Chrysanthemum seedling responses to low-light conditions
On day 21 of low-light exposure, chrysanthemum plants of the GF cultivar exhibited thinner leaves and weaker growth relative to corresponding control (CK) plants, whereas no such morphological changes were evident for plants of the XF cultivar (Fig. 1). The low-light-sensitive GF plants exhibited increased internode and petiole lengths, in contrast to low-light-resistant XF (Fig. 2).
The response curves for different chrysanthemum varieties grown under low light treatment conditions are shown in Fig. 3. When photosynthetically active radiation was low (PAR ≤ 200 µmol∙m− 2∙s− 1), the net photosynthetic rate of leaves exhibited an almost linear increase with changing light intensity, and the net photosynthetic rate of XF CK plants was significantly higher than that of plants under low light treatment (p < 0.05). Similarly, GF CK plants exhibited a net photosynthetic rate that was significantly higher than that of plants under low light conditions (p < 0.01). When the photosynthetically active radiation was 1200–1500 µmol∙m− 2∙s− 1, the net photosynthetic rate of XF LL plants tended to plateau and to reach the light saturation point. This trend manifested earlier in GF LL plants, indicating that GF plants were more significantly affected by low light.
Chrysanthemum gene expression profiles in response to low light
Relative to the low-light-resistant XF cultivar, the low-light-sensitive GF cultivar exhibited substantially more DEGs (both upregulated and downregulated) upon exposure to LL conditions (Fig. 4). When comparing shared DEGs between XF and GF plants (LL vs. CK conditions), 343 and 182 commonly up- and downregulated genes were identified, respectively (Figs. 4b-c).
GO classification of common DEGs
We next evaluated the transcriptomic responses of XF and GF plants to better understand how they respond to LL conditions. Overall, similar LL-induced DEG profiles were observed in leaf samples from both cultivars, with a slightly higher fraction of genes involved in extracellular responses, transcription regulator activity, and signal transduction activity being observed in GF plants relative to XF plants. A number of resistance-associated genes were among DEGs identified in both XF and GF plants (Fig. 5). Resistance-related genes that were preferentially expressed in XF plants included those encoding light-harvesting proteins and protein phosphatases. Enrichment analyses revealed upregulated genes to be primarily associated with photosynthesis, including 9 proteins related to photosystem I (GO: 0009522), 7 with pectatelyase activity (GO: 0030570), 9 with photosystem II-related functions (GO: 0009523), 9 associated with chlorophyll binding (GO: 0016168), 8 photosynthesis-related proteins (GO: 0009765), 9 involved in chromophore linkage (GO: 0018298), 9 associated with the chloroplast thylakoid membrane (GO: 0009535),7 involved in pectin catabolic processes (GO: 0045490), 2 with ferroxidase activity (GO: 0004322), 2 capable of ferric iron binding (GO: 0008199), 2 with sigma factor activity (GO: 0016987), 2 linked to iron-ion transport (GO: 0006826), 10 involved in metal-ion binding (GO: 0046872), 2 pigment binding-related proteins (GO: 0031409), 2 cell-related proteins (GO: 0005623), 2 related to cellular iron-ion homeostasis (GO: 0006879), 2 plastoglobule-associated proteins (GO: 0010287), 2 with isocitratelyase activity (GO: 0004451), 3 extracellular proteins (GO: 0005576), and 2 involved in light harvesting in photosystem I (GO: 0009768) (Table 1). Proteins encoded by the downregulated genes were primarily related to glycometabolism and amino acid metabolism, including 12 linked to chorismate biosynthetic processes (0009423) and aromatic amino acid family biosynthetic processs (GO:0009073), 6 with 3-deoxy-7-phosphoheptulonate synthase activity (GO: 0003849), 20 chloroplast-related proteins (GO: 0009507), 4 with chorismate synthase activity (GO:0004107), 7 involved in FMN binding (GO: 0010181), 4 proteins with glycolipid biosynthetic process (GO: 0009247), 4 with prephenate dehydratase activity (GO: 0004664), 4 associated with L-phenylalanine biosynthetic processes (GO:0009094), 5 with phosphatase activity (GO: 0016791), 3 exhibiting strictosidine synthase activity (GO: 0016844), 4 with arogenatedehydratase activity (GO: 0047769), 8 related to biosynthetic processes (GO: 0009058), 2 associated with UDP-glucose metabolic processes (GO: 0006011), 2 sulfolipid biosynthetic process-related proteins (GO: 0046506), 3 with glucose-1-phosphate uridylyltransferase activity (GO: 0003983), 5 with transferase activity (GO: 0016758), 2 with 3-dehydroquinate synthase activity (GO: 0003856), 2 with receptor activity (GO: 0004872), and 2 hormone-related proteins (GO: 0009725) (Table 2).
Table 1
GO enrichment analysis of common up-regulated gene in both XF and GF
Gene Ontology Term | Gene Num | -log10(P value) |
photosystem I (GO: 0009522) pectate lyase activity (GO: 0030570) | 9 7 | 12.5341 |
photosystem II (GO: 0009523) | 9 | 12.1079 |
chlorophyll binding (GO: 0016168) | 9 | 11.7762 |
photosynthesis, light harvesting (GO: 0009765) | 8 | 11.6658 |
protein-chromophore linkage (GO: 0018298) | 9 | 9.7478 |
chloroplast thylakoid membrane (GO: 0009535) | 9 | 9.6017 |
pectin catabolic process (GO: 0045490) | 7 | 9.5221 |
ferroxidase activity (GO: 0004322) | 2 | 8.1500 |
ferric iron binding (GO: 0008199) | 2 | 4.1121 |
sigma factor activity (GO: 0016987) | 2 | 4.1121 |
iron ion transport (GO: 0006826) | 2 | 4.1121 |
metal ion binding (GO: 0046872) | 10 | 3.8241 |
pigment binding (GO: 0031409) | 2 | 3.8286 |
cell (GO: 0005623) | 2 | 3.7338 |
cellular iron ion homeostasis (GO: 0006879) | 2 | 3.5206 |
plastoglobule (GO: 0010287) | 2 | 3.3802 |
isocitrate lyase activity (GO: 0004451) | 2 | 3.2669 |
extracellular region (GO: 0005576) | 3 | 2.6369 |
photosynthesis, light harvesting in photosystem I (GO: 0009768) | 2 | 2.6269 |
*Pvalue of all GO terms are lower than 0.05. Conversely, -log10(Pvalue) values of all GO terms are greater than 1.3010, that is, the greater -log10(Pvalue) value, the better significance. |
Table 2
GO enrichment analysis of common down-regulated gene in both XF and GF
Gene Ontology term | Gene Num | -log10(P value) |
chorismate biosynthetic process GO:0009423 | 12 | 20.33677 |
aromatic amino acid family biosynthetic process GO:0009073 | 12 | 19.80942 |
3-deoxy-7-phosphoheptulonate synthase activity GO:0003849 | 6 | 10.08535 |
Chloroplast GO:0009507 | 20 | 7.367669 |
chorismate synthase activity GO:0004107 | 4 | 6.505752 |
FMN binding GO:0010181 | 7 | 5.897949 |
glycolipid biosynthetic process GO:0009247 | 4 | 5.310687 |
prephenate dehydratase activity GO:0004664 | 4 | 4.714618 |
L-phenylalanine biosynthetic process GO:0009094 | 4 | 4.840234 |
phosphatase activity GO:0016791 | 5 | 4.703904 |
strictosidine synthase activity GO:0016844 | 3 | 4.759647 |
arogenate dehydratase activity GO:0047769 | 4 | 4.714618 |
biosynthetic process GO:0009058 | 8 | 4.356696 |
UDP-glucose metabolic process GO:0006011 | 2 | 3.894192 |
sulfolipid biosynthetic process GO:0046506 | 2 | 3.894192 |
UTP:glucose-1-phosphate uridylyltransferase activity GO:0003983 | 2 | 3.832329 |
transferase activity, transferring hexosyl groups GO:0016758 | 5 | 3.777114 |
3-dehydroquinate synthase activity GO:0003856 | 2 | 3.358706 |
receptor activity GO:0004872 | 2 | 3.358706 |
response to hormone GO:0009725 | 2 | 3.122524 |
*Pvalue of all GO terms are lower than 0.05. Conversely, -log10(Pvalue) values of all GO terms are greater than 1.3010, that is, the greater -log10(Pvalue) value, the better significance. |
KEGG pathway enrichment analysis of common DEGs
To explore the roles of DEGs in low-light resistance, we next conducted KEGG pathway enrichment analyses (Table 3). Upregulated genes were associated with 17 distinct metabolic pathways, including plant hormone signal transduction, MAPK signaling, purine metabolism, pyrimidine metabolism, phenylpropanoid biosynthesis, photosynthesis antenna proteins, galactose metabolism, RNA polymerase activity, circadian rhythms, porphyrin and chlorophyll metabolism, the pentose phosphate pathway, glutathione metabolism, nicotinate and nicotinamide metabolism, glycosylphosphatidylinositol-anchor biosynthesis, the synthesis and degradation of ketone bodies, and brassinosteroid biosynthesis. Processes associated with downregulated DEGs included phenylpropanoid biosynthesis, amino acid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, phenylalanine/tyrosine/tryptophan biosynthesis, stilbenoid and phenylalanine metabolism, stilbenoid/diarylheptanoid/gingerol biosynthesis, phenylalanine metabolism, RNA degradation, isoflavonoid biosynthesis, circadian rhythms, monoterpenoid biosynthesis, ubiquinone and other terpenoid–quinone biosynthesis, the pentose phosphate pathway, anthocyanin biosynthesis, flavone and flavonol biosynthesis, diterpenoid biosynthesis, and glucosinolate biosynthesis (Table 4).
Table 3
KEGG pathway enrichment analysis of common up-regulated gene in both XF and GF
Pathway ID | Pathway Name Gene Num -log10(P value) |
ko00196 | photosynthesis antenna proteins | 16 | 10.3448 |
ko04075 | plant hormone signal transduction | 39 | 7.246061 |
ko00240 | pyrimidine metabolism | 21 | 2.952804 |
ko00402 | benzoxazinoid biosynthesis | 4 | 3.079756 |
ko00860 | porphyrin and chlorophyll metabolism | 9 | 3.007799 |
ko04712 | circadian rhythm | 13 | 2.935614 |
ko00052 | galactose metabolism | 15 | 2.746596 |
ko03020 | RNA polymerase | 14 | 2.565113 |
ko00030 | pentose phosphate pathway | 10 | 2.387964 |
ko00230 | purine metabolism | 22 | 2.336557 |
ko00760 | nicotinate and nicotinamide metabolism | 5 | 2.022957 |
ko00940 | phenylpropanoid biosynthesis | 17 | 1.880869 |
ko00563 | glycosylphosphatidylinositol (GPI)-anchor biosynthesis | 4 | 1.720203 |
ko04016 | MAPK signaling pathway | 23 | 1.5111 |
ko00480 | glutathione metabolism | 8 | 1.472649 |
ko00072 | synthesis and degradation of ketone bodies | 3 | 1.376332 |
ko00905 | brassinosteroid biosynthesis | 2 | 1.306046 |
*P value of all terms are lower than 0.05. Conversely, -log10(Pvalue) values of all terms are greater than 1.3010, that is, the greater -log10(Pvalue) value, the better significance. |
Table 4
KEGG pathway enrichment analysis of common down-regulated gene in both XF and GF
Pathway ID | Pathway Name | Gene Num | -log10(P value) |
ko00941 | Flavonoid biosynthesis | 43 | 28.56404 |
ko00945 | Stilbenoid, diarylheptanoid and gingerol biosynthesis | 27 | 19.0251 |
ko00940 | Phenylpropanoid biosynthesis | 56 | 17.62388 |
ko00400 | Phenylalanine, tyrosine and tryptophan biosynthesis | 29 | 12.48904 |
ko00943 | Isoflavonoid biosynthesis | 16 | 12.05271 |
ko00360 | Phenylalanine metabolism | 25 | 11.59716 |
ko00902 | Monoterpenoid biosynthesis | 15 | 9.850463 |
ko00942 | Anthocyanin biosynthesis | 12 | 9.275998 |
ko01230 | Biosynthesis of amino acids | 48 | 5.176359 |
ko00130 | Ubiquinone and other terpenoid-quinone biosynthesis | 14 | 5.040118 |
ko00944 | Flavone and flavonol biosynthesis | 6 | 4.746465 |
ko00500 | Starch and sucrose metabolism | 32 | 3.080086 |
ko00750 | Vitamin B6 metabolism | 6 | 3.003794 |
ko00030 | Pentose phosphate pathway | 13 | 2.338327 |
ko00906 | Carotenoid biosynthesis | 10 | 2.269926 |
ko04712 | Circadian rhythm | 15 | 2.132154 |
ko00904 | Diterpenoid biosynthesis | 6 | 1.883375 |
ko03018 | RNA degradation | 23 | 1.77389 |
ko00966 | Glucosinolate biosynthesis | 4 | 1.691318 |
ko00710 | Carbon fixation in photosynthetic organisms | 14 | 1.643104 |
ko00052 | Galactose metabolism | 16 | 1.473552 |
ko00740 | Riboflavin metabolism | 4 | 1.317761 |
*P value of all terms are lower than 0.05. Conversely, -log10(Pvalue) values of all terms are greater than 1.3010, that is, the greater -log10(P value) value, the better significance. |
Analysis of low-light-related DEGs
To identify regulatory pathways related to low-light responses in these two chrysanthemum cultivars, heat map analyses were next conducted, revealing significant downregulation of the basic helix–loop–helix domain genes (unigene 4998) and elongation factor (CL1856) in XF plants (Fig. 6), suggesting a role for these genes in the maintenance of normal stem length.
Candidate DEG validation. To validate our RNA-seq data, we next selectively assessed gene expression patterns for five DEGs via qPCR, including the plant hormone signal transduction-related genes XP_021983913.1 (phytochrome interacting factor 7, PIF7), XP_022023437.1 (elongated hypocotyls 5, HY5), and XP_022017432.1 (auxin-responsive protein IAA1-like, IAA) (KEGG Orthology), the mitochondrial electron transport-related gene XP_022017432.1 (succinate dehydrogenase assembly factor 1, SDH1) (Gene Orthology), and the dormancy-related gene DOR (Swissprot) (Fig. 6). We observed PIF7 upregulation in response to LL conditions in both cultivars, although it was expressed at levels roughly four-fold higher in GF plants relative to XF plants (Fig. 7a). HY5 was significantly downregulated in LL plant samples relative to CK plant samples (Fig. 7b), with low-light similarly significantly downregulating IAA (Fig. 7c) and SDH1 (Fig. 7d) in both tested cultivars. DOR-associated gene expression was increased in response to LL conditions in both cultivars, with the increase in GF plants being roughly 32-fold higher than that observed in XF plants (Fig. 7e).