 65:74–80. https://doi.org/10.6165/tai.2020.65.74
Pfister B, Zeeman SC (2016) Formation of starch in plant cells. Cell Mol Life Sci 73:2781–2807. https://doi.org/10.1007/s00018-016-2250-x
Qi Y, Wang X, Lei P, Li H, Yan L, Zhao J, Meng J, Shao J, An L, Yu F et al (2020) The chloroplast metalloproteases VAR2 and EGY1 act synergistically to regulate chloroplast development in Arabidopsis. J Biol Chem 295:1036–1046. https://doi.org/10.1016/S0021-9258(17)49913-3
Riverón-Giró FB, Damon A, García-González A, Solís-Montero L, Aguilar-Romero O, Ramírez-Marcial N, Nieto G (2017) Anatomy of the invasive orchid Oeceoclades maculata: ecological implications. Bot J Linn Soc 184:94–112. https://doi.org/10.1093/botlinnean/box014
Ruzin SE (1999) Plant microtechnique and microscopy. Oxford University Press, New York
Sakamoto W (2003) Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana. Genes Genet Syst 78:1–9. https://doi.org/10.1266/ggs.78.1
Sakamoto W, Uno Y, Zhang Q, Miura E, Kato Y, Sodmergen (2009) Arrested differentiation of proplastids into chloroplasts in variegated leaves characterized by plastid ultrastructure and nucleoid morphology. Plant Cell Physiol 50:2069–2083. https://doi.org/10.1093/pcp/pcp127
Semiarti E, Nopitasari S, Setiawati Y, Lawrie MD, Purwantoro A, Widada J, Ninomiya K, Asano Y, Matsumoto S, Yoshioka Y (2020a) Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids. Indones J Biotechnol 25:61–68. https://doi.org/10.22146/ijbiotech.39485
Semiarti E, Perdana NGA, Rozikin R, Kurniawan FY (2020b) In vitro culture and characterization of the HSP70 gene on Vanda tricolor Lindley var. Suavis ‘Queen Maxima’. In: BIO web of conferences, vol 28. https://doi.org/10.1051/bioconf/20202803004
Setiawati Y, Nopitasari S, Lawrie MD, Purwantoro A, Widada J, Sasongko AB, Ninomiya K, Asano Y, Matsumoto S, Yoshioka Y et al (2020) Agrobacterium-mediated transformation facillitates the CRISPR/Cas9 genome editing system in Dendrobium macrophyllum A. Rich orchid. In: AIP Conf Proc, vol 2260. https://doi.org/10.1063/5.0016200
Sheue CR, Pao SH, Chien LF, Chesson P, Peng CI (2012) Natural foliar variegation without costs? the case of Begonia. Ann Bot 109:1065–1074. https://doi.org/10.1093/aob/mcs025
Shih TH, Lin SH, Huang MY, Huang WD, Yang CM (2019) Transcriptome profile of the variegated Ficus microcarpa c.v. milky stripe fig leaf. Int J Mol Sci 20:1–14. https://doi.org/10.3390/ijms20061338
Stern WL (2014) Anatomy of the monocotyledons: orchidaceae, 1st edn. Oxford University Press, Oxford
Subchan AN, Nopitasari S, Setiawati Y, Zubaidah L, Nikmah N, Lawrie MD, Sasongko AB, Matsumoto S, Yoshioka Y, Semiarti E (2022) Agrobacterium-mediated transformation by using immerse and agroinfiltration method for genome editing of orchids with CRISPR/Cas9. Acta Hortic 1:263–272. https://doi.org/10.17660/ActaHortic.2022.1334.32
Toshoji H, Katsumata T, Takusagawa M, Yusa Y, Sakai A (2012) Effects of chloroplast dysfunction on mitochondria: white sectors in variegated leaves have higher mitochondrial DNA levels and lower dark respiration rates than green sectors. Protoplasma 249:805–817. https://doi.org/10.1007/s00709-011-0325-y
Tsai CC, Wu YJ, Sheue CR, Liao PC, Chen YH, Li SJ, Liu JW, Chang HT, Liu WL, Ko YZ et al (2017) Molecular basis underlying leaf variegation of a moth orchid mutant (Phalaenopsis aphrodite subsp. formosana). Front Plant Sci 8:1–11. https://doi.org/10.3389/fpls.2017.01333
Tsukaya H, Okada H, Mohamed M (2004) A novel feature of structural variegation in leaves of the tropical plant Schismatoglottis calyptrata. J Plant Res 117:477–480. https://doi.org/10.1007/s10265-004-0179-x
Wstzel CM, Jiang C, Meehan LJ, Voytas DF, Rodermel SR (1994) Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis. Plant J 6:161–175. https://doi.org/10.1046/j.1365-313X.1994.6020161.x
Wu G, Liu H, Hua L, Luo Q, Lin Y, He P, Feng S, Liu J, Ye Q (2018) Differential responses of stomata and photosynthesis to elevated temperature in two co-occurring subtropical forest tree species. Front Plant Sci 9:1–8. https://doi.org/10.3389/fpls.2018.00467
Yang Y, Chen X, Xu B, Li Y, Ma Y, Wang G (2015) Phenotype and transcriptome analysis reveals chloroplast development and pigment biosynthesis together influenced the leaf color formation in mutants of Anthurium andraeanum ‘Sonate.’ Front Plant Sci 6:1–16. https://doi.org/10.3389/fpls.2015.00139
Yang S, Sun M, Yang Q, Ma R, Zhang J, Zhang S (2016) Two strategies by epiphytic orchids for maintaining water balance: thick cuticles in leaves and water storage in pseudobulbs. AoB Plants 8:1–11. https://doi.org/10.1093/aobpla/plw046
Yu F, Fu A, Aluru M, Park S, Xu Y, Liu H, Liu X, Foudree A, Nambogga M, Rodermel S (2007) Variegation mutants and mechanisms of chloroplast biogenesis. Plant Cell Environ 30:350–365. https://doi.org/10.1111/j.1365-3040.2006.01630.x
Zhang JH, Zeng JC, Wang XM, Chen SF, Albach DC, Li HQ (2020) A revised classification of leaf variegation types. Flora Morphol Distrib Funct Ecol Plants 272:1–14. https://doi.org/10.1016/j.flora.2020.151703
Acknowledgements
This research was supported by the Final Project Recognition grant (RTA), Universitas Gadjah Mada in 2022 no. 1525/UN1/DITLIT/Dit-Lit/PT.01.05/2022 to ES as PI. The authors would like to express our gratitude to Titi Orchid Nursery for providing variegated mutant P. ‘Sogo Vivien’ for this research. One of the authors, FYK also thanked LPDP; Ministry of Finance; the Republic of Indonesia for funding support in the master studies at Universitas Gadjah Mada.
Author information
Authors and Affiliations
Contributions
FYK, MDL, and ES determined the ideas and designed this research structure. FYK, SUP, and AL conducted observations and data collection in the laboratory. MDL conducted data verification. AP and ES encouraged and supervised this research. All authors discussed the results and contributed to the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors have declared no conflict of interest.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Kurniawan, F.Y., Putri, S.U., Linggabuwana, A. et al. Mutation of the VAR2 gene plays a role as one of the causes of leaf variegation in the moth orchid Phalaenopsis ‘Sogo Vivien’. Hortic. Environ. Biotechnol. 65, 119–130 (2024). https://doi.org/10.1007/s13580-023-00543-w
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13580-023-00543-w