Cytological, physiological, and transcriptomic analyses of golden leaf coloration in Ginkgo biloba L

Abstract

Ginkgo biloba is grown worldwide as an ornamental plant for its golden leaf color. However, the regulatory mechanism of leaf coloration in G. biloba remains unclear. Here, we compared G. biloba gold-colored mutant leaves and normal green leaves in cytological, physiological and transcriptomic terms. We found that chloroplasts of the mutant were fewer and smaller, and exhibited ruptured thylakoid membranes, indistinct stromal lamellae and irregularly arranged vesicles. Physiological experiments also showed that the mutant had a lower chlorophyll, lower flavonoid and higher carotenoid contents (especially lutein). We further used transcriptomic sequencing to identify 116 differentially expressed genes (DEGs) and 46 transcription factors (TFs) involved in chloroplast development, chlorophyll metabolism, pigment biosynthesis and photosynthesis. Among these, the chlorophyll biosynthesis-related PPO showed down-regulation, while chlorophyll degradation-related NYC/NOL had up-regulated expression in mutant leaves. Z-ISO, ZDS, and LCYE, which are involved in carotenoid biosynthesis were up-regulated. Quantitative real-time PCR (RT-qPCR) further confirmed the altered expression levels of these genes at three stages. The alteration of PPO and NYC/NOL gene expression might affect chlorophyll biosynthesis and promote degradation of chlorophyll b to chlorophyll a, while the up-regulated genes Z-ISO, ZDS and LCYE enhanced carotenoid accumulation. Consequently, changes in the ratio of carotenoids to chlorophylls were the main factors driving the golden leaf coloration in the mutant G. biloba.

References

[1] Feild, T. S., Lee, D. W. & Holbrook, N. M. Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood. Plant Physiol. 127, 566-574 (2001).

[2] Nagata, N., Tanaka, R., Satoh, S. & Tanaka, A. Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of prochlorococcus species. Plant Cell 17, 233-240 (2005).

[3] Adhikari, N. D. et al. GUN4-porphyrin complexes bind the ChlH/GUN5 subunit of Mg-Chelatase and promote chlorophyll biosynthesis in Arabidopsis. Plant Cell 23, 1449-1467 (2011).

[4] Wu, Z. et al. A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol. 145, 29-40 (2007).

[5] Wang, L. et al. Biochemical and transcriptome analyses of a novel chlorophyll-deficient chlorina tea plant cultivar. BMC Plant Biol. 14, 352-365 (2014).

[6] Zhu, G. et al. Transcriptome characterization of Cymbidium sinense ‘Dharma’ using 454 pyrosequencing and its application in the identification of genes associated with leaf color variation. PLoS One 10, e0128592 (2015).

[7] Dong, H. et al. A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants. Plant Physiol. 162, 1867-1880 (2013).

[8] Li, Y. et al. Comprehensive transcriptome analysis discovers novel candidate genes related to leaf color in a Lagerstroemia indica yellow leaf mutant. Genes Genom. 37, 851-863 (2015).

[9] Li, C. F. et al. Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in Anji Baicha (Camellia sinensis). BMC Plant Biol. 16, 195-212 (2016).

[10] Wang, L. et al. An embryological study and systematic significance of the primitive gymnosperm Ginkgo biloba. J. Syst. Evol. 49, 353-361 (2011).

[11] Matile, P., Flach, B. M. & Eller, B. M. Autumn leaves of Ginkgo biloba L.: optical properties, pigments and optical brighteners. Plant Biol. 105, 13-17 (1992).

[12] Liu, X. et al. Comparative proteomic and physiological analysis reveals the variation mechanisms of leaf coloration and carbon fixation in a Xantha mutant of Ginkgo biloba L.. Int J. Mol. Sci. 17, 1794-1818 (2016).

[13] Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat Methodol 57, 289-300 (1995).

[14] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCt method . Methods 25, 402-408 (2001).

[15] Eckhardt, U., Grimm, B. & Hörtensteiner, S. Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant Mol. Biol. 56, 1-14 (2004).

[16] Hörtensteiner, S. Update on the biochemistry of chlorophyll breakdown. Plant Mol. Biol. 82, 505-517 (2013).

[17] Yang, Y. et al. 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, 139-155 (2015).

[18] Lonosky, P. M. et al. A proteomic analysis of maize chloroplast biogenesis. Plant Physiol. 134, 560-574 (2004).

[19] Li, Y. H. et al. Morphological structure and genetic mapping of new leaf-color mutant gene in rice (Oryza sativa). Rice Sci. 19, 79-85 (2012).

[20] Maekawa, S. et al. Pale-green phenotype of atl31atl6 double mutant leaves is caused by disruption of 5-aminolevulinic acid biosynthesis in Arabidopsis thaliana. PLoS One 10, e0117662 (2015).

[21] Wu, Z. M., Zhang, X., Wang, J. L. & Wan, J. M. Leaf chloroplast ultrastructure and photosynthetic properties of a chlorophyll-deficient mutant of rice. Photosynthetica 52, 217-222 (2014).

[22] Yoo, S. C. et al. Ricevirescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development. Plant Physiol. 150, 388-401 (2009).

[23] Yang, H. Y. et al. Identification of genes involved in spontaneous leaf color variation in Pseudosasa japonica. Genet. Mol. Res. 14, 11827-11840 (2015).

[24] Seo, T. S. et al. Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry. Proc. Natl. Acad. Sci. USA 101, 5488-5493 (2004).

[25] Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57-63 (2009).

[26] Fromme, P. et al. Structure and function of photosystem I: interaction with its soluble electron carriers and external antenna systems. FEBS Lett. 555, 40-44 (2003).

[27] Meier, S. et al. A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana. BMC Syst. Biol. 5, 77-96 (2011).

[28] Kumar, A. M. & Söll, D. Antisense HEMA1 RNA expression inhibits heme and chlorophyll biosynthesis in Arabidopsis. Plant Physiol. 122, 49-56 (2000).

[29] Koch, M. et al. Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis. EMBO J. 23, 1720-1728 (2004).

[30] Lai, B. et al. Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis. BMC Genom. 16, 225-243 (2015).

[31] Kusaba, M. et al. Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence. Plant Cell 19, 1362-1375 (2007).

[32] Sato, Y. et al. Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J. 57, 120-131 (2009).

[33] Jia, T., Ito, H. & Tanaka, A. The Chlorophyll b, reductase NOL participates in regulating the antenna size of photosystem II in Arabidopsis thaliana. Procedia. Chemistry 14, 422-427 (2015).

[34] Waters, M. T., Moylan, E. C. & Langdale, J. A. GLK transcription factors regulate chloroplast development in a cell-autonomous manner. Plant. J. 56, 432-444 (2008).

[35] Hall, L. N. et al. GOLDEN 2: A novel transcriptional regulator of cellular differentiation in the maize leaf. Plant Cell 10, 925-936 (1998).

[36] Fitter, D. W. et al. GLK gene pairs regulate chloroplast development in diverse plant species. Plant J. 31, 713-727 (2002).

[37] Waters, M. T. et al. GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis. Plant Cell 21, 1109-1128 (2009).

[38] Schmitz, A. J., Glynn, J. M., Olson, B. J., Stokes, K. D. & Osteryoung, K. W. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development. Mol. Plant 2, 1211-1222 (2009).

[39] Goral, T. et al. Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis. Plant J. 69, 289-301 (2012).

[40] Zhao, X. et al. Non-photochemical quenching plays a key role in light acclimation of rice plants differing in leaf color. Front. Plant Sci. 7, 1968 (2016).

[41] Kim, E. H. et al. The multiple roles of light-harvesting chlorophyll a/b-protein complexes define structure and optimize function of Arabidopsis chloroplasts: a study using two chlorophyll b-less mutants. BBA 1787, 973-984 (2009).

[42] Shi, L. X. et al. Photosystem II, a growing complex: updates on newly discovered components and low molecular mass proteins. BBA 1817, 13-25 (2012).

[43] Klodawska, K. et al. Morphological and physiological characterization of the delta-psaL mutant of cyanobacterium Synechocystis. Polyhedron 18, 289-297 (2013).

[44] Cazzonelli, C. I. & Pogson, B. J. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant. Sci. 15, 266-274 (2010).

[45] Song, L. et al. Molecular link between leaf coloration and gene expression of flavonoid and carotenoid biosynthesis in Camellia sinensis cultivar ‘Huangjinya’. Front Plant Sci. 8, 803 (2017).

[46] Dong, H. et al. The Arabidopsis Spontaneous Cell Death1 gene, encoding a zeta-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signalling. Cell Res. 5, 458-470 (2007).

[47] Yuan, H. et al. Carotenoid metabolism and regulation in horticultural crops. Hortic. Res. 2, 15036 (2015).

[48] Liu, J. X. et al. RNA interference-based gene silencing of phytoene synthase impairs growth, carotenoids, and plastid phenotype in Oncidium hybrid orchid. SpringerPlus 3, 478 (2014).

[49] Xu, W. J., Dubos, C. & Lepiniec, L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 20, 176-186 (2015).

[50] Appelhagen, I. et al. Leucoanthocyanidin dioxygenase in Arabidopsis thaliana: characterization of mutant alleles and regulation by MYB-BHLH-TTG1 transcription factor complexes. Gene 484, 61-68 (2011).