Abstract
Background
Nitrogen (N) is an essential component of photosynthetic apparatus.
However, the mechanism that photosynthetic capacity is suppressed by N
is not completely understood. Photosynthetic capacity and
photosynthesis-related genes were comparatively analyzed in a
shade-tolerant species Panax notoginseng grown under the levels of low
N (LN), moderate N (MN) and high N (HN).
Results
Photosynthetic assimilation was significantly suppressed in the LN- and
HN-grown plants. Compared with the MN-grown plants, the HN-grown plants
showed thicker anatomic structure and larger chloroplast accompanied
with decreased ratio of mesophyll conductance (g[m]) to Rubisco content
(g[m]/Rubisco) and lower Rubisco activity. Meanwhile, LN-grown plants
displayed smaller chloroplast and accordingly lower internal
conductance (g[i]). LN- and HN-grown individuals allocated less N to
light-harvesting system (N[L]) and carboxylation system (N[C]),
respectively. N surplus negatively affected the expression of genes in
Car biosynthesis (GGPS, DXR, PSY, IPI and DXS). The LN individuals
outperformed others with respect to non-photochemical quenching. The
expression of genes (FBA, PGK, RAF2, GAPC, CAB, PsbA and PsbH) encoding
enzymes of Calvin cycle and structural protein of light reaction were
obviously repressed in the LN individuals, accompanying with a
reduction in Rubisco content and activity. Correspondingly, the
expression of genes encoding RAF2, RPI4, CAB and PetE were repressed in
the HN-grown plants.
Conclusions
LN-induced depression of photosynthetic capacity might be caused by the
deceleration on Calvin cycle and light reaction of photosynthesis, and
HN-induced depression of ones might derive from an increase in the form
of inactivated Rubisco.
Keywords: Photosynthesis, Rubisco, Chloroplast, Non-photochemical
quenching, Nitrogen, Panax notoginseng
Background
Nitrogen (N) is a major limiting factor in natural ecosystems and in
most agricultural systems [[29]1, [30]2]. N is regarded as a necessary
component of numerous biomolecules, such as DNA, RNA, proteins,
chlorophyll (Chl) and cell envelope [[31]3, [32]4]. N shortage results
in enormous changes in plant morphology and even destroys the balance
of biological process, including N metabolism and photosynthesis
[[33]5, [34]6]. N-deficient crops show the premature of leaves, and
reduce leaf area expansion, plant height and ultimately yield of their
own [[35]5–[36]9]. On the other hand, excessive N supply makes leaves
dark green and stems frail and immature, and consequently cause an
imbalance between the vegetative and reproductive growth
[[37]10–[38]12]; For example, excessive N supply considerably reduces
the biomass of cucumber (Cucumis sativus) [[39]13] and of tomato
(Lycopersicon esculentum) [[40]14] . However, N surplus in plants
receives relatively little attentions in comparison with N deficiency
over the past decades.
It has been commonly accepted that photosynthesis is highly influenced
by leaf anatomy and chloroplast ultrastructure. HN-grown Arabidopsis.
thaliana displays thicker upper epidermises, lower epidermises, spongy
tissue and palisade tissue, and increased thickness of anatomic
structure would not facilitate CO[2] diffusion in the liquid phase of
mesophyll cells [[41]15]. N deficiency exhibits small chloroplast with
lower internal conductance (g[i,]) [[42]16], and a large chloroplast
with well-developed grana under high-N application has been reported in
summer maize [[43]17]. Indeed, Photosynthesis-related components are
strongly regulated by leaf N and photosynthetic capacity is closely
related to N content since more than 50% of total leaf N is allocated
to photosynthetic machinery and proteins of Calvin cycle represent the
majority of leaf N [[44]18–[45]21]. In leaves of developing maize (Zea
mays), N deficiency results in an obvious decrease in photosynthesis
with an reduction in activities of phosphor enolpyruvate carboxylase
(PEPC), pyruvate orthophosphate di-kinase (PPDK) and ribulose 1,
5-bisphosphate carboxylase (Rubisco) [[46]17, [47]22–[48]24]. A
reduction in content of Rubisco and in effective and maximum quantum
yield of photosystem II (ΔF[v]/F[m] & F[v]/F[m]) has been recorded in
A. thaliana and Oryza sative grown under N deficiency condition
[[49]25, [50]26]. Likewise, Rubisco carboxylase activity considerably
declines in spinach (Spinacia oleracea) and cassava (Manihot esculenta)
due to N deficiency [[51]27, [52]28]. On the other hand, negative
responses of photosynthetic capacity to excess N, including decreased
Rubisco activity, lower N allocation to light-harvesting system (N[L])
and lower photosynthetic efficiency, have been observed in field-grown
wheat [[53]29], rice [[54]30] and cotton [[55]31]. In general, the
relationship between N levels and photosynthesis is nonlinear in a
sufficiently broad range of leaf N content [[56]32, [57]33]. However,
relatively little is known about its molecular mechanism on the
nonlinear relationship between leaf N and photosynthesis.
N-suboptimal plants would suffer from greater excess of light energy,
and this could produce excessive reactive oxygen species (ROS)
[[58]34]. Plants has employed a series of photoprotective mechanism to
survive long periods of no-optimal N regimes. Non-photochemical
quenching (NPQ) of excess light energy within the light-harvesting
antennae of PSII (LHCII) are believed as an effective photoprotective
mechanisms, as observed in A. thaliana plants subjected to low nitrogen
(LN) [[59]26, [60]35, [61]36] and in Coffea Arabica [[62]37] and
Lavandula angustifolia [[63]38]. Excess N supply in benthic diatom
(Entomoneis paludosa) [[64]39] have revealed an up-regulation of
xanthophyll cycle, which reduce the efficiency of PSII photochemistry
and enhance NPQ. In addition, several studies have highlighted a
positive effect of glycolytic pathway and pentose phosphate pathway
(PPP) on energy and carbon balance in N-stressed plants [[65]40,
[66]41]. Unexpectedly, molecular mechanisms of photoprotection are not
completely clear in the N-stressed non-model species, especially in a
shade-tolerant plant.
Comparative transcriptomes have revealed that unigenes expression of
Chl biosynthesis, Calvin cycle and ribosomal proteins were decreased in
Scenedesmus acuminatus under high N (HN) supply [[67]42]. Up-regulated
gene transcripts are predominantly matched in kinds of amino acid
metabolism, transport and stress, whereas repressed transcripts are
overrepresented in categories of hormone metabolism and redox control
in roots of A. thaliana under N deficiency [[68]43]. During periods of
N-limitation, gata transcription factor (GNC), a gene regulating carbon
(C) and N metabolism, operates to support A. thaliana survival by
elevating Chl biosynthesis [[69]44]. Genes encoding enzymes for C
skeleton production are down-regulated in spinach plants under
N-starvation, and plants also significantly show low contents of amino
acid and high levels of glucose and consequently decelerate growth
[[70]45]. Light reaction center of photosynthesis by extrinsic proteins
labled as PsbO, PsbP, PsbQ, PsbR, PsbU and PsbV are suppressed in
Synechocystis under N stress [[71]46]. PsbS protein is activated by the
acidity of thylakoid lumen in A. thaliana plants under N-stressed
condition [[72]47]. In addition, the expression of NR and GOGAT was
dramatically up-regulated in the cucumber exposed to HN [[73]48].
Surprisingly, relatively less investigation has been conducted to
elucidate the correlation of photosynthesis-related genes expression
with photosynthetic performance in the context of N.
Panax notoginseng (Burkill) F. H. Chen (Sanqi in Chinese) is a
typically shade-tolerant species from the family of Araliaceae
[[74]49–[75]51], In our previous researches, P. notoginseng is believed
to be highly sensitive to high light, and 10% of full sunlight is
suitable for its growth [[76]50, [77]52]. Besides, the development and
growth of P. notoginseng is highly sensitive to high N [[78]53–[79]55].
HN application considerably enhance rust cracking, root decay and
mortality rate of P. notoginseng [[80]56]. Indeed, significant
decreases in root, stem and leaf biomass have been observed in P.
notoginseng grown under LN, along with narrow and yellow leaves
[[81]57, [82]58]. However, these previous studies have mainly focused
on effects of N input on agronomic traits, yield, and plant growth.
Nowadays, the molecular mechanism of the sensitivity of P. notoginseng
to N is still unclear.
Different N levels were applied to P. notoginseng, and photosynthetic
capacity, photoprotection and photosynthetic pigments were
comparatively analyzed in the plants grown under low N (LN), moderate N
(MN) and high N (HN). Meanwhile, a comprehensive transcriptome was
conducted to elucidate the expression of photosynthesis-related gene.
The objective of our study was to elucidate the photosynthetic
performance and the expression of photosynthesis-related genes in the
typically shade-tolerant and N-sensitive plant P. notoginseng under
different levels of N, and it was anticipated that photosynthetic
performance might be coordinated with the expression of
photosynthesis-related genes.
Results
Effect of N regimes on plant growth and leaf gas change
HN-grown leaves were pretty dark-green, and LN-grown leaves were
significantly smaller and yellowish (Additional file [83]1: Figure
S1a). HN-grown plants possessed a low survival rate (Additional file
[84]1: Figure S1b). LN-grown leaves were dramatically reduced in the
thickness of upper epidermis, lower epidermis, spongy tissue and
palisade tissue, and biomass of leaf was significantly reduced in LN
and HN treatments (Table [85]1). On the other hand, LN significantly
decreased the size of chloroplasts accompanied with a reduction in
chloroplast exposed to intercellular air space per unit leaf area
(S[c]), and correspondlingly an increase in the size of chloroplasts
was observed in P. notoginseng under excessive N supply (Fig. [86]1 c;
Table [87]2). The LN plants and HN plants showed 52.7 and 96.8% lower
liquid phase (g[lip]) than the MN plants, respectively. g[lip] can be
expressed as g[lip] = C[lip] × S[C], therefore, conductance per unit of
exposed chloroplast surface area (C[lip]) is one of a determinant of
g[lip]. C[lip] were reduced in HN-grown plants as compared to the
MN-grown individuals. Internal CO[2] condutance (g[i]) is mainly
determined by g[lip], and LN-and HN-grown plants was decreased in g[i]
(Table [88]2)[.]
Table 1.
Effects of nitrogen regimes on the leaf morphology, anatomy and biomass
in a shade-tolerant plant Panax notoginseng
Variables Nitrogen level
LN MN HN
Upper epidermis (μm) 11.209 ± 0.024 c 17.694 ± 1.927 a 14.738 ± 0.269 b
Lower epidermis (μm) 10.590 ± 1.027 c 13.177 ± 2.186 a 11.420 ± 0.918 b
Spongy tissue (μm) 42.551 ± 2.194 c 70.378 ± 0.182 a 56.518 ± 0.189 b
Palisade tissue (μm) 17.069 ± 1.283 c 32.867 ± 0.173 a 24.490 ± 1.825 b
Palisade/spongy 0.401 ± 0.002 0.467 ± 0.016 0.433 ± 0.016
Leaf length (cm) 6.484 ± 1.980 c 7.515 ± 1.068 a 6.795 ± 1.238 b
Max width (cm) 2.777 ± 0.698 c 3.114 ± 0.621 ab 3.273 ± 0.519 a
Leaf length/max width 2.341 ± 1.339 2.413 ± 0.8445 2.076 ± 0.879
Leaf dry weight (g plant^− 1) 0.413 ± 0.040 c 0.545 ± 0.025 a
0.496 ± 0.064 b
Total dry weight (g plant^− 1) 1.068 ± 0.294 c 1.649 ± 0.181 a
1.524 ± 0.088 b
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Values are means ± SD. (n = 7). Different letters among nitrogen
regimes indicate significant difference (P ≤ 0.05)
Fig. 1.
[90]Fig. 1
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Electron micrograph of chloroplast with low(a), moderate(b) and high(c)
nitrogen level were taken at 5000, 5000 and 5000 times, respectively
Table 2.
Effects of N regimes on leaf photosynthesis in Panax notoginseng
Variables LN MN HN
g[s] (mol CO[2] m^− 2·s^− 1) 0.05 ± 0.02 a 0.03 ± 0.02 ab 0.03 ± 0.02
ab
g[m] (mol CO[2] m^− 2·s^− 1) 0.09 ± 0.01 c 0.26 ± 0.04 b 0.36 ± 0.02 a
R[d] (μmol CO[2] m^− 2·s^− 1) 1.0 ± 0.04 a 0.52 ± 0.02 b 0.57 ± 0.03 b
g[lip] (mol CO[2] m^− 2·s^− 1) 2.61 ± 0.08 b 5.52 ± 0.03 a 0.183 ± 0.09
c
C[c] (μmol CO[2] m^− 2·s^− 1) 199.53 ± 8.27 b 265.45 ± 7.31ab
291.58 ± 9.15 a
S (mol mol^− 1) 844.15 ± 7.56 b 1057.25 ± 5.41 a 860.16 ± 3.89 b
S*(mol mol^− 1) 739.39 ± 95.61b 983.75 ± 67.32 a 753.41 ± 90.34 b
Rubisco activity (nmol/min/g) 0.643 ± 0.24 c 40.51 ± 5.39 a
22.51 ± 4.89 b
Rubisco content (μg/g^−1) 6.931 ± 0.36 c 10.057 ± 0.67 b 70.494 ± 0.32
a
S[c](m^2 m^− 2) 8.42 ± 1.25 b 12.01 ± 1.65 a 13.15 ± 0.56 a
C[lip] (mol CO[2] m^− 2·s^− 1) 0.31 ± 0.06 ab 0.46 ± 0.02 a 0.02 ± 0.01
b
g[i] (mol CO[2] m^− 2·s^− 1) 0.13 ± 0.01 b 0.35 ± 0.04 a 0.12 ± 0.05 b
g[m]/Rubisco content 12.99 ± 1.23 b 25.81 ± 1.90 a 5.12 ± 0.78 c
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Values are means ± SD. (n = 7). Different letters among nitrogen
regimes indicate significant difference (P ≤ 0.05). g[s]: stomatal
conductance; g[m]: mesophyll conductance; R[d]: dark respiration rate;
g[lip]: liquid phase; C[c]: chloroplastic CO[2] concentration; S: the
specificity factor of Rubisco for O[2] and CO[2]; S*: apparent Rubisco
specificity; S[c]: chloroplast exposed to intercellular air space per
unit leaf area; C[lip]: conductance per unit of exposed chloroplast
surface area; g[i]: internal CO2 condutance
N-induced changes in photosynthetic capacity
The leaf exhibited a significant difference in a response of net
photosynthetic assimilation (A[net]) to incident photosynthetic photon
flux density (PPFD) and to internal leaf CO[2] concentrations (C[i])
within N regimes (Fig. [93]2). The maximum net photosynthetic
assimilation (A[max]), CO[2] response curves and carboxylation
efficiency (CE), maximum electron transfer rate (J[max])and maximum
carboxylation efficiency (V[cmax)] were highest in MN- grown plants;
however, these variables did not show apparent differences between LN
and HN individuals except for A[max] (Table [94]3). N allocation to the
photosynthetic system (N[photo]) is the sum of N allocation to the
carboxylation system (N[C]), the bioenergetics component (N[B]) and the
light-harvesting system (N[L]). N content per unit leaf area (SLN) was
increased significantly with the increase in N application (Table
[95]3). HN treatment caused a significant increase in N[L], whereas
there is a significant reduction in N[C] in HN-grown plants (Fig. [96]3
a). Most importantly, photosynthetic N use efficiency (PNUE) was
significantly decreased from 45.2 to 20.3% with an increase in N supply
(Fig. [97]3 b). These results support that high N[photo] did not
trigger an increase in PNUE.
Fig. 2.
[98]Fig. 2
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a Response of net photosynthetic rate (A[net]) to photosynthetic photon
flux density (PPFD) in Panax notoginseng grown under low nitrogen (LN),
moderate nitrogen (MN), high nitrogen (HN). b The change of net
photosynthetic assimilation (A[net]) with intercellular CO[2]
concentration (C[i]) in Panax notoginseng grown under different
nitrogen levels. Values for each point were means ± SD (n = 7).
Significant differences are indicated by asterisks (ANOVA; P values
≤0.05)
Table 3.
Steady-state photosynthetic-related traits in Panax notoginseng under
different levels of nitrogen
Variables Nitrogen level
LN MN HN
A[max] (μmol·m^− 2·s^− 1) 2.378 ± 0.261c 3.437 ± 0.241a 2.600 ± 0.165 b
CE (mol·mol^−1) 0.017 ± 0.002 ab 0.022 ± 0.003 a 0.018 ± 0.005 ab
Γ*(μmol·mol^− 1) 124.399 ± 8.014 a 99.259 ± 10.957 b 122.121 ± 21.084
ab
J[max] (μmol·mol^− 1) 66.558 ± 6.123 b 74.518 ± 15.599 a
63.334 ± 23.251b
V[cmax] (μmol·mol^− 1) 16.480 ± 1.821b 20.771 ± 2.939 a 16.830 ± 5.058
b
J[max]/V[cmax] 4.059 ± 0.127 ab 3.527 ± 0.337 b 4.329 ± 0.106 a
SLN (g m^− 2) 0.890 ± 0.130 c 1.245 ± 0.006 b 2.178 ± 0.348 a
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Values are means ± SD. (n = 7). Different letter among nitrogen
treatments represents a significant level (P ≤ 0.05). A[max]: maximum
photosynthetic assimilation at the saturating light; CE: carboxylation
efficiency; Γ*: carbon dioxide compensation point; J[max]: maximum
electron transfer rate; V[cmax]: maximum carboxylation efficiency; SLN:
nitrogen content per unit leaf area
Fig. 3.
[101]Fig. 3
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Effects of different nitrogen levels on nitrogen distribution (n = 7)
and photosynthetic nitrogen use efficiency (n = 7) in Panax notoginseng
leaves. N[photo]: Photosynthetic apparatus; N[C]: Carboxylation system;
N[B]: Bioenergetics; N[L]: Light harvesting system; PNUE:
Photosynthetic nitrogen use efficiency. Data are mean with bars
depicting standard deviation (± SD). Significant differences are
indicated by letters (ANOVA; P values ≤0.05)
SLN and Rubisco content were greater when plants were exposed to high N
as compared with ones to moderate and low N (Additional file [103]2:
Figure S2; Tables [104]2, [105]3). MN treatment exhibited 44.4–98.4%
more Rubisco activity than two other treatments (Table [106]2). There
were no significant differences in stomatal conductance (g[s]) within
treatments, but g[m] were higher in HN-grown individuals (Table
[107]2). HN-grown plants had a decreased ratio of g[m]/Rubisco content
and a lower Rubisco specific activity than two other treatments (Table
[108]2).
Photosynthetic electronic transport
The responses of photosynthetic electronic transport to continuous
steady-state light were markedly different among N regimes
(Additional file [109]3: Figure S3; Fig. [110]4). In the light response
curves, the minimum values of PSII maximum quantum
efficiency(F[v]`/F[m]`), PSII photochemical quantum yield (Φ[PSII]),
photochemical quenching(qP) as well as PSII total electron transport
rate (J[T]), rate of electron transport for oxidation reaction (J[O]),
carboxylation reaction (J[C]) and the maximum values of
non-photochemical quenching (NPQ) were generally recorded in the LN
individuals, the maximum value of F[v]`/F[m]`, Φ[PSII], qP as well as
J[T], J[O], J[C] were obtained in the MN ones (Additional file [111]3:
Figure S3; Fig. [112]4).
Fig. 4.
[113]Fig. 4
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Responses of PSII maximum quantum efficiency (F[v]`/F[m]`, a), PSII
photochemical quantum yield (Φ[PSII], b), photochemical quenching (qP,
c), non-photochemical quenching (NPQ, d) to photosynthetic photon flux
density (PPFD) in Panax notoginseng grown under different levels of
nitrogen. Values for each point were means ± SD (n = 7). Significant
differences are indicated by asterisks (ANOVA; P values ≤0.05)
Changes in photosynthetic-related pigments
The amounts of β-carotene (β- Cars) and the ratio of (V + A + Z)/Chl
were enhanced in LN individuals, whereas total Chl decreased
(Table [115]4). LN resulted in a decrease in neoxanthin (N) and lutein
(L), and an increase in violaxanthin(V), antheraxanthin (A), and
zeaxanthin(Z). Violaxanthin de-epoxide activity ((A + Z)/(V + A + Z))
was greatest in the LN ones (Table [116]4).
Table 4.
Photosynthetic-related pigment in a shade-tolerant plant Panax
notoginseng grown under different levels of nitrogen, means ± SD were
given (n = 7)
Variables Nitrogen Level
LN MN HN
N (μg·cm^−2) 0.362 ± 0.129 c 0.865 ± 0.265 b 1.643 ± 0.332 a
V (μg·cm^− 2) 0.913 ± 0.124 a 0.267 ± 0.195c 0.493 ± 0.458b
A (μg·cm^− 2) 0.213 ± 0.019 a 0.043 ± 0.072c 0.153 ± 0.079b
L (μg·cm^− 2) 1.284 ± 0.352 c 3.018 ± 0.970 b 5.852 ± 0.926a
Z (μg·cm^− 2) 0.194 ± 0.023 a 0.032 ± 0.048 c 0.073 ± 0.201b
Chl(g·cm^− 2) 12.270 ± 1.783 c 31.618 ± 2.356 b 60.101 ± 2.455 a
β-Car(g·cm^− 2) 4.08 ± 2.14 a 1.59 ± 0.69 c 2.95 ± 0.69 b
V + A + Z (g·cm^− 2) 1.314 ± 0.023 a 0.332 ± 0.035 c 0.712 ± 0.043 b
(A + Z)/(V + A + Z) 0.309 ± 0.015 ab 0.226 ± 0.017 b 0.317 ± 0.037 a
(V + A + Z)/Chl 0.107 ± 0.018 a 0.011 ± 0.027 b 0.012 ± 0.028 b
[117]Open in a new tab
Different letter among nitrogen treatments represents a significant
level (P ≤ 0.05). V violaxanthin; A antheraxanthin; Z Zeaxanthin; L
Lutein; N Neoxanthin; β-Car: β-Carotene
Gene expression identification
Compared to the MN individuals, 1391 and 895 genes were classified as
differentially expressed genes (DEGs) in the LN and HN groups. Whereas,
there were 428 DEGs in both LN- and HN- treatments (Fig. [118]5). In
the LN group, 467 DEGs were up-regulated, and 924 DEGs were
down-regulated. Two hundred ninety-four genes were up-regulated and 601
genes were suppressed in HN individuals (Additional file [119]4: Figure
S4). Moreover, 963 and 467 DEGs were typically detected in LN, HN
groups. Two DEG sets were subjected to 34 Gene ontology (GO) classes
(Fig. [120]6). Under the classification of molecular function,
“catalytic activity” were largely represented, followed by “binding”
(Fig. [121]6 a). The GO enrichment was further analyzed to identify
specific GO enrichment terms among DEG sets. Based on Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis,
the DEGs in LN ones were categorized into photosynthesis, carbon
fixation, N metabolic, plant hormone signal transduction, starch and
sucrose metabolism and galactose metabolism, the DEGs in HN ones were
significantly overrepresented in pathway of citrate cycle (TCA cycle),
alpha-Linolenic acid metabolism, carbon fixation in photosynthetic
organism, N metabolism and galactose metabolism (Fig. [122]6 b). In
addition, the first 13 pathways widely related to the mechanism about
photosynthesis and photo-protection were explored among KEGG enrich
analysis of all annotated unigenes (Additional file [123]5: Table S1).
Fig. 5.
Fig. 5
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Venn diagrams of differentially expressed genes (DEGs) in response to
varied nitrogen level
Fig. 6.
[125]Fig. 6
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Functional annotation and enrichment analysis of differentially
expressed genes (DEGs) responsive to low nitrogen and high nitrogen. a
Gene annotation of DEGs. b KEGG enrichment analysis for DEGs
Transcriptional changes
GO enrichment analysis was presented and elucidated in Fig. [127]7.
Enriched GO terms of further induced genes between two pairwise
comparisons (MN vs LN., MN vs HN.) embraced photosynthesis, pigment
metabolic process, carbohydrate catabolic process, thylakoid and so on.
Common DEGs with suppressed expression were significantly enriched in
cellular amino acid catabolic process, alpha-amino acid catabolic
process, proline metabolic process and glutamine family amino acid
metabolic (Fig. [128]7). KEGG pathway analysis was further certified
distinct functional enrichments in biological process among common DEGs
(Fig. [129]7 b, c), revealing that these induced expression of common
DEGs were richen in TCA cycle, photosynthesis, carbon fixation in
photosynthetic organism, glycolysis/gluconeogenesis and carbon
metabolism, and down-regulated DEGs were primarily related to N
metabolism, glutathione metabolism, biosynthesis of amino acids and
starch and sucrose metabolism. In addition, a large number of specific
DEGs involved in diverse biological processes were detected in the MN
vs. HN ones, and response patterns in the LN and HN level also
exhibited differences.
Fig. 7.
Fig. 7
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Functional annotation and enrichment analysis of common differentially
expressed genes (DEGs) between moderate- (MN) vs. low- (LN) nitrogen
and high-nitrogen (HN) vs. LN comparisons. a Heat clustering of common
DEGs based on the expression profiles. Blue indicates lower expression,
and red indicates higher expression. b KEGG pathway analysis of
up-regulated DEGs. (c) KEGG pathway analysis of down-regulated DEGs
Genes expression related to Calvin cycle and light reaction
Interestingly, the expressions of the majority of genes encoding
enzymes in Calvin cycle were down-regulated between LN and HN
individuals, furthermore, the transcript levels of a substantial number
of genes were reduced in LN ones (Additional file [131]6: Figure S5;
Fig. [132]8). Expression of unigenes involved in photosystems II (e.g.,
PsbA, PsbE, PsbF, and PsbH) and photosystems I (e.g., PsaN) were
down-regulated between LN and HN individuals (Additional file [133]7:
Figure S6a; Fig. [134]8), while the unigenes involved in PsbS and PetE
were up-regulated (Additional file [135]7: Figure S6b; Fig. [136]8).
Fig. 8.
[137]Fig. 8
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Expression profiles of differentially expressed genes (DEGs) that
regulate photosynthesis and photoprotection under different nitrogen
level
Photoprotection-related genes
Both zeaxanthin epoxidase (ZEP) and violaxanthinde-epoxidase (VDE)
genes are positively induced by LN level (Additional file [139]8:
Figure S7a). Glucose-6-phosphate dehydrogenase (G6PDH) and glutathione
S-transferase (GSTs) involved in glucose metabolism was up-regulated in
the LN individuals (Additional file [140]8: Figure S7b). Genes were
found to be enriched in Chl degradation, and genes encoding chlorophyll
b reductase (NYC) and red chlorophyll catabolite reductase (RCCR) were
up-regulated in the LN groups (Additional file [141]8: Figure S7c).
Under LN levels, transcript levels of genes involved in nitrate
reduction were up-regulated (Additional file [142]8: Figure S7d).
Expressions of genes encoding phytoene synthase (PSY), geranyl
pyrophosphate synthase (GGPS), 1-deoxy-D-xylulose-5-phosphate
reductoisomerase (DXR), isopentenyl pyrophosphate isomerase (IPI) and
1-deoxy-D-xylulose-5-phosphate synthase (DXS) were up-regulated in LN
groups (Additional file [143]8: Figure S7e). In addition, a substantial
number of pathway regulating photosynthesis and photoprotection were
activated by N-induction, including glutathione metabolism, N
metabolism, carotenoid biosynthesis, photosynthesis-antenna proteins
(Fig. [144]8).
Real-time quantitative PCR (RT-qPCR) of photosynthetic-related genes
In RT-qPCR analyses, melting curves of actin and 19
photosynthetic-related genes were clear and every curve all held a
single and sharp peak (Additional file [145]9: Figure S8), indicating
that the primer pairs could positively amplify specific products of 19
genes (Additional file [146]10: Table S2). The expressions of 19 genes
were approximately similar to the results from RNA-Seq data in leaves
(Additional file [147]11: Figure S9).
Discussion
N-driven changes in photosynthesis is in part explained by leaf anatomy and N
allocation
Photosynthetic capacity is at least in part determined by leaf anatomy
and chloroplast ultrastructure [[148]59] and A[net] is limited by the
rate of CO[2] diffusion from the atmosphere to the chloroplast
[[149]60]. C[lip] has a close relationship with mesophyll cell
thickness, and a thick tissue is always accompanied with a low C[lip]
[[150]30]. Nevertheless, C[lip] is positively related to the rate of
CO[2] diffusion from the atmosphere to the chloroplast [[151]61]. N
deficiency obviously reduce the size of chloroplasts and consequently
lead to a low chloroplast surface area exposed to S[c] [[152]16].
Correspondingly, a large chloroplast has been documented in HN-grown
rice, and the large chloroplasts would enhance chloroplastic CO[2]
concentration (C[c]) and g[m] [[153]62]. Thicker upper epidermis, lower
epidermis, spongy tissue and palisade tissue (Table [154]1) would
reduce liquid phase diffusion of CO[2] in mesophyll cells, evidencing
by lower C[lip] in the HN individuals (Table [155]2), and this might
partly explain the fact that a significant decrease in A[net] was
observed in the HN individuals. The increase in size of chloroplast and
in g[m] would contribute to the increase in C[c] (Fig. [156]1 c; Table
[157]2), as has also been observed in Tosens & Laanisto [[158]63]. C[c]
positively reinforce photosynthetic capacity [[159]64–[160]66].
Nevertheless, HN supply resulted in a decline in A[net] (Fig. [161]2),
it has been thought that the increase in g[m] is not enough to provide
sufficient CO[2] to activate the increased Rubisco, the imbalance
between g[m] and Rubisco content contributes to the reduction in A[net]
in the HN individuals (Table [162]2) as has also been observed in Yin &
Struik [[163]67]. LN-grown plants displayed small chloroplast and low
S[c] (Fig. [164]1; Table [165]2) and these characteristics suppress
g[i]and A[net], as has been proposed by Onoda et al [[166]68] .
30–40% of leaf N is allocated to photosynthetic carboxylation and
photosynthetic efficiency is determined by the proportion of N
allocated to N[C] [[167]69]. SLN is significantly increased, but
V[cmax], CE, Rubisco activity and N[C] is obviously decreased, and
consequently photosynthetic efficiency is suppressed in wheat and rice
grown in excessive N environment [[168]29, [169]30]. The LN plants
exhibited lower SLN, Chl and N[L] (Tables [170]3, [171]4; Fig. [172]3
a), this would only limit the synthesis of light-trapping chlorophyll
a/ b-protein complexes and effectively prevent absorption of excessive
light energy [[173]70]. Correspondingly, lower N[C] and activity of
Rubisco were observed in the HN individuals (Fig. [174]3 a), this would
suppress photosynthetic carboxylation and reduce photosynthetic
efficiency.
The dark and light reaction of photosynthesis under non-optimal N regimes
Rubisco is the key CO[2]-fixing enzyme in Calvin cycle. The depressed
photosynthesis in the LN plants might be the consequence of low Rubisco
catalytic capacity and CE [[175]30, [176]71]. Rubisco activity and CE
were decreased in the LN individuals with down-regulated expression of
genes encoding Rubisco (Tables [177]2, [178]3; Additional file [179]6:
Figure S5). N deficiency in maize led to the decline in photosynthetic
capacity and Rubisco catalytic capacity [[180]22]. According to classic
theories [[181]72], V[cmax] positively reflects potential carboxylation
capacity of Rubisco and regeneration rate of ribulose-bisphosphate
(RuBP). V[cmax] and Rubisco activity were considerably reduced in the
HN individuals, whereas Rubisco content were increased (Tables [182]2,
[183]3). On the other hand, the expressions of Rubisco-catalyzing genes
(CPN60A1, Os02g079470025 and RAF2) were down-regulated in the HN
individuals (Tables [184]2, [185]3; Additional file [186]6: Figure S5).
Overall, these results support the view that under HN supply, the
majority of Rubisco function as N storage rather than as catalyzing
enzyme [[187]30], and the proportion of inactive Rubisco is greater in
the HN leaves. However, high Rubisco activity is recorded in the
HN-grown maize with high A[max] [[188]73]. The difference might be
explained by the fact that P. notoginseng is a shade-tolerant C3
species and highly sensitive to excess N [[189]54, [190]55], and maize
is a high N- and sun-demanding C4 plant [[191]74, [192]75]. In the
atmospheric environment, Rubisco has a low affinity with CO[2],
resulting in inferior catalytic capacity [[193]76]. The potential
catalytic capacity of Rubisco in C3 plants is much lower than in C4
plants [[194]77], suggesting that relatively less Rubisco operate on
photosynthesis, and that a high proportion of Rubisco serves as a
storage of N in HN-grown C3 plants.
The expression of genes encoding enzymes involved in regeneration of
RUBP was reduced in the LN individuals, including phosphor ribulokinase
(PRK), phosphor glycerate kinase (PGK), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), triosephosphate isomerase (TPI),
fructose-bisphosphate aldolase (FBA), transketolase (TKT) and
sedoheptulose 1,7-bisphosphatase (SBP, Additional file [195]6: Figure
S5), and correspondingly A[net], Φ[PSII], F[v]`/F[m]` and J[T] were
reduced. Likewise, N deficiency suppress photosynthetic capacity and
inhibit the expression of genes involved in dark reaction of
photosynthesis in barley (Hordeum vulgare) [[196]78] and rice
[[197]79]. Our findings are in general agreement with several previous
researches that down-regulated expressions of genes encoding enzymes
involved in the regeneration of Rubisco is associated with abiotic
stress [[198]80, [199]81].
Photosynthetic assimilation and photosynthetic electronic transport was
considerably decreased in the LN and HN individuals (Figs. [200]2,
[201]4; Additional file [202]3: Figure S3), and it is believed to
derive from an interruptive synthesis of photosynthetic proteins
[[203]82]. The expression of genes encoding structural proteins of
photosystems, including PsbA, PsbE, PsbF, PsbH and PsaN, were decreased
in the LN- and HN-grown individuals, while the expression of genes
encoding subunits of the PsbS and PetE was increased as compared with
the MN individuals (Additional file [204]7: Figure S6a). The expression
of genes encoding structural proteins of photosystems are positively
correlated with photosynthetic capacity [[205]83, [206]84]. In
addition, the down-regulation of genes involved in porphyrin and Chl
metabolism and photosynthesis-antenna proteins might also in part
explain the depressed photosynthetic capacity in the LN individuals
(Fig. [207]8).
Photoprotection in N deprivation
Lower A[max], V[cmax] and J[max] were recorded in the LN and HN
individuals when compared with the MN individuals (Table [208]3).
No-optimal N application induces suppressions of photosynthesis as has
been recorded in the sun-demanding species Viciafaba [[209]85], Lemna
minor [[210]86], Z. mays [[211]87] and C. sativus [[212]48], and in the
shade-tolerant species Abies fabri [[213]88], Brassica juncea [[214]89]
and polypodiopsida [[215]90]. On the other hand, Φ[PSII] was lower in
the LN individuals than in the HN individuals (Fig. [216]4 b),
indicating that a greater proportion of light energy absorbed by PSII
would have to be expended by non-photochemical process in the LN
individuals. The consumption of electrons by non-photochemical process
would facilitate the formation of trans-thylakoid ΔpH [[217]86]. ΔpH is
also a precondition for the activation of VAZ cycle and NPQ [[218]91].
This indicates that NPQ and V cycle pool might be reinforced in the LN
individuals as confirmed by the present study (Fig. [219]4 d; Table
[220]4).
The degradation of Chl has been believed to be a photoprotective
mechanism for plants to cope with stress and to prevent photodamage
[[221]92, [222]93]. Chl was considerably reduced in LN-grown plants
(Table [223]4). Genes involved in Chl degradation were found to be
enriched in LN-grown plants, and genes encoding NYC and RCCR were
up-regulated (Additional file [224]8: Figure S7c). The results obtained
in our study indicate that low Chl in LN-grown plants might be caused
by up-regulation of genes involved in Chl degradation. Similarly, the
expression of genes involved in Chl degradation are also elevated in a
shade-tolerant plant Neoregelia cruenta when grown under N-limited
condition, together with the obvious decrease in leaf Chl [[225]40].
LN-grown Spinach showed low Chl content and the corresponding elevation
in expression of genes involved Chl degradation [[226]94, [227]95].
Car is not comprised of N atoms and their accumulation is beneficial
for protecting photosystem from photodamage [[228]42, [229]96]. The
genes involved in Car biosynthesis (GGPS, DXR, PSY, IPI and DXS) were
down-regulated in the HN individuals (Additional file [230]8: Figure
S7e), as has also been observed by Vidhyavathi et al [[231]97].
Correspondingly, the expression of genes involved in Car biosynthesis
were up-regulated in Haematococcus pluvialis by a combination of light
and N-deprivation [[232]98]. Further, a greater xanthophyll pool size
(V + A + Z) and higher de-epoxidation state ((A + Z)/(V + A + Z)) were
observed in the LN individuals (Table [233]4), this suggests that LN
cloud effectively improve the capacity of heat dissipation. The PsbS
protein and violaxanthin de-epoxidase is believed to involve in
regulating energy dissipation [[234]93]. The latter catalyses the
de-epoxidation of V to Z [[235]99]. ΔpH-dependent quenching (qE) is
activated by PsbS and the xanthophyll cycle [[236]100]. The expression
of genes encoding ZEP and VDE (key enzymes in xanthophyll cycle) were
up-regulated in the LN individuals with the enhanced expression of PsbS
genes (Additional file [237]8: Figure S7a), as has been observed in
LN-induced maize where genes involved de-epoxidation state were
up-regulated [[238]34].
Nitrate assimilation is a process highly sensitive to N stresses
[[239]101]. Nitrite assimilation consumes six electrons from reduced
ferredoxin [[240]102, [241]103]. This reaction is a strong alternative
sink for photosynthetic electron transport chain. The expressions of
genes encoding nitrite reductase (NIR) and nitrate reductase (NIA) were
increased in the LN-grown individuals (Additional file [242]8: Figure
S7d). It has been reported that maize [[243]74], A. thaliana [[244]104]
and apple [[245]105] show high activity of NIR and NIA in leaves in
presence of suboptimal N application. The previous investigations and
the present study both strongly evidence that LN-induced enhancement in
nitrate assimilation might mitigate the accumulation of excess energy.
Conclusion
The non-optimal N supply significantly suppresses photosynthetic
capacity in a typically shade-tolerant and N-sensitive species such as
P. notoginseng. Thick leaf limits liquid phase diffusion of CO[2] in
mesophyll cells and accordingly reduces internal conductance. Moreover,
large chloroplast with low N[c] results in an imbalance between the
increases in gm and in Rubisco content, consequently causing the
decreased A[net] in the HN individuals. In addition, the expression of
genes involved in Calvin cycle, Chl biosynthesis and antenna proteins
are obviously repressed in the LN individuals; correspondingly, the
expression of genes (e.g. RAF2, CAB and PetE) involved in Calvin cycle
and light reaction is also inhibited, however, photosynthetic capacity
might be primarily inhibited by the inactivated Rubisco in the HN
individuals. Overall, our results indicate that photosynthetic
performance and photosynthesis-related genes expression is coordinated
in a shade-tolerant and N-sensitive plant grown along an N gradient.
Methods
Plant materials and growth conditions
Experimental plots were conducted at the teaching and experimental farm
of Yunnan Agricultural University in Kunming, Yunnan, China. P.
notoginseng is a perennial herb; farmers have cultivated this medicinal
crop for more than 400 years. One-year-old P. notoginseng seedlings
were collected from the Wenshan Miao Xiang P. notoginseng Industrial
Co., Ltd., China (Longitude 104°32 ‘, latitude 23°53 ‘). 1-year-old
healthy rootstalks of P. notoginseng were selected in our experiments
and transplanted to a white plastic pot (30 cm in diameter and 40 cm in
depth) on January 2017, and 3 individuals per pot, 120 pots per
treatment were arranged.
The soil had the following chemical characteristics: organic mater
0.573%, total N 0.201%, pH (H[2]O) 6.42, total phosphorus (P)
0.727 g/kg, ammonium N 39.93 mg/kg, available potassium (K) 0.019 mg/g,
available P 4.88 mg/kg, soil water regime 12%. Pots were placed in
environmentally controlled growth permeable black plastic net with
growth irradiance of 10% full sunlight. Three N-fertilizer levels were
applied in our experiments: (1) LN without N addition, (2) MN with
225 kg·ha^− 1 N addition in four applications, (3) HN with
450 kg·ha^− 1 N addition in four applications. N was supplied on April
22, June 22, July 22, August 22, 2017 respectively, along with 225
P[2]O[5]kg· ha^− 1 (superphosphate) and 450 kg·ha^− 1 K[2]O (potassium
sulphate) in four applications.
P. notoginseng grown for 8 months were used to determine plant
mortality, leaf morphology and photosynthetic performance, and to
collect leaves for comparative transcritome, chlorophyll and elemental
N analyses. Five biological replicates were quickly frozen in liquid N
and stored at − 80 °C for RNA extraction.
Leaf anatomy and chloroplast ultrastructure
The juvenile leaves achieved for morphological and anatomical traits
were used after 8 months of the N regimes. Leaf anatomical properties
were performed in the method of paraffin section, and then the leaves
were dehydrated in an alcohol series. Leaf tissues were embedded in
paraffin (Thermo Scientific Histostar™) and transversely sectioned at
10 mm thickness by means of microtome (Microm HM325, Walldorf,
Germany). Finally, sections were stained with hematoxylin observed
under a bright field Microscope (Zeiss Axio Cam HRC, Oberkochen,
Germany).
A small piece of 1 ~ 2 mm^2 was cut between the middle leaf vein and
leaf edge and fixed with 2.5% glutaraldehyde and 1% osmic acid.
According to the conventional series of ethanol dehydration, epoxy
resin embedding, ultra-thin slicer sectioning, sectioning was stained
with uranyl acetate and then stained with lead citrate, the chloroplast
ultrastructure was observed under JEM100CX-II transmission electron
microscope. For the estimation of Sc, 700 nm-thick sections were used
by the method of Hanba et al [[246]59].
Steady-state gas exchange rate
Steady-state gas exchange measurements were carried out using the
photosynthesis system (Li-6400-40, Li-Cor, USA) with the 2 cm^2
fluorescence leaf chamber. The leaf temperature and CO[2] in the
chamber were maintained at 25 °C and 400 μmol mol^− 1 during
measurements, respectively. Subsequently, Photosynthetic light response
curves and photosynthetic CO[2] response curves were performed. Based
on photosynthetic light response curves, full induction was complete,
an automatic program of light response curves was run to measure the
change in gas exchange rate with a set of PPFD. The level of PPFD was
listed in the following order: 800, 500, 400, 300, 200, 100, 80, 60,
40, 20 and 0 μmol m^− 2 s^− 1, each light intensity stabilized for
5 min. The relationship between A[net] and PPFD was fitted,
A[net] = A[max] - A[max]C[0]e^-αPPFD/ Amax, where A[max] is the maximum
net photosynthetic assimilation under saturating light, α is the
apparent quantum efficiency (AQY), where AQY was estimated by the slope
of the linear region of the light response curve. C[0] is the index to
measure the net photosynthetic rate approaching 0 in low light.
According to the parameters in the formula, dark respiration rate
(R[d]) = A[max]- A[max]C[0].
A[net] and C[i] were evaluated at a range of reference CO[2]
concentrations (400, 300, 200, 150, 100, 50, 400, 600, 800, 1000 and
1200 1500 μmol mol^− 1). CO[2] response curves and CE were achieved by
fitting the data to a nonrectangular hyperbola and the slope the linear
region of the CO[2] response curve, respectively. V[cmax] and J[max]
was gained according to the idea offered by Buckley and Diazespejo
[[247]106], this calibration requires measurements under low O[2].
Chlorophyll fluorescence of PSII
At predawn, minimum and maximum Chl fluorescence yield (F[O] and F[m])
was measured in the fully dark-adapted leaves. Minimum, maximum and
steady-state fluorescence intensity (F[O]`, F[V]`, F[m]` and F[s]) were
made in the process of light response curves. F[v]`/F[m]` was estimated
as (F[m]` – F[O]`)/ F[m]`; Φ[PSII] as (F[m]`– F[s])/F[m]`;
J[T] = PPFD×Φ[PSII] × α[leaf] × β, commercial fluorometers usually
provide an estimate of PSII total electron transport rate (J[T]) by
assuming that 400–700 nm (PAR) leaf absorptance (α[leaf]) equals 0.84
[[248]107] and that absorbed photons (β) are equally distributed
between the two photosystems (β = 0.5) [[249]108]. This approximation
is reasonable for comparison of J[T] between optically similar samples
such as leaves of cultivars of a single plant species [[250]109].
Moreover, there was a curvilinear relationship between α[leaf] and
chlorophyll content, whereas the curvature was extremely low when the
chlorophyll content was > 0.4 mmol m^− 2 [[251]30, [252]110]. According
to Evans and Poorter [[253]110], the calculation of α[leaf]
demonstrated that α[leaf] (0.84, 0.85, and 0.85, in leaves with low,
moderate, and high N content, respectively) was similar to the value of
0.84 [[254]111–[255]113]. Therefore, in this study, α[leaf] also
assumed to be 0.84, and β was assumed to be 0.5 [[256]108, [257]114].
NPQ as (F[m] – F[m]`)/F[m]`, and qP as (F[m]` – F[s])/(F[m]` – F[0]`).
Jc and Jo was calculated according to the method of Valentini et al
[[258]115], J[O] = 2/3 × (J[T] − 4 × (A[net] + R[d])),
J[C] = 1/3 × (J[T] + 8 × (A[net] + R[d])). According to the methods of
Manter and Kerrigan [[259]116], g[m] and C[lip] were calculated as
g[m] = A[net]/{C[i]-Γ* × [J[T] + 8(A[net] + R[d])]/[J[T]-4(A[net] + R[d
])]}, C[lip] = Γ* × [J[T] + 8(A[net] + R[d])]/[J[T]-4(A[net] + R[d])],
where Γ* is the CO[2] compensation point. C[c] was calculated as
C[c] = C[i] × S*/S. The initial slope of the regression of Jc/Jo to
C[i]/O was used to S*(Additional file [260]12: Figure S10), O[2]
concentration (210 mmol CO[2] mol^− 1). S was calculated as follow:
S=O/2Γ*. g[lip] can be showed that g[lip] = C[lip] × S[c]. g[i] was
calculated by g[i] = A[max]/(C[i]-C[c]) .
Calculation of N allocation in photosynthetic components
Leaf N was determined with Kjeldahl. SLN was calculated.
Photosynthetic-related pigments were determined by the method of Xu et
al. [[261]91] and Thayer & Björkman [[262]117]. N[C], N[B] and N[L]
were determined according to the method of Niinemets et al [[263]118].
N[photo] is the sum of N[C], N[B] and N[L]. PNUE is the ratio of leaf N
used for C fixation per unit leaf area. The formula is as follows:
[MATH: NC=Vcmax/6.25×Vcr×SLN×SLN :MATH]
1
[MATH: NB=Jmax/8.06×Jmc×SLN×
SLN :MATH]
2
[MATH: NL=Cc/CB×SLN×SLN :MATH]
3
[MATH: Nphoto=<
mi mathvariant="normal">NC+NB+NL
:MATH]
4
[MATH:
PNUE=AmPNUE
=Amax/SLNax/SLN :MATH]
5
V[cr] is the Rubisco specific activity with a value of 20.8 μmol
CO[2]·g^− 1 Rubisco·s^− 1. J[mc] is the maximum electron transfer rate
per unit cytochrome f (Cyt f) with a value of 155.6 μmol
electrons·μmol-1 Cyt f·s^− 1. Cc is the leaf chlorophyll content
(mmol·m-^2), C[B] is the combined light system I (PSI), photosystem II
(the chlorophyll in PSII) and PSII light-harvesting pigment complex
(LHCII) with a value of 2.15 mmol·g^− 1 N.
Leaf Rubisco content and activity
The Rubisco content was determined according to Makino et al
[[264]119]. Briefly, newly expanded leaves were stored at − 80 °C.
0.5 g frozen leaves were ground in a solution containing 50 mM Tris-HCl
(pH =8.0), 5 mM β-mercaptoethanol, and 12.5% glycerol (v/v), and then
centrifuged at 1500 g for 15 min at 4 °C. The supernatants were mixed
with a solution containing 2% (w/v) SDS, 4% (v/v) β-mercaptoethanol and
10% (v/v) glycerol, boiled in a water bath for 5 min before SDS-PAGE
using a 4% (w/v) stacking gel, and a 12.5% (w/v) separating gel. After
electrophoresis, the gels were stained with 0.25% Commassie Blue for
12 h, and destained. Gel slices containing the large subunits and small
subunits of Rubisco were transferred to a 10 mL cuvette containing 2 ml
of formamide and incubated at 50 °C in a water bath for 6 h. The
absorbance of the wash solution was measured at 595 nm. Protein
concentrations were determined using bovine serum albumin as a
standard. Bovine serum albumin (BSA) was measured at 595 nm as standard
protein.
Rubisco activity was measured according to Parry et al [[265]120] with
minor modifications. The extraction solution contained: 50 mM Tris-HCl
(pH = 7.5), 10 mM β-mercaptoethanol, 12.5% (v/v) glycerol, 1 mM
EDTA-Na[2], 10 mM MgCl[2] and 1% (m/v) PVP-40. Extracts were clarified
by centrifugation (8000 g at 4 °C for 10 min) and the supernatant was
immediately assayed for Rubisco activity.
RNA extraction and library construction, sequencing
RNA samples were extracted using RNA pre-pure Plant Kit (Tiangen,
Beijing, China). After total RNA was extracted, mRNA was enriched by
Oligo (dT) bads, and then the enriched mRNA was fragmented into short
fragments using fragmentation buffer and reverse transcripted into cDNA
with random primers. Second-strand cDNA were synthesized by DNA
polymerase I, RNase H, dNTP and buffer. The cDNA fragments were
purified with QiaQuick PCR extraction kit, end repaired, poly(A) added,
and ligated to Illumina sequencing adapters. The ligation products were
selected by agarose gel electrophoresis, PCR amplified, and sequenced
using Illumina HiSeqTM 4000 by Gene Denovo Biotechnology Co.
(Guangzhou, China).
Raw reads filtering and de novo assembly
Low quality reads containing adapters, more than 10% of unknown
nucleotides (N), were eliminated. Transcriptome de novo assembly was
carried out with short reads assembling program-Trinity. The redundancy
was eliminated by the TGICL software and further assembled into a set
of non-redundant unigenes.
105G sequencing data were obtained and de novo assembled into 93,162
unigenes (Additional file [266]13: Table S3) with an average length of
790 bp (Additional file [267]14: Table S4). Collectively, 41,569
(44.62%) unigenes were functionally annotated in accordance with their
parallels with known genes/proteins in the databases. The particular
statistics of the functional annotation are emerged as in
Additional file [268]15: Figure S11. After eliminating adaptors,
unknown nucleotides and low quality reads, the data generated
43,588,606, 46,978,940, 43,177,242 paired-end 125-bP reads in the LN,
MN and HN treatments, respectively, coinciding with approximately 6.48
Gb data (Additional file [269]16: Table S5). Q20 percentages exceeded
98%, uncalled base (“N”) percentage was equal to 0% per sample
(Additional file [270]16: Table S5). The GC contents were almost
identical for all 15 leaves tissues, ranging from 43.08 to 44.20%
(Additional file [271]16: Table S5). In general, between 83.23 and
84.79% of clean reads could be mapped on full gene set
(Additional file [272]17: Table S6). A Pearson’s correlation analysis
revealed high correlations between biological replicates (R^2 = 0.8671
to 0.9769, Additional file [273]18: Figure S12).
Basic annotation of unigenes
To annotate the unigenes, we used BLASTx program
([274]http://www.ncbi.nlm.nih.gov/BLAST/) with an E-value threshold of
1e^− 5 to NCBI non-redundant protein (Nr) database
([275]http://www.ncbi.nlm.nih.gov), the Swiss-Prot protein database
([276]http://www.expasy.ch/sprot), the Kyoto Encyclopedia of Genes and
Genomes (KEGG) database ([277]http://www.genome.jp/kegg), and the
COG/KOG database ([278]http://www.ncbi.nlm.nih.gov/COG). Protein
functional annotations are obtained according to the best alignment
results.
Analysis of DEGs
To identify DEGs within N regimes, the normalized read counts from five
replicates of each sample were analyzed and the edge R package
([279]http://www.r-project.org) was used. We identified genes with a
fold change ≥2 and a false discovery rate (FDR) < 0.05 in a comparison
as significant DEGs. DEGs were then subjected to enrichment analysis of
GO functions and KEGG pathways.
GO enrichment analysis and pathway enrichment analysis
All DEGs were mapped to GO terms in the Gene Ontology database
([280]http://www.geneontology.org), gene numbers were calculated for
each term, significantly enriched GO terms in DEGs comparing to genome
background were defined by hyper geometric test. KEGG enrichment
analysis was carried out through Genomes database (g"
[281]http://www.genome.jp/kegg). P-value of GO terms and KEGG pathway
was gone through FDR Correction, taking FDR ≤ 0.05 as a threshold.
RT-qPCR assay
To validate the expression of 19 significant DEGs observed in RNA-Seq
data, reaction was carried out using EvaGreen 2X qPCR MasterMix Kit
(abm, Vancouver, Canada) in a Quanstudio™ 5 Real-Time PCR Intruments
(Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized
using the RevertAid™First strand cDNA Synthesis Kit (TransGen Biotech,
Beijing, China). DEGs primers were designed using the Primer-Blast (/"
[282]https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized
commercially (Shuoqing, Kunming, China). Actin were selected as
reference genes [[283]121]. The primers used in qRT-PCR analyses are
listed in Table [284]S1. Amplification reaction mixtures were made of
10 μL of Eva Green 2X qPCR Master Mix, 0.5 μL of each forward and
reverse primer (10 mM), and 1 μL of cDNA template, and ddH[2]O was
added to a final volume of 20 μL. The amplification cycling program was
as follows: enzyme activation was operated at 95 °C for 10 mins,
moreover, 40 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for
30 s. The results were analyzed using the software accompanying the
Quanstudio™ 5 Real-Time PCR instruments. The relative expression values
were obtained by using the 2^-ΔΔCt method [[285]122].
Statistical analyses
Statistical analyses were performed with SPSS software package
(Chicago, IL, USA) and SigmaPlot 10.0, where the data were tested to
confirm their normality and the variables were present as the mean ± SD
(n = 5–7). We obtained 7 repetiotions that studied physiological
parameter for N- cultivated plants, and we generally obtained 5
repetions for bioinformatic analyse. Differences were considered
significant when P < 0.05 according to the ANOVA F-test. The Ct values
derived from qPCR were normalized and the relative fold changes in
transcripts were calculated using the relative expression software
tool, REST.
Supplementary information
[286]12870_2020_2434_MOESM1_ESM.pdf^ (206.9KB, pdf)
Additional file 1: Figure S1. Leaf phenotypic traits (a) and plant
mortality (b) of Panax. notoginseng under nitrogen regimes.
[287]12870_2020_2434_MOESM2_ESM.pdf^ (254.9KB, pdf)
Additional file 2: Figure S2. Detection of Rubisco large and small
subunits in the leaves of Panax notoginseng.
[288]12870_2020_2434_MOESM3_ESM.pdf^ (314.6KB, pdf)
Additional file 3: Figure S3. Responses of PSII total electron
transport rate (J[T], a), rate of electron transport for oxidation
reaction (J[O], b) and carboxylation reaction (J[C], c) to
photosynthetic photon flux density (PPFD) in Panax notoginseng grown
under different levels of nitrogen. Values for each point were means ±
SD (n = 7). Significant differences are indicated by asterisks (ANOVA;
P values ≤0.05).
[289]12870_2020_2434_MOESM4_ESM.pdf^ (217.1KB, pdf)
Additional file 4: Figure S4. Common differentially expressed genes
(DEGs) and their expression profile between moderate- (MN) vs. low-
(LN) nitrogen and MN vs. high-nitrogen (HN). Red number indicates
up-relation, green number indicates down-relation.
[290]12870_2020_2434_MOESM5_ESM.pdf^ (186.5KB, pdf)
Additional file 5: Table S1. KEGG enrichment analysis of the first 13
pathways related to the protective mechanism.
[291]12870_2020_2434_MOESM6_ESM.pdf^ (245.8KB, pdf)
Additional file 6: Figure S5. Calvin cycle pathways of Panax
notoginseng and hierarchical cluster analysis of genes that were
differentially expressed under different nitrogen level. Red indicates
that the gene has a high expression in the nitrogen level; green
indicates that the gene has a lower expression in the nitrogen level.
[292]12870_2020_2434_MOESM7_ESM.pdf^ (364KB, pdf)
Additional file 7: Figure S6. Differentially expressed genes (DEGs)
participating in light reaction under varied nitrogen level. (a) MN vs
LN and MN vs HN differential gene of photosynthesis pathway for samples
of control group, the red box labeled for raising genes, green box
labeled as the blue box labeled as there are raised and lowered genes
at the same time, the box numbers for the number of the enzyme,
suggests that the corresponding gene is associated with the enzyme, and
the whole passage is there are many different forms through complex
biochemical reactions, an enzyme that differences in genes associated
with this pathway are marked by different color box. (b) The expression
pattern of DEGs involved in photosynthesis pathway. Red indicates that
the gene has a high expression in the nitrogen level; green indicates
that the gene has a lower expression in the nitrogen level.
[293]12870_2020_2434_MOESM8_ESM.pdf^ (461.5KB, pdf)
Additional file 8: Figure S7. The pathway and genes encoding for the
photoprotection. In heat map, firebrick indicates that the gene has a
high expression in the nitrogen level; navy indicates that the gene has
a lower expression in the nitrogen level. (a) The expression pattern of
DEGs involved in Lutein cycle. (b) The expression pattern of DEGs
involved in Antioxidant pathway. (c) The expression pattern of DEGs
involved in Chlorophyll degradation pathway. (d) The expression pattern
of DEGs involved in nitrate assimilation. (e) The expression pattern of
DEGs involved in Carotenoid metabolism.
[294]12870_2020_2434_MOESM9_ESM.pdf^ (673.6KB, pdf)
Additional file 9: Figure S8. Melt curve of 19 differentially expressed
genes (DEGs) and house-keeping gene (Actin).
[295]12870_2020_2434_MOESM10_ESM.pdf^ (360.7KB, pdf)
Additional file 10: Table S2. Primers for the RT-qPCR assays of the
twenty RNA-Seq libraries used in this study.
[296]12870_2020_2434_MOESM11_ESM.pdf^ (475.2KB, pdf)
Additional file 11: Figure S9. Quantitative real-time PCR validation of
19 differentially expressed genes (DEGs) (n = 5). Data are mean with
bars depicting standard deviation (± SD). Significant differences are
indicated by letters (ANOVA; P values ≤0.05). The columns represent
relative expression obtained by RT-qPCR.
[297]12870_2020_2434_MOESM12_ESM.pdf^ (239.1KB, pdf)
Additional file 12: Figure S10. The curvilinear relationships between
J[c]/J[o] and C[i]/O. Every data point represents the mean value of
five individual replicates, and small error bars indicate the standard
deviation. Initial slopes of (a), (b), and (c) represent S* of Panax
notoginseng grown at low, moderate, and high N concentration,
respectively.
[298]12870_2020_2434_MOESM13_ESM.xlsx^ (27.6MB, xlsx)
Additional file 13: Table S3. De novo assembled genes of P. notoginseng
grown under nitrogen regimes.
[299]12870_2020_2434_MOESM14_ESM.xlsx^ (3.4MB, xlsx)
Additional file 14: Table S4. Assembly quality statics of P.
notoginseng.
[300]12870_2020_2434_MOESM15_ESM.pdf^ (230.7KB, pdf)
Additional file 15: Figure S11. Statistics of the annotation of
unigenes in public databases.
[301]12870_2020_2434_MOESM16_ESM.pdf^ (199.9KB, pdf)
Additional file 16: Table S5. Summary of sequencing data quality for P.
notoginseng.
[302]12870_2020_2434_MOESM17_ESM.pdf^ (190.9KB, pdf)
Additional file 17: Table S6. Summary ofmapping rate and statistics of
expression genes based on the RNA-Seq data.
[303]12870_2020_2434_MOESM18_ESM.pdf^ (112.4KB, pdf)
Additional file 18: Figure S12. Pearson’s correlation analysis of the
RNA-Seq data.
Acknowledgements