Abstract
Plants with partial or complete loss of chlorophylls and other pigments
are frequently occurring in nature but not commonly found. In the
present study, we characterize a leaf color mutant ‘arly01’ with an
albino stripe in the middle of the leaf, which is an uncommon
ornamental trait in Anoectochilus roxburghii. The albino “mutant”
middle portion and green “normal” leaf parts were observed by
transmission electron microscopy (TEM), and their pigment contents were
determined. The mutant portion exhibited underdevelopment of plastids
and had reduced chlorophyll and other pigment (carotenoid, anthocyanin,
and flavonoid) content compared to the normal portion. Meanwhile,
comparative transcript analysis and metabolic pathways mapping showed
that a total of 599 differentially expressed genes were mapped to 78
KEGG pathways, most of which were down-regulated in the mutant portion.
The five most affected metabolic pathways were determined to be
oxidative phosphorylation, photosynthesis system, carbon fixation &
starch and sucrose metabolism, porphyrin and chlorophyll metabolism,
and flavonoid biosynthesis. Our findings suggested that the mutant
‘arly01’ was a partial albinism of A. roxburghii, characterized by the
underdevelopment of chloroplasts, low contents of photosynthetic and
other color pigments, and a number of down-regulated genes and
metabolites. With the emergence of ornamental A. roxburghii in southern
China, ‘arly01’ could become a popular cultivar due to its unique
aesthetics.
Subject terms: Biochemistry, Metabolomics
Introduction
Anoectochilus roxburghii (Wall.) Lind. is one of the classical Chinese
medicinal herbs belonging to the Orchidaceae family, whose wild
populations are now endangered and extremely rare. In traditional
Chinese medicine, A. roxburghii is used to clear heat (relieve the
latent dryness-heat of the body), relieve coughing, relieve swelling,
and detoxication^[32]1. Modern research shows that A. roxburghii has
antitumor, antidiabetic, antihyperglycemic, antioxidant,
immunostimulatory, and hepatoprotective activities^[33]1–[34]4.
Moreover, the A. roxburghii plant leaf is velvety and dark green in
color with red-golden veins. These special and unique features make it
an attractive ornamental plant. With the developments of cultivation
techniques, the commercialization of A. roxburghii has quickly grown
into a burgeoning industry, including facility cultivation, wild
imitation planting, product processing, and sales^[35]5–[36]7.
Leaf color mutations occur frequently in nature, but are rare in A.
roxburghii. Our laboratory-bred variety ‘arly01’ is a leaf color mutant
originating from the A. roxburghii wild type (WT). The discernible
dissimilarity in appearance between ‘arly01’ and A. roxburghii (WT) is
that there is a light yellow-green stripe in the middle of the ‘arly01’
leaf, which increases the aesthetic and commercial appeal of A.
roxburghii.
There are numerous categories of leaf color mutant
classifications^[37]8. Basically, it can be classified into the
following phenotypes: albinism, yellowing, mottled or stripe,
virescent, and the intermedium or mixed type of these phenotypes.
Previous investigations have demonstrated that genetic changes in plant
cells can directly or indirectly affect pigment synthesis, degradation,
and proportions, leading to diverse types of leaf color
mutations^[38]9–[39]11. However, there have been few reports on the
molecular mechanisms of A. roxburghii leaf color mutations, and the
mechanism remains unclear.
The Pacific Biosciences (PacBio) Sequel II, providing circular
consensus sequencing (CCS) and continuous long-read (CLR) sequencing
modes, is the latest generation of a long-read sequencer platform. The
PacBio Sequel II significantly improved the long-read sequencing and
the discovery of candidate genes involved in biological processes,
especially for plants without a reference genome (de novo sequencing)
^[40]12,[41]13, such as A. roxburghii (improving the identified number
of candidate genes associated with biological processes and their
accuracy).
In the present study, we quantified and compared the pigment
proportions in the leaves of the ‘arly01’ mutant. Subsequently, the
PacBio Sequel II system was employed to conduct a transcriptome
sequencing analysis (results were polished by the Illumina sequencing)
of mixed RNA extracted from the different portions of ‘arly01’ (normal
and mutated portions). After data analysis, specific genes and
metabolic pathways associated with the leaf color mutation in A.
roxburghii were identified and mapped. These transcriptome analyses
have offered valuable insights into the molecular mechanisms underlying
leaf color mutations in A. roxburghii and provided a valuable resource
for improving and adapting ornamental plants.
Materials and methods
Plant materials
Anoectochilus roxburghii ‘Changtai’, wild type from Fujian, China, and
the color mutant variety ‘arly01’, were grown in a greenhouse at the
Institute of Subtropical Agriculture (ISA), Fujian Academy of
Agricultural Sciences (FAAS, Zhangzhou, China, 117.738° E, 24.553° N).
A voucher specimen of A. roxburghii ‘Changtai’ was also stored in the
ISA of FAAS, voucher ID: ArCT20150321.
The ‘arly01’ was naturally mutated and got after 5 years of tissue
culture and propagation using the ‘Changtai’ (WT) plant stems. Within
the tissue culture and propagation process, axillary buds were used as
propagules, which guaranteed their genetic backgrounds were consistent.
Plant materials for experiments, ‘Changtai’ (WT) and ‘arly01’ (as shown
in Fig. [42]1), were planted under 25 ± 2 °C with 3000–5000 Lux light
intensity for 4 months. Experimental samples with three biological
replicates were rapidly collected and frozen in liquid nitrogen and
were immediately transferred and stored at − 80 °C for pigment
extraction and subsequent RNA-seq analyses.
Figure 1.
[43]Figure 1
[44]Open in a new tab
Phenotypes of the mutant ‘arly01’ (A) and wild type ‘Changtai’ (B) of
A. roxburghii in the greenhouse. Overview of the chloroplast in the
mutated portion (D) and normal portion (E) of the ‘arly01’ leaf (C) by
transmission electron microscopic (TEM).
Transmission electron microscopy (TEM) observations
The ‘arly01’ leaves were generally divided into two parts, the mutated
portion (M, with yellow-green color) and the normal portion (N, with
dark green color), as shown in Fig. [45]1C. The observation of the
chloroplast ultrastructure was conducted using the M portion and N
portion of the ‘arly01’ leaf. Leaves were first cut into 1 mm × 1 mm
pieces and fixed in 2.5% (w/v) glutaraldehyde for 24 h at 4 °C,
followed by 0.1 mol L^−1 phosphate buffer (pH 7.4) washing for three
times. Leaf samples were post-fixed in 1% (w/v) OsO[4] at 4 °C for 2 h
and washed with 0.1 mol L^−1 phosphate buffer for three times. Samples
were then dehydrated through a graded ethanol series (50%, 70%, 80%,
90%, 100%, and 100%, v/v), embedded in pure SPI-Pon 812 resin (Epon 812
substitute), and polymerized at 60 °C for 48 h. Ultrathin sections with
60–80 nm thickness were obtained using the Leica EM UC7 ultramicrotome
(Leica, Germany). Samples were stained with 0.2% (w/v) aqueous uranyl
acetate followed by lead citrate, for 15 min, respectively. The
ultrastructure of leaf samples was examined using a Tecnai G2 20 TWIN
TEM (FEI, USA).
Pigment extraction and measurement
Approximately 0.2 g of M and N portions of leaf material were used to
determine the chlorophyll and carotenoid contents. Briefly, fresh
leaves were ground with clean quartz sands in 1 mL 80% (v/v) acetone
(pre-cooled to 4 °C). The pasty was transferred to a 15 ml tube, and
more acetone was added to a final volume of 10 mL. The tube was wrapped
up in tin foil and put in darkness. The extraction process was
continued till the pasty turned white. The absorption at 470, 646, and
663 nm was measured using an L5S UV–Vis spectrometry (INESA, China).
The total chlorophyll and carotenoid content was calculated using the
following Eqs. ^[46]14:
[MATH:
Ca=12.21A663-2.81A6
46 :MATH]
,
[MATH:
Cb=20.13A646-5.03A6
63 :MATH]
,
[MATH:
Cx+c=1000A470-3.27Ca-
104Cb229 :MATH]
, where C[a], C[b], C[x+c] represent the contents of chlorophyll a,
chlorophyll b, and total carotenoids (xanthophylls and carotenes),
respectively.
Approximately 0.1 g of leaf material was used to determine the
anthocyanin content. Leaves were ground in 10 mL acidified methanol (1%
HCl, v/v) and extracted twice in darkness at 25 °C for 2 h. The
homogenates were centrifuged at 4 °C, 12,000 r/min for 15 min. The
absorbance of the supernatant was measured at 530 and 657 nm. The
anthocyanin content was calculated using the following formula:
[MATH:
C=A530
-0.3A657×V
m :MATH]
, where V represents the volume of the extract (ml), and m represents
the weight of the fresh sample^[47]15. Acidified methanol (1% HCl, v/v)
was used as the blank control.
Fresh leaf material was first dried at 60 °C to consistent weight. Dry
samples were ground to fine powder followed by adding 95% (v/v) ethanol
and ultrasonic for 30 min to extract flavonoids. The homogenates were
centrifuged at 4 °C, 12,000 r/min for 15 min. Total flavonoid contents
were determined by the NaNO[2]-Al(NO[3])[3]-NaOH method. Briefly, 1 mL
of the extracts was added with 0.3 mL 5% (w/v) NaNO[2] solutions and
incubated for 5 min, followed by adding 0.3 mL 10% (w/v) Al (NO[3])[3]
solutions for 6 min. Then 4 mL 4% (w/v) NaOH solution was added to the
mixture, and the volume was adjusted to 10 mL with 70% (v/v) ethanol.
After 15 min, the absorbance of the solution was measured at 510 nm.
The total flavonoid content was determined using a standard curve with
rutin (Aladdin, China) as the standard.
RNA extraction, cDNA library construction, and de novo sequencing
The M and N portions of the leaves were ground into powder in liquid
nitrogen, respectively. Total RNA extractions were done using the
RNAprep Pure Plant Kit (DP441, Tiangen, China) following the
manufacturer’s instructions. RNA integrity and quantity were assessed
using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA), making
sure all the RNA integrity numbers (RIN) were at least 8.0. RNA
concentration was measured using Nanodrop 2000 (Thermo Fisher
Scientific, USA). To ensure obtaining enough RNA for sequencing, each
RNA sample was mixed from five individual plant leaves. RNA extraction,
DNA library construction, and transcriptome sequencing were completed
by Biomarker Technologies Corporation (Beijing, China) using Illumina
HiSeq4000 (next-generation sequencing, NGS) and Pacific Biosciences
(PacBio) Sequel II (third-generation sequencing, TGS) platform. Quality
checks of the cDNA libraries were done by Qubit (> 20 ng/μL), and
Agilent 2100 (fragment range 250–390 bp, without impurity peaks).
Transcriptome assembly, gene annotation and KEGG pathway mapping
To acquire high-quality clean reads for assembly and analysis, data
from the two platforms were combined and analyzed^[48]16. Raw reads
obtained from the NGS were used to polish errors in the long reads from
the PacBio Sequel II platform, resulting in a more accurate and
complete assembly of the transcriptome. The PacBio Sequel II
transcriptome sequencing mainly includes the following three steps.
First, raw reads were combined into circular consensus sequences (CCS)
according to the adaptor. Next, full-length non-chimeric (FLNC)
transcripts were determined by searching for the polyA tail signal, the
5′ and 3′ cDNA primers in CCS. Third, all full-length sequences from
the same transcript and similar full-length sequences were clustered
using the Iso-seq module in SMRT Link v7.0, and a consensus isoform
sequence was obtained from each cluster. To get high-quality
full-length transcripts an optimized CD-HIT^[49]17,[50]18 (identity
> 0.99) was set to remove redundancy.
Function annotation of assembled genes was performed using BLAST
(version 2.2.26) based on public databases, including NR (NCBI
non-redundant protein sequences), Swiss-Prot (A manually annotated and
reviewed protein sequence database), GO (Gene Ontology), Pfam (Protein
family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins),
and KEGG (Kyoto Encyclopedia of Genes and Genomes).
To identify the metabolic pathways genes and their biological functions
in leaf color mutation of A. roxburghii, assembled unigenes were
assigned to KEGG Automatic Annotation Server (KAAS,
[51]https://www.genome.jp/tools/kaas/) for ortholog assignment and
pathway mapping based on sequence similarity.
Differentially expressed genes (DEGs) analysis
Gene expression levels were estimated based on the FPKM (fragments per
kilobase of transcript per million fragments mapped) method, as derived
by the following equation:
[MATH:
FPKM=cDNAFragmentsM<
mi>appedFragmentsMillions×T
ranscri<
mi>ptLengthkb
:MATH]
Identification of the differentially expressed genes (DEGs) was
performed with DESeq2^[52]19. DESeq2 provides statistical routines for
determining differential expression in digital gene expression data
using a model based on a negative binomial distribution. The resulting
p-values were adjusted using Benjamini and Hochberg’s approach to
control the false discovery rate (FDR). Genes with an FDR < 0.01 and
|log[2](fold change)|≥ 1 were assigned as differentially expressed. GO
enrichment and KEGG pathway enrichment analysis were performed using
GoSeq R and KOBAS^[53]20, respectively.
Validation of DEGs by quantitative real‑time PCR
The RT-qPCR was performed using the SYBR®Green Premix Pro Taq HS qPCR
Kit (No. AG11701, Accurate, China) in a Roche lightcycle®96 real-time
PCR system (Roche, Germany), with 3 technical replicates. The qPCR
volume was 20 μL in total, including 3 μL template cDNA, 1 μL forward
primer and 1 μL reverse primer, 10 μL SYBR®Green Pro Taq HS Premix, and
5 μL RNase-free water. Six DEGs were selected for the validation of the
transcript data. A housekeeper gene, A. roxburghii β-Actin, was used as
the internal reference ^[54]21, which provided a basis for the relative
quantification assays. The two-step cycling qPCR method was used in our
experiment. Briefly, the denaturation was 95 °C for 30s, following
62 °C, 30s of annealing for 40 cycles. All primers used in this assay
are listed in Table [55]1.
Table 1.
Primers of DEGs for real-time fluorescent quantitative PCR.
Gene name Forward primer (5′-3′) Reverse primer (5′-3′)
HemH AAAGCGGAGATGGAGGAGTG ATTCCACAGGTCCAACTCGG
ChlM CCAAGCAGCCCTATTCGTTC CGAGCTGGACCTTATTGACG
GLU AGGTGGGATGTCTTTGGGAG CAAAACGCCCTGAAGCAACC
bHLH CTCCATGCTCCTCTGATTG ATGAGCACTATTTGTGGGTC
YABBY CCAAGCCAGACATTCCTCAC ACTTGGACCGCTACTGTTGG
LOB ACGTGGCGAAACTCCTGAAC TAGCCAACGCATCCGTAGAC
β-Actin AGATGAGGCACAGTCCAAGA GCTGGAACATTGAAGGTCTC
[56]Open in a new tab
Statistical analysis
The statistical analyses of pigment contents were conducted with the
one-way ANOVA LSD test (p-value < 0.01) using the IBM SPSS Statistics
(version 25) statistical software.
Ethical approval
All materials in this study are comply with relevant institutional,
national, and international guidelines, legislation, and sub-section
ethical approval and consent to participate.
Results
Chloroplast observations
Ultrastructure of the plastids were observed in both portions
(Fig. [57]1D,E) by transmission electron microscopy (TEM). The TEM
images revealed that plastids in the M portion leaf tissues were
underdeveloped compared to the chloroplasts in the N portion, and their
number was lower, no ultrastructural changes were observed.
Qualitative and quantitative pigments analyses
To determine the important pigments responsible for the leaf color
mutation phenotype, pigment profile measurement was performed based on
the M portion and N portion of ‘arly01’ leaves. Results showed that
color pigment profiles were significantly different between the M
portion and the N portion. As shown in Fig. [58]2, pigments in the N
portion, chlorophyll a, chlorophyll b, carotenoid, anthocyanin, and
total flavonoids contents were 2.179 mg g^−1, 0.758 mg g^−1,
0.597 mg g^−1, 3.844 mg g^−1, and 6.497 mg g^−1, respectively.
Significant declines of 71.5%, 72.4%, 66.6%, 66.6%, and 40.1% were
measured, respectively, in the M portion compared to the N portion.
However, the value of (chlorophyll a/chlorophyll b) in the M portion
was slightly higher than that of the N portion, which is not
significant (p-value > 0.05).
Figure 2.
[59]Figure 2
[60]Open in a new tab
Chlorophyll (A) and other pigment contents (B) in the mutated portion
(M) and the normal portion (N) of ‘arly01’ leaves (p-value < 0.01).
Overview of the transcriptome data and functional annotation
As shown in Supplementary Table [61]S1, in the Illumina HiSeq
sequencing, a total of 65.74 Gb clean data was obtained from the M and
N portion leaves (three biological replicates for each portion). The
dataset of each library ranged from 9.97 to 12.48 Gb. The GC contents
of each sample ranged from 48.37 to 48.85%, and the percentages of base
sequencing quality scores that reach 30 (referred to here as % ≥ Q30)
of each library were greater than 94.73%.
A total of 537,890 polished CCS reads were obtained from the PacBio
Sequel II sequencing platform (polished by the Illumina HiSeq
sequencing data, all clean reads were merged), including 405,580
full-length non-chimeric (FLNC) reads which accounts for 75.40%.
130,098 consensus isoforms were obtained from each cluster by
clustering of the FLNC reads, 130,057 of which were high-quality
consistent sequences. CD-HIT^[62]17,[63]18 (identity > 0.99) was set to
remove the redundancy of the high-quality consistent sequences.
Ultimately, the sequence and expression information of 82,215 genes was
obtained for subsequent analysis. All these analysis algorithms ensure
that our sequencing quality was sufficient for further analysis.
A total of 72,666 annotated unigenes were obtained based on the
database searching against COG, GO, KEGG, KOG, Pfam, Swiss-Prot,
EggNOG, and Nr, as shown in Supplementary Table [64]S2.
Functional analysis of DEGs between mutated and normal portions
FPKM method was used to evaluate the unigene expression levels. A total
of 982 DEGs were identified, including 366 up-regulated and 616
down-regulated genes (M portion vs. N portion), as shown in Fig. [65]3.
A heatmap of all the DEGs based on the FPKM values (normalized by the
z-score method) showed that all the replicates exhibited similar
expression patterns (Supplementary Figure [66]S1). To further confirm
these observations, six genes were selected for qRT-PCR validation
(Fig. [67]4). The results of the qRT-PCR analysis were consistent with
our transcriptome data and thus validated its reliability.
Figure 3.
[68]Figure 3
[69]Open in a new tab
Volcano plots of gene expression profile between M portion versus N
portion. Red points represent up-regulated DEGs. Blue points represent
down-regulated DEGs. Gray points represent not changed genes. The
identification thresholds were set as FDR < 0.01 and |log[2](fold
change)|≥ 1. (A) 982 identified DEGs, including 366 up-regulated and
616 down-regulated genes (M portion vs. N portion); (B) 645 GO
annotated DEGs, including 197 up-regulated and 448 down-regulated genes
(M portion vs. N portion). The top three abundant GO terms were
metabolic process (387 DEGs), cellular process (298 DEGs), and
single-organism process (239 DEGs).
Figure 4.
[70]Figure 4
[71]Open in a new tab
Comparison of the expression levels of six selected genes detected by
transcriptome and RT-qPCR experiment. Results were presented as the
mean of three repeated experiments.
GO and KEGG annotation analysis
A total of 40,206 unigenes and 645 DEGs were assigned in GO annotation.
GO enrichment analysis of all unigenes and DEGs were conducted to
illustrate the distribution of genes involved in the following
classifications: biological process (BP), cellular component (CC), and
molecular function (MF), as shown in Fig. [72]5.
Figure 5.
[73]Figure 5
[74]Open in a new tab
GO enrichment analysis of the genes of mutation portion vs. normal
portion from ‘arly01’ leaves. A total of 40,206 unigenes and 645 DEGs
were divided into three categories: biological process (BP), cellular
component (CC), and molecular function (MF). Red columns represent the
number of unigenes involved in the GO terms, blue columns represent the
number of DEGs.
Within the biological process category, the top three abundant GO terms
were metabolic process (387 DEGs, 60% accounted for all the 645
annotated DEGs), cellular process (298 DEGs, 46.20%), and
single-organism process (239 DEGs, 37.05%). In the cellular component
category, 333 and 250 DEGs (51.63%, 38.76%) were distributed in
membrane and membrane part, 312 and 311 DEGs (48.37%, 48.21%) in cell
and cell parts, 226 and 148 DEGs (35.03% and 22.95%) in organelle and
organelle part. In terms of molecular function category, catalytic
activity (347 DEGs, 53.80%), binding (283 DEGs, 43.87%), and
transporter activity (87 DEGs, 13.49%) were the top three abundant GO
terms.
High percentage of DEGs in these GO terms implied significant shifts or
differences in the chemical and biological system between the N portion
and M portion, such as in metabolite production, energy utilization,
molecule transportation and movement, cell growth, signal transduction,
and ligand-receptor interaction, etc. This was consistent with the
results mentioned previously, such as the underdevelopment of plastids,
decease of chlorophyll and other pigment contents and in the M portion.
To determine the significance of the mapped KEGG pathway and the DEGs
enrichment, pathway significance enrichment analysis was conducted. A
total of 599 DEGs were mapped to 78 KEGG pathways and were further
classified into five classifications, including metabolism, genetic
information processing, environmental information processing, cellular
processes, and organismal systems. As shown in Fig. [75]6, the higher
value of − log[10] (p-value) for a certain pathway in our study
indicated a higher significance of the pathway correlated to the
conditions. Rich factors that represented the ratio of DEG numbers
located in the same pathway were also calculated.
Figure 6.
[76]Figure 6
[77]Open in a new tab
Enriched and classified KEGG pathways of the DEGs between the M portion
versus N portion of the ‘arly01’ leaves. Color and size of the dots
indicated the p-value and the number of DEGs (numbers noted next to the
dots) mapped to a certain pathway, respectively. The rich factor
(x-axis) is the ratio of the DEG number to the total gene number in the
same pathway, represented as dots position. The pathways were further
classified into five major groups: metabolism, genetic information
processing, environmental information processing, cellular processes,
and organismal systems.
Metabolic pathways and mapping
Based on the overall analysis of DEGs identification and KEGG
classification, we picked the top 30 pathways and constructed five
metabolic pathway maps influenced by the leaf color mutation, as shown
in Fig. [78]7. DEGs involved in the pathways of oxidative
phosphorylation (ko00190), photosynthesis system (ko00195 and ko00196),
carbon fixation (ko00710) & starch and sucrose metabolism (ko00500),
porphyrin and chlorophyll metabolism (ko00860), and flavonoid
biosynthesis (ko00941) were sorted out (Supplementary Table [79]S3).
Figure 7.
[80]Figure 7
[81]Open in a new tab
Overview of the differentially expressed genes (DEGs) that were
involved in top-influenced metabolic pathways from the leaf color
mutant ‘arly01’. Genes in red boxes and green boxes represented
up-regulated genes and down-regulated genes, respectively. The
metabolic processes include (A) photosynthesis system, (B) oxidative
phosphorylation, (C) carbon fixation and starch and sucrose metabolism,
(D) porphyrin and chlorophyll metabolism, and (E) flavonoid
biosynthesis. Gene information is listed in Supplementary Table [82]S3.
Due to the underdevelopment of plastids, the photosynthesis antenna
protein genes in light-harvesting complex I (includes chlorophyll a
binding proteins of LHCa2, LHCa3, and LHCa4) and light-harvesting
complex II (includes chlorophyll b binding protein of LHCb1, LHCb2,
LHCb3, LHCb4, LHCb5, and LHCb6) were down-regulated. Eight genes of
Photosystem I protein subunits (PsaD, PsaE, PsaF, PsaG, PsaH, PsaK,
PsaL, and PsaO), and eight genes of Photosystem II protein subunits
(PsbA, PsbE, PsbK, PsbO, PsbP, PsbQ, PsbR, and PsbS) were
down-regulated. The PetE (plastocyanin), and PetF (ferredoxin) of
photosynthetic electron transport genes were down-regulated
(Fig. [83]7A). Influenced by this, in the oxidative phosphorylation
pathway, the V-type H^+-transporting ATPase subunit E(ATPeV1E) and
H^+-transporting ATPase(PMA1) were up-regulated, while three
NAD(P)H-quinone oxidoreductase subunits genes (NDHD, NDHF, and NDHH)
were down-regulated (Fig. [84]7B). A total of 10 down-regulated DEGs
were identified in the carbon fixation & starch and sucrose metabolism
pathway map, including SCRK, GLGC, FBP, RPE, GAPA, ALDO, RPIA, TPI,
PPDK, and PK (Fig. [85]7C). Furthermore, nine genes of porphyrins and
chlorophyll metabolisms such as GLU, HemA, HemH, ChlM, ChlE, POR,
HMOX1, FECH, and HCAR were involved. Among the other down-regulated
DEGs, the HCAR was the only up-regulated gene (Fig. [86]7D). Unlike the
above-mentioned pathway, we identified three up-regulated DEGs that
influenced many flavonoid biosynthesis in the flavonoid biosynthesis
pathway, including CYP75B1, F3H, and FLS (Fig. [87]7E).
Discussion
Pigments and leaf color
The relationship between different pigments and leaf color is complex
in plant biology. The pigment profiles between the M and N portions
suggested that the mutation of ‘arly01’ leaves had significant impacts
on the pigments and biochemical compositions.
Chlorophyll a (Chl a) and chlorophyll b (Chl b) are essential for
photosynthesis, and their levels are crucial for determining leaf
greenness^[88]22. The decline of 71.5% Chl a and 72.4% Chl b in the M
portion significantly reduced the portion’s green color.
Carotenoids^[89]23 (Car) can appear yellow, orange, or red;
anthocyanins^[90]24 are responsible for red, purple, and blue; while
flavonoids often appear yellow or white in plants^[91]25. Collectively,
the decrease in photosynthetic pigments (chlorophylls and carotenoids)
and color-related pigments (anthocyanins and flavonoids) resulting in a
yellow-green stripe on the leaves (Fig. [92]1C).
Photosynthesis
Chloroplast is the organelle where the plant photosynthesis process and
energy conversion occur^[93]26,[94]27. It is composed of three parts:
chloroplast membrane, thylakoid and stroma. There are light-harvesting
complexes I and II, photosystem I and II, and photosynthetic electron
transfer systems located on the thylakoid membrane, together responding
for light capture and energy transfer from light energy to produce ATP
and NADH.
In plant cells^[95]28,[96]29, the photosystem I is composed of 16
proteins (PsaA ~ X), the photosystem II is composed of 27 proteins
(PsbA ~ 28), the photosynthetic electron transfer process is composed
of 4 proteins (PetE ~ J), and the light-harvesting complexes I and II
are composed of 5 (LHCa1 ~ 5) and 7 (LHCb1 ~ 7) proteins, respectively.
The assembly of these proteins forms the PS I-LHC I supramolecular
complex and PS II-LHC II supramolecular complex, with electron and
energy transfers from PS II to PS I. The down-regulation of these genes
(Fig. [97]7A) in the mutated portion of the ‘arly01’ leaf, is expected
to reduce PS I-LHC I and PS II-LHC II supramolecular complexes, likely
resulting in the underdevelopment of plastids, which was consistent
with the results of electron microscope observation(Fig. [98]1D,E). The
resulting lower efficiency of light absorption, energy transfer, and
photosynthetic activity in the M portion consequently is expected to
result in reduced production of glucose and other organic compounds.
Electrons from PS II are ultimately passed to PS I, where
re-energization occurs with another photon of light energy^[99]30.
Energized electrons from PSI are then used to reduce NAD^+ to NADH,
which is used in carbon fixation & starch and sucrose metabolism.
However, in the current study, energy production genes were
up-regulated, while the NADH oxidoreduction was down-regulated
(Fig. [100]7B). A total of 10 genes were down-regulated in the carbon
fixation & starch and sucrose metabolism pathway, as shown in
Fig. [101]7C, which significantly reduced the storage of organic
compounds in the mutated portion of ‘arly01’.
Chlorophyll metabolism
Chlorophyll a, chlorophyll b, and carotenoids are the main
photosynthetic pigments of higher plants, which also determine the
color of plant leaves. Leaf color mutants are usually accompanied by
changes of chlorophyll (Chl) and carotenoid levels ^[102]31,[103]32.
The Ornamental crabapple (Malus sp.) delayed-green leaf color mutant
had lower Chl, Car, and flavonoid contents^[104]33. A study on the
chlorophyll-deficient leaf color mutant of tree peony (Paeonia
suffruticosa) showed that the mutant had lower pigment contents, but
increased Chl a/b ratio and Car to Chl ratio^[105]34. In the present
study, the content of Chl and Car in the mutated portion of ‘arly01’
was significantly decreased (p-value < 0.01), and the decreased ratio
of Chl was higher than that of Car (p-value < 0.01). However, there was
no significant difference found in the Chl a/b value between the M and
the N portion, suggesting that the decrease of Chl a and Chl b was
uniform.
As shown in Fig. [106]7D, the entire process of chlorophyll synthesis
can be roughly categorized into three main steps^[107]35: the first
step is the conversion of L-Glutamine into protoporphyrin IX (occurs in
the cytoplasm), the second step is the insertion of magnesium ions into
protoporphyrin IX (Mg-proto IX, completed in the middle of the
chloroplast stroma), and the third step takes place in chloroplast
membrane for the transformer process of Mg-proto IX to chlorophyll.
Differential expression of any genes in the three steps leads to
changes in enzyme function and activity that affect the whole
chlorophyll synthesis process^[108]36, such as the accumulation of
intermediates^[109]37, metabolism of chlorophyll^[110]8, and the
distribution of chlorophyll pigment in plant cells^[111]37,[112]38,
which causes leaf color mutations, consequently.
In this study, the down-regulated expression of gene GLU and HemA
(first step), CHLM and CHLE (second step), and POR (third step) reduced
the efficiency of the Chl a and Chl b production. In addition,
cross-conversion can happen between Chl a and Chl b in green plants,
and we detected an up-regulated HCAR gene that could affect the
efficiency of the transformation of Chl b to Chl a, possibly leading to
the slight increase of chlorophyll a/b value (no significant
difference) detected between the M portion and the N portion
(Fig. [113]2).
Flavonoid biosynthesis
Flavonoids play pivotal roles in plant development^[114]39,[115]40,
such as cell wall synthesis, pigmentation, pest resistance, and
protecting plants from UV irradiation and oxidative stress. Flavonoids
in A. roxburghii were well studied^[116]41–[117]43. In the current
study, three up-regulated genes were identified in the flavonoid
biosynthesis pathway (Fig. [118]7E). However, the total flavonoid
content of the M portion was significantly decreased (Fig. [119]2B).
This paradox might be attributed to the decrease of photosynthesis
efficiency, stimulating the production of plant secondary metabolites
like flavonoids and phenolics^[120]44. However due to the lack of
intermediate substances for flavonoid synthesis, which derived from the
decreased photosynthesis and substances storage, the total flavonoid
content was decreased^[121]45. Despite this, the ‘arly01’ mutant
necessitates flavonoids in favor of leaf development and protection.
Thus, a few flavonoid biosynthesis related genes were found
up-regulated. Future efforts should be conducted to test this
hypothesis and demonstrate the broad roles of flavonoids in A.
roxburghii.
Conclusions
In the present study, we have gained a comprehensive understanding of
the leaf color mutation mechanism in the A. roxburghii variant,
‘arly01’ by using TEM, pigment profile analysis, comparative transcript
analysis, and metabolic pathway mapping. Underdevelopment of plastids
and significant declines of chlorophyll and other pigment contents were
detected in the mutated portion. A series of negatively affected
metabolic pathways were found and mapped, including the photosynthesis
system, oxidative phosphorylation, carbon fixation & starch and sucrose
metabolism, porphyrin and chlorophyll metabolism, and flavonoid
biosynthesis. In conclusion, the variation of this mutant ‘arly01’ is
characterized by the underdevelopment of plastids, low contents of
photosynthetic and other color pigments, and several down-regulated
genes and metabolites.
Further research can focus on strategies that control leaf color and
improve functional ingredient products, such as deeper exploration of
plastid gene mutations influencing leaf color^[122]46, breeding novel
color variations in ornamental plants, and increasing flavonoid content
in A. roxburghii.
Supplementary Information
[123]Supplementary Information.^ (106.8KB, docx)
Author contributions
L.J. devised the project and the main conceptual ideas. H.H. wrote and
revised the main manuscript text. Z.H. contributed to sample
preparation. L.H. revised the manuscript. D.Y. supervised the project.
All authors discussed the results and contributed to the final
manuscript.
Funding
Funding was provided by Fujian Academy of Agricultural Sciences (Grant
No. CXTD2021001-2) and Fujian Provincial Department of Science and
Technology (Grant No. 2020R1030003, 2021R1030001).
Data availability
All materials and related data in this study are available upon
request. The datasets generated during and/or analyzed during the
current study are available in the NCBI Sequence Read Archive (SRA)
repository, [124]https://www.ncbi.nlm.nih.gov/bioproject/PRJNA973204.
Competing interests
The authors declare no competing interests.
Footnotes
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Supplementary Information
The online version contains supplementary material available at
10.1038/s41598-023-50352-5.
References