Early transcriptomic adaptation to Na2CO3 stress altered the expression of a quarter of
the total maize (Zea mays) genes and exhibited common and distinctive profiles with
NaCl and high pH stresses
Li-Min Zhang1,2+, Xiang-Guo Liu1+, Xin-Ning Qu2, Yin Yu2, Si-Ping Han1, Yao Dou2,
Yao-Yao Xu2, Hai-Chun Jing3*, Dong-Yun Hao1,2*
1
Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences,
Changchun 130124, China;
2
Key Laboratory for Molecular Enzymology and Engineering of the Ministry of
Education, Jilin University, Changchun 130012, China;
3
The Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of
Sciences, Beijing 100093, China
Running title: Maize root early transcriptomic adaptation to Na2CO3 stress
+
These authors contributed to the work equally.
*Corresponding authors: [email protected]; [email protected]
1
Abstract
Sodium carbonate (Na2CO3) presents a huge challenge to plants by the combined
damaging effects of Na+, high pH and CO32-. Little is known about the cellular
responses to Na2CO3 stress. In this study, the transcriptome of maize (Zea mays L. cv.
B73) roots exposed to Na2CO3 stress for five hours were compared with those of
NaCl and NaOH stresses. The expression of 8319 genes, representing over a quarter
of the total number of genes in the maize genome, was altered by Na2CO3 stress, and
the genes down-regulated (5232) out-numbered those up-regulated (3087). The effects
of Na2CO3 differed from those of NaCl and NaOH primarily by down-regulating
different categories of genes. Pathways commonly altered by Na2CO3, NaCl and
NaOH were enriched in phenylpropanoid biosynthesis, oxidation of unsaturated fatty
acid, ATP-binding cassette (ABC) transporters as well as metabolism of secondary
metabolites. Genes for brassinosteriod biosynthesis were specifically up-regulated by
Na2CO3, whiles genes in ascorbate and aldarate metabolism, protein processing in
endoplasmic reticulum and by N-glycosylation, fatty acid biosynthesis and circadian
rhythm were down-regulated. This work provided a first holistic picture of the early
transcriptomic adaptation to Na2CO3 stress and highlighted potential molecular
pathways to manipulate for the improvement of tolerance in maize.
Key words: maize (Zea mays) roots; saline and alkaline stress; Na2CO3;
transcriptomic adaptation; RNA-Seq
2
Introduction
Soil salinization and alkalinisation is a major abiotic stress to agriculture production
worldwide. In China, there are in total about 3.5×107hm2 saline soil spreading across
Northern China. A large proportion of saline soil is the inland sodic alkaline soil, a
soil with a high pH (>9.0) due to the presence of excessive sodium carbonate
(Na2CO3). An estimated area of 3.73×106hm2 is located at the Song-Nen Plain in
Northeast China, which is one of the top three saline-alkali fields in the world and the
area is still expanding at a rate of 1.4% annually. Different from NaCl stress, Na2CO3
stress is the combination of Na+, high pH and CO32- stress. Intensive effort has been
devoted to dissect the adaptive responses of plants to NaCl stress and three major
mechanisms have been identified: maintaining ion and osmotic homeostasis,
detoxification and growth inhibition (Zhu, 2002). Evidence is also provided to show
that high soil pH (>9.0) could directly affect nutrient uptake, organic acid balance,
and ion homeostasis, especially the pH stability at cellular and whole plant levels
(Chen et al., 2009; Miller et al., 2010).
Na2CO3 stress, as a combination of excessive Na+, high pH and CO32-, imposes much
severe damage to plants (Sheng et al., 1999; Shi and Sheng, 2005; Shi and Wang,
2005). A couple of studies have been carried in the halophytic plant Puccinellia
tenuiflora and shown that energy consumption for CO2 assimilation suppressed by
Na2CO3 stress led to reactive oxygen species (ROS) imbalance, which would cause
oxidative damage to enzymes and thus the photosynthetic apparatus (Badger and
Price, 1994; Parida and Das, 2005). The activities of key enzymes in Calvin cycle
were reduced by Na2CO3 stress, which implied that the decrease in photosynthesis
was due to the less efficient carbon fixation under Na2CO3 stress (Badger and Price,
1994). Lipid peroxidation was readily observed under Na2CO3 stress, as envisaged by
the significantly increased electrolyte leakage and malondialdehyde (MDA) contents,
indicating that plasma membrane was an initial site of damage (Miller et al., 2010).
Other ions including Ca2+, Mg2+, and Si, have also been found to increase in
3
concentrations on the P. tenuiflora leaf surface with the increasing in Na2CO3
concentrations (Sun et al., 2005). The increased contents of K+ and Na+ on the leaf
surface associated with the increase of external Na2CO3 concentrations support the
hypothesis that P. tenuiflora leaves could exude salts through stomata or together with
wax secretion (Sun et al., 2005; Yu et al., 2013). Previous studies have revealed that P.
tenuiflora can remarkably accumulate citric acid in leaves and roots when exposed to
alkaline stress (Wu et al., 2007; Qing et al., 2009). The vacuolar Na+/H+ antiporters
were induced by NaHCO3 in P. tenuiflora, suggesting its key role in pH regulation
under alkaline conditions (Kobayashi et al., 2012). These studies indicated that a
range of physiological and molecular responses are induced to adapt to Na2CO3 stress
in halophytes. However, cereal crops such as maize belonging to glycophytes may
differ from halophytes in the adaptive responses to Na2CO3 stress. So far, little is
known on this regard, particularly at the transcriptomic level. Furthermore, none
studies have focused on comparing the global gene expression profiles amongst
Na2CO3, NaCl and high pH stresses.
Maize (Zea mays L.) has in recent years become the dominant crop in China in terms
of production and yield, owing to its considerable agricultural and economic value as
a crop for food, feed and fuel. In Northeast China, maize production contributes to
nearly 40% of the total national production (http://www.stats.gov.cn/). Na2CO3 stress
has increasingly become a limiting factor for maize to achieve yield potential in this
area due to irrigation-induced secondary salinization and alkalisation. Na2CO3 stress,
as a result of capillary upward flow of ground water at the early spring, affects maize
seed germination and post-germination seedling growth (Shi, 2007). However, little is
known about responses of maize to such sodic saline stress, particularly the early
adaptation responses in the root systems of maize seedlings. Furthermore, it is also
not well examined the differences in plants’ responses to stresses imposed by different
saline and alkaline salts. Here, we took the advantage of the next-generation
sequencing technology and carried out high-resolution RNA-Seq studies to compare
4
the global gene expression profiles amongst Na2CO3, NaCl and high pH stresses
during the early transcriptomic adaptation in maize roots. Our results indicated that
over one quarter of the total number of the maize genes changed significantly their
expression levels upon Na2CO3 stress, much more than the numbers of genes found
under NaCl and high pH stresses. Na2CO3 stresses shared to a certain degree common
pathways with NaCl and high pH stresses, but possessed more distinctive
transcriptomic shifts in genes involved in brassinosteroid biosynthesis, protein
processing, nitrogen and amino acid metabolism, and circadian rhythm.
Results
Rationale of experimental design for dissecting the effects of Na2CO3 stress
We are interested in dissecting crop’s adaptive responses to sodic alkaline stresses in
general. The forms of CO32- in soil mainly depend on the effects of solution pH values;
HCO3- is the primary form when solution pH values are between 6.5 and 10.5, while
CO32- is the dominate form when pH values exceed 10.5 (Lindsay, 1979). To mimic
the field conditions in Northeast China, we focused on understanding the effects of
Na2CO3 stress, which is a combination of stresses of high sodium, high pH and high
carbonate. As an attempt to separate the effects of CO32- from those of Na+ and high
pH, we included two extra treatments of NaCl and NaOH. Hence, in the end, the
experiments were designed in a way such that the effects of CO32-, Na+ and high pH
could be compared by using three treatments of Na2CO3, NaCl and NaOH
independently (Figure 1A). In such a design, the effects of Na+ stress in Na2CO3
treatment were compared with that of NaCl treatment, while the effects of high pH
with that of NaOH. Intuitively, the effects of CO32- could be analysed in this manner.
We are interested in the early transcriptomic adaptation to sodic alkaline stress and
decided to use maize root systems of young seedlings as the objectives to study a
sudden shock stress, since germination and seedling establishment are crucial stages
5
in the maize life cycle. It has been shown in maize roots the response of the
transcriptome to sub-lethal salt stress (150mM NaCl) was rapid and transient, leading
to a burst of changes at the 3-hour time point (Wang, et al. 2003). In a similar maize
transcriptomic study, a 5-hour treatment of 100mM NaCl was used to examine the
early stage of stress responses in the roots of maize seedling (Qing, et al. 2009). In
comparison with Na+ stress, however, there has rarely been report on the
transcriptomic responses to CO3-2 or HCO3-1 stresses in maize. In wild soybean
(Glycine soja), previous studies on roots response 50mM NaHCO3 stress shown that
the number of significantly stress regulated genes increased dramatically 3 hours after
stress treatment and peaked at 6-hour (Ge et al., 2010). Taking the relevant evidence
together, to mimic the early alkaline stress conditions, we set the treatment time at
five hours for 2-week-old seedlings and tested the proper sodium concentrations and
pH values by observing the root morphologic changes under various combinations
(Figure 1B) and, in the end, Na2CO3 treatment was set at 50mM, which gave a final
concentration of Na+ at 100mM and pH value at 11.39. Concomitantly, 100mM NaCl
and NaOH with pH at 11.39 were used for comparisons and, double-distilled water as
control (Figure 1C). Maize inbred line B73 was used as the testing genotype for its
draft whole genome sequences have been published (Schnable et al., 2009).
Generation of RNA-Seq data and the quality assessment
Roots of 10 two-week-old seedlings from each treatment were harvested and pooled
for RNA extraction, cDNA conversion and RNA-Seq library preparation. Ten
seedlings continuously grown in double distilled water for 5 hours were used as
controls. Two independent experiments were carried out. In the end, 8 RNA-Seq
datasets were obtained with over 98% clean reads (Figure 2A). When the clean reads
were over 3000k, the gene coverage reached 80% of the total numbers of predicted
genes in the B73 genome (Figure 2B). The reads mapped to the reference genome
exhibited an adequate distribution manner in both genes and chromosomes (Figure 2C,
D). Table 1 shows the stats of the RNA-Seq reads mapped to the reference B73
6
genome. Roughly, about six million total reads were generated for each treatment, of
which 65-85% was mapped to the genome including 57-72% uniquely matched ones.
The RPKM ratio [false discovery rate (FDR) <0.001 and |log2 RPKM Ratio|≥1] test
showed that the homogeneity and consistence of the two biological experiments was
high and indicated that our RNA-Seq data were reproducible, reliable and suitable for
further analysis (Additional File 1, Figure S1).
The expression profiles of 8319 genes were altered by Na2CO3 stress
The generated datasets were mined for genes with significantly altered expression
profiles under the three stress conditions using the expression levels of the
corresponding genes in the double-distilled water control as the reference. A striking
total number of 8319 genes were found with changed expression profiles under
Na2CO3 stress, which was substantially greater than those of NaCl (3226) and high
pH (734) stress imposed by NaOH treatment (Figure 3, Additional File 1, Figure S2).
In the B73 genome the total number of predicted high-confidence protein-coding
genes under high stringency is 32,540 (Schnable et al., 2009), which means that
Na2CO3 stress altered the expression levels of over a quarter of the total number of
genes in maize, such a global and profound effect on transcriptomic profiles has so far
rarely reported. Interestingly, the number of down-regulated genes under Na2CO3
stress was larger than that of up-regulated ones, whereas in the other two stress
conditions the numbers of down-regulated genes were smaller than the corresponding
up-regulated ones. Another surprising result was the substantially low number of
genes with altered expression profiles under high pH stress at this early stage. The
number of genes with altered expression levels in the NaCl treatment was comparable
with other reports. Hence, we reckon that the huge differences in the number of genes
affected reflected the true situation in the three treatments and that Na2CO3 stress was
the strongest effector causing profound changes in the gene expression profiles in
maize roots.
7
This notion was further supported by the analysis of the numbers of genes shared or
different in the three stresses. In the Na2CO3 treatment, 1709 up-regulated genes and
4198 down-regulated genes were unique to the stress itself, representing over 55%
and 90% of the total up and down regulated genes, respectively (Figures 3A and B)
On the contrary, the percentages of the numbers of genes unique to NaCl stress were
28% in the up-regulated category and 32% in the down-regulated category,
respectively; the percentages of the numbers of genes unique to NaCl stress were 26%
in the up-regulated category and 20% in the down-regulated category, respectively.
Thus, the effects of Na2CO3 treatment differed from those of NaCl and high pH
stresses not only in the total number of genes changed but also in the ratios of up- and
down-regulated genes and shared and unique genes. We selectively examined seven
genes whose expression was up-regulated in Na2CO3 stress but down-regulated in
NaCl and NaOH treatments using quantitative PCR and found the gene patterns were
consistent with RNA-Seq (Figure 4).
WEGO analysis (Ye et al., 2006) was carried out to examine the cellular components,
molecular functions and biological processes involved in the genes with altered
expression levels. Figure 3C shows the distribution of the major categories. Although
the total number of affected genes varied substantially in the three treatments, these
genes fell into similar categories of the cellular components, molecular functions and
biological processes, indicating the commonality of these abiotic stresses imposed to
plant cells. Furthermore, the categories of the up- and down-regulated genes were
pretty similar and differed only in the molecular functions, where genes involved in
the structural molecular activities were only up-regulated, and those activities of
transporters and electron carriers only down-regulated. We performed gene ontology
enrichment analyses for these genes and found that many of up-regulated genes in
Na2CO3 and NaCl treatments were enriched in similar categories, but the
down-regulated genes were enriched in very different categories using agriGO (Du et
8
al., 2010) (Tables 2 and 3). Enrichment was not found for most of genes affected by
NaOH treatment.
Biological pathways commonly altered by Na2CO3, NaCl and high pH stresses
The KEGG analyses were carried out to examine the biological pathways represented
by the genes whose expression profiles were altered in responses to the three stresses.
As shown in Table 4, within the 333 genes whose expression levels were commonly
changed by the three stresses, enrichment was found in seven pathways. The
prominent features of these enriched pathways were stress- and/or defence-related.
The expression of genes for the phenylpropanoid biosynthesis and the metabolism of
its substrate amino acid phenylalanine was the most significantly changed. As shown
in Figure 5, genes encoding for the enzymes in the entire pathway network were
found to have altered expression profiles, suggesting a global impact on all the
subsequent products of phenylalanine. Phenylpropanoids are known to serve as
essential components of structural polymers for plant cell growth (e.g., spermidine)
and secondary metabolites (e.g., coumarine, flavonoid and stilbenoid), and provide
protection from ultraviolet light, defend against herbivores and pathogens. In plants
the concentrations of phenylpropanoids within plants are also known altered by
changes in resource availability (Yu et al., 2013). The cytochrome P450 genes
involved in xenobiotic metabolism were significantly up-regulated, presumably to
drive the pathways for the removal of ‘alien’ toxic compounds. Furthermore,
substantial changes in the expression levels were also found in genes responsible for
the alpha-linolenic acid and linoleic acid metabolism, indicative of the potential
production of phyto-oxylipins by the oxidation of unsaturated fatty acids (Blée, 2002).
Overall, these results implicated that at the early stage the maize root cells are under
high oxidative stress imposed by the Na2CO3, NaCl and NaOH treatments. The
enrichment
of
genes
in
ABC
transporters,
suggested
a
stimulation
of
energy-dependent transmembrane transportation activities. Interestingly, genes for
nitrogen metabolism were down-regulated under Na2CO3 treatment, but up-regulated
9
in NaCl and NaOH treatments (Additional File, Figure S3). The down-regulation of
nitrogen metabolism genes was reflected in the genes involved in the amino acid
metabolism, a detailed examination showed that in Na2CO3 treatment genes for nine
of the 13 amino acid metabolic processes were down-regulated, whereas in NaCl and
NaOH treatments most of them were up-regulated (Table 5).
Besides the pathway enrichment for the genes commonly shared by the three
treatments, we also analysed those shared by two of the three treatments. An obvious
commonality between Na2CO3 and NaCl was the enrichment of genes in the
secondary metabolic pathways including benzoxazinoid biosynthesis, flavone and
flavonol biosynthesis and pro-vitamin A carotenoids (retinol) metabolism. These
compounds, particularly the benzoxazinoids (hydroxamic acids), are secondary
metabolites which are for host resistance against microbial pathogens and insects and
for allelopathic effects (Yu et al., 2013). Consistently, genes were also found enriched
in the pathways for plant-pathogen interaction. In the Na2CO3 and NaOH treatments
genes were found commonly enriched in two pathways: the methane metabolism and
3-chloroacrylic acid degradation, while in NaCl and NaOH treatments, genes were
enriched in cysteine and methionine metabolism and in the biosynthesis of secondary
metabolites including stilbenoids, diarylheptanoids, gingerols and flavonoids. Thus,
although the defence and stress-related pathways were commonly enriched, different
secondary metabolites were involved depending on the stresses imposed by Na2CO3,
NaCl or high pH.
Biological pathways unique to Na2CO3, NaCl and high pH stresses
Our experimental design allowed us to compare and define unique biological
pathways altered by Na2CO3 in comparison with NaCl and NaOH. As shown in Table
6, genes were enriched in six pathways in Na2CO3 treatment. The only uniquely
up-regulated pathway was brassinosteriod biosynthesis. As shown Figure 6, almost all
10
the genes encoding the key enzymes in the brassinosteriod biosynthesis pathway were
up-regulated in Na2CO3 treatment. Genes in five pathways were down-regulated
including ascorbate and aldarate metabolism, protein processing in endoplasmic
reticulum membranes (RE) and by N-glycan biosynthesis pathway, fatty acid
biosynthesis and circadian rhythm. For fatty acid biosynthesis (Figure 7) and the
protein processing (Additional File 1, Figure S4), almost all the genes encoding the
key enzymes were down-regulated, showed a global knock-down of the pathways. In
the ascorbate and aldarate metabolism pathway, the genes with down-regulated
profiles primarily resided at the L-ascorbate and vitamin C biosynthesis such as those
encoding VTC4,
D-threo-aldose-1-dehydrogenase
and
L-galactono-1,4-lactone
dehydrogenase (Additional File 1, Figure S5). Similarly, the genes for circadian
rhythm regulation also displayed down-regulation, implicating the disruption of the
light-regulated growth in the early Na2CO3 treatment.
Six enriched pathways unique to NaCl stress were up-regulated. They were primarily
involved in the degradation of organic compounds such as polyhalogenated
compound cyclohexane (hexachlorocyclohexane, similar to pesticides), naphthalen
and anthracene. These suggested that toxic compounds harmful to cell function were
probably produced at the early NaCl stress. Interesting, NaCl stress suppressed the
expression of genes involved in the formation of the ribosomes, possibly directly
affecting the protein synthesis machinery. In high pH stress imposed by NaOH
treatment genes involved in glutathione, arginine and proline metabolism were
uniquely altered.
Discussion
Soil salinization and alkalisation presents a huge challenge to agricultural production.
An estimate from FAO Land and Plant Nutrition Management Service showed that
over 6% of the world's land is affected by either salinity or sodicity
11
(http://www.fao.org/nr/aboutnr/nrl/en/). One of the ways to tackle such challenge is to
breed new crop varieties with enhanced tolerance, in addition to best land
management practice. Understanding the molecular basis for plant responses to salt
stress is a first step towards successful breeding effort. While the effects of the NaCl
stress have been extensively examined in crops, little is known about the responses to
carbonate stress at the molecular level. In this study, the effects of Na2CO3 on gene
expression were examined in comparison with those of NaCl and high pH in maize
root system. Our results showed that the early transcriptomic adaptation to Na2CO3
stress involved huge numbers of genes in the maize genome and differed from that of
NaCl and high pH stresses in a number of distinctive pathways.
A holistic picture of the early transcriptomic adaptation to Na2CO3 stress
Salt stress primarily results from the excessive Na+ content in the soil, which could
competitively replace K+ and paralyse the enzymes in a plant cell. So far, Na2CO3 is
known with the most sever damaging effect on plants (Shi and Yin, 1993; Shi and
Sheng, 2005). This is because Na2CO3 has the combined effects of excessive Na+,
high pH and CO32-. This was reflected in our current RNA-Seq work, which allowed
us to present a holistic picture of the early transcriptomic adaptation to Na2CO3 stress.
A number of distinctive features different from Na+ and high pH stresses were
identified in this study. First of all, over a quarter of the predicted genes in the maize
genome altered the expression levels, in contrast to about 10% in NaCl stress and 2%
in high pH stress. Early studies using EST microarrays also showed that indeed NaCl
stress affected genes were in the range of 10% of the total probes printed (Wang et al.,
2003). In rice and tomato, NaCl was also shown to influence about 10% of the total
numbers of genes in the genomes (Kawasaki et al., 2001; Sun et al., 2010). Thus, we
believe that our results reflected the true differences between the Na2CO3 stress and
NaCl and high pH stress. Secondly, the genes whose expression was down-regulated
out-numbered the up-regulated ones under Na2CO3 stress. In this study, we found that
5232 genes were down-regulated and 3087 genes were up-regulated. A recent report
12
showed that NaHCO3 stress could down-regulate more numbers of genes in wild
soybean (Glycine soja) roots (Ge et al., 2010). Thus, it seems to be a rule rather than
an exception that carbonate stress tends to knock-down the expression of most genes,
which is also in contrast to NaCl and high pH stresses. Thirdly, although the total
number of genes affected by the three stress conditions differed substantially, the gene
ontology categories did not differ. Furthermore, the up-regulated gene sets in Na2CO3
and NaCl stresses overlapped in many biological processes and molecular functions,
while the down-regulated genes sets showed quite divergent enrichment. Hence, it
seems that one of the unique features of carbonate stress is to cause down-regulation
of more genes; this is rarely seen in other stress factors. This feature is unique to
carbonate since only limited numbers of genes showed altered expression profiles in
the high pH stress and did not show any enrichment. The fact that the effects of high
pH were limited is count-intuitive, considering the global effects of high pH could
affect the availability of multiple micronutrients. One explanation for this could be
that we selected an early time point of 5-hours post-stress when the effects of high pH
were lagged behinds, which requires further experiments to validate by extending the
duration of stress treatment.
Distinctive and shared cellular responses to Na2CO3, NaCl and high pH stresses
A two-phase growth model has been proposed in response to salinity in plants (Munns
and Termaat, 1986): an initial short term response to osmotic stress and a long term
response to ionic toxicity. In our current research, we focused on the early responses
to the salt stress and RNA-Seq was carried out on samples collected 5-hours
post-stress treatment. At this stage, we reckon that the responses in maize root cells
were primarily to the osmotic stress, which imposes ‘drought’ to cells due to water
scarcity caused by the excessive salt concentrations in the environments. Previous
work showed that the early NaCl-imposed osmotic stress involves signalling and
metabolic pathways for oxidative burst, reactive oxygen species scavenge system,
shift from primary metabolism to the biosynthesis of secondary metabolites (Zörb et
13
al., 2004; Zörb et al., 2010). Although genes for these cellular responses were
amongst the list of genes with altered expression under the three stress conditions
tested, the paramount features of such early cellular responses appeared using quite
different pathways to fight against the stress. The most significant changes were those
genes involved in biosynthesis of structural polymers phenylpropanoids via the amino
acid phenylalanie, the oxidation of unsaturated fatty acids (alpha-linolenic acid and
linoleic acid) metabolism, and the detoxification of the xenobiotics by cytochrome
P450. Biosynthesis of phenylpropanoids is a key step for the generation of an array of
enormous secondary metabolites (Blée, 2002) and the suppression of the genes in this
pathway by Na2CO3 implies the involvement of secondary metabolites in cellular
adaption. Genes encoding ABC-transporters were up-regulated under the three stress
conditions. ABC transporters are transmembrane proteins involved in the
translocation of various substrates across membranes including metabolic products,
lipids and sterols, and drugs. They are also involved in non-transport-related
processes such as translation of RNA and DNA repair. Shift in the expression of
genes for nitrogen metabolism was found to be common to the three stresses.
However, Na2CO3 stress tended to down-regulate the expression of the genes,
opposite to the effects of NaCl and high pH stresses. This was reflected in genes for
the amino acid metabolism. While most of the genes displayed down-regulated
expression under Na2CO3 stress, they were up-regulated by NaCl and high pH stresses.
This contrasting effect between Na2CO3 stress and NaCl or high pH stresses was not
reported previously and deserves further investigation.
The commonality between Na2CO3 and NaCl and high pH stresses was also reflected
in the biosynthesis of secondary metabolites such as flavones, flavonols and
flavonoids. We also observed that benzoxazinoids (hydroxamic acids) were
significantly up-regulated under Na2CO3 and NaCl stresses. Hydroxamic acids are
plant secondary metabolites important for host resistance against microbial pathogens
and insects and for allelopathic effects (Yang et al., 2010). In maize, benzoxazinoid
14
biosynthesis is achieved through a series of so-called BX genes (BX1-5) to form
DIMBOA, which is then glucosylated by the UDP-glucosyltransferase (BX8/BX9),
the glucoside is further processed to form DIMBOA-glucoside and stored in the
vacuoles (Qing et al., 2009). It is not clear why the genes in this BX defence pathway
for biotic stresses were up-regulated under salt stress.
Besides sharing these features with NaCl and high pH stresses, Na2CO3 possessed
distinctive gene expression profile unique to itself. Similar to the situation that more
genes were down-regulated than up-regulated, gene enrichment occurred in more
biological pathways which were down-regulated rather than up-regulated. The only
up-regulated pathway was the brassinosteriod biosynthesis. It is known that maize
possesses the complete set of genes for brassinosteriod biosynthesis and recent
genetic analysis shows that disruption of genes in the pathway causes dwarfness in
maize (Makarevitch et al., 2012). Presumably, the early up-regulation of genes for
brassinosteriod biosynthesis is a mechanism to enhance tolerance by stimulating cell
growth. Furthermore, the genes for circadian rhythm responses were down-regulated
under Na2CO3 stress. Thus, it appears that active adjustment in cellular and
whole-plant grow this important for adaptation to Na2CO3 stress. Genes for protein
processing in RE and N-glucan biosynthesis were simultaneously down-regulated in
Na2CO3 stress. These two pathways are important for a range of processes including
protein transportation to Golgi and export out of the cell, protein targeting and
degradation as well as protein glycosylation and glucosylation. Thus, down-regulation
of these genes may imply that disruption of protein traffic and accumulation of
damaged protein in the cells. Concomitantly, genes for ascorbate and fatty acid
metabolism were down-regulated. All the evidence points to the fact that Na2CO3
stress presents a highly harmful and stressful condition to maize root cells.
In summary, our RNA-Seq work using next-generation sequencing technology
demonstrates that Na2CO3 imposes considerable stress to maize root cells, as
15
envisaged by the alteration in the expression of over one quarter of the total maize
genes. Although Na2CO3 stress shares commonality to NaCl and high pH stresses, it
has more distinctive effects, particularly by causing the down-regulation of more
numbers of genes than up-regulation and the suppression of more biological pathways.
This work provides a holistic picture of the early transcriptomic adaptation to Na2CO3
stress, which will be helpful for further targeted dissection of specific pathways for
the improvement of tolerance in maize.
Materials and Methods
Plant growth and experimental treatments
The root of the maize inbred line B73 was used as the experimental material in this
study. This line is cultivated in the city of Changchun in Jilin province and is
considered to be tolerant to Na2CO3, NaCl and NaOH stresses. Eighty to 100 seeds
from the B73 lines were pre-germinated in the dark at 27°C. The germinated seeds
were then transferred to small pots containing half-strength Hoagland's nutrient
solution in a growth chamber, where the light cycle was set at 16h light (200 μEm−2
s−1, 25°C) and 8h darkness and the temperature kept at 20°C, the relative humidity at
50%. Seedlings grown for fortnight under such conditions were then kept for five
hours in pots containing double-distilled water supplemented with 50mM Na2CO3,
100mM NaCl, or 1.28×10-4mM NaOH (pH=11.39), respectively. The plants grown in
double distilled water were used as controls. For harvesting, 10 uniform seedlings
from each treatment were selected for root sampling. The experiments were repeated
once, in the end, two biological samples for each treatment, in total eight root samples
were obtained for RNA-Seq analysis.
Sample preparation and total RNA extraction
The roots of the control and stressed seedlings were harvested for gene expression
analysis. The collected root samples were immediately frozen in liquid nitrogen and
16
stored at −80°C until further use. Total RNA was isolated using an RNAprep Pure
Plant kit (Tiangen, China) following the manufacturer's protocol. The yield and
quality of the total RNA samples were determined using agrose gel electrophoresis
and NanoDrop Spectrometer methods.
Library construction and deep sequencing
For each biological repeat, a total RNA amount of 20μg was used for construction of
library for RNA sequencing. Magnetic beads with oligos (dT) attached were used for
purifying and enriching the mRNA from the total RNA. The mRNA was then cleaved
into small fragments with fragmentation buffer at elevated temperature. The
fragments were used to synthesise first-strand cDNA using random hexamer adaptors
and reverse transcriptase (Invitrogen, USA). Second-strand cDNA was synthesised
with RNaseH (Invitrogen, USA) and DNA polymerase I (NEB, USA). Fragments of
300-bp with 200-bp insertions were isolated on separation gels. Read lengths were
produced using an IlluminaHiSeq™ 2000 following the manufacture’s protocol
(Wang et al., 2009).
Sequencing data analysis
The original image data is transferred into sequence data by base calling, which is
defined as raw reads and saved as FastQ files. Before data analysis, the dirty raw
reads were filtered to get the clean reads with removing adaptors, tags of reads
(unknown bases are more than 10%) and low quality reads (the percentage of the low
quality bases of quality value ≤5 is more than 50% in a read). Clean reads were
mapped to reference sequences using SOAP aligner/soap2 (Li et al., 2009).
Mismatches no more than 2 bases were allowed in the alignment. Subsequently,
quantity of sequencing was assessed including reads quality, sequencing saturation,
distribution of reads on reference genes and genome (Mortazavi et al., 2008).
17
Differential expression of genes
A statistical analysis of the frequency of each reads in the different cDNA libraries
was performed to compare the expression profiles of the genes in both stress and
control conditions. The gene expression level is calculated by using RPKM (Reads
Per Kb per Million reads) method (Mortazavi et al., 2008), and the formula is shown
as follows: RPKM=106C/NL*103. A strict algorithm was used to identify
differentially expressed genes between two samples (Audic and Claverie, 1997).
Based on hypergeometric distribution, the P-value corresponding to the differential
gene expression was tested using Fisher exact test. False discovery rate (FDR) is a
method to determine the threshold of the P-value in multiple tests and analyses and is
obtained by manipulating the FDR value (Benjamini and Yekutieli, 2001). We used
FDR≤0.001 and the absolute value of log2 ratio>1 as the threshold to judge the
significance of gene expression differences. More stringent criteria with smaller FDR
and bigger fold-change value were used to identify differentially expressed genes.
Gene Ontology (GO) analysis was done for biological process, cellular components
and molecular function by BGI WEGO (Ye et al., 2006) (Web Gene Ontology
Annotation Plotting, http://wego.genomics.org.cn/cgi-bin/wego/index.pl) and agriGO
(Du et al., 2010) (GO Analysis Toolkit and Database for Agricultural Community,
http://bioinfo.cau.edu.cn/agriGO/index.php), pathways which were statistically
significantly (Q value≤0.05) enriched with KEGG (Kanehisa et al., 2008).
Quantitative real-time PCR (qRT-PCR) analysis
The expression of seven candidate genes from the DGE libraries was validated using
quantitative real time PCR (qRT-PCR) using the same RNA samples as in the DGE
library construction. The first strand cDNA fragments were synthesized from total
RNA using RNAprep Pure Plant kit (Tiangen, China). Seven gene-specific primer
pairs were designed based on the target gene sequences using the Primer 5 software.
The qRT-PCRs were performed with a ABI7500 in final volumes of 25 μl, each
18
containing 2 μl of cDNA, 12.5 μl 2× SYBR premix Ex taq™ (Takara, Japan) and 10
μM of the forward and reverse primers. The thermal cycling conditions were as
follows: 40 cycles of 95°C denaturation for 5s and 56°C annealing and extension for
20 s. The maize actin gene was used as an internal control. The relative expression
levels were calculated as 2−(ΔCt of treatment−ΔCt of control).
Acknowledgements
We thank the rest members of the D.Y.H. lab and H.C.J lab for the help in setting up
the experiments. This work is financially supported in part by grants from the
National Natural Science Foundation of China (No. 31170731) and the National
Special Program-New Varieties Breeding of GM maize (No. 2011ZX08003-005).
References
Audic S, Claverie J-M
(1997) The significance of digital gene expression profiles. Genome Res.
7:
986-995
Badger MR, Price GD
45:
Benjamini Y, Yekutieli D
(1994) The role of carbonic anhydrase in photosynthesis. Annu. Rev. Plant Biol.
369-392
(2001) The control of the false discovery rate in multiple testing under
:
dependency. Annals of statistics
1165-1188
Blée E
Chen W, Cui P, Sun H, Guo W, Yang C, Jin H, Fang B, Shi D
(2002) Impact of phyto-oxylipins in plant defense. Trends Plant Sci.
7:
315-322
(2009) Comparative effects of salt and alkali
stresses on organic acid accumulation and ionic balance of seabuckthorn (< i> Hippophae
30:
Du Z, Zhou X, Ling Y, Zhang Z, Su Z
38:
Ge Y, Li Y, Zhu Y-M, Bai X, Lv D-K, Guo D, Ji W, Cai H
rhamnoides</i> L.). Ind. Crops Prod.
351-358
(2010) agriGO: a GO analysis toolkit for the agricultural community.
Nucleic Acids Res
W64-W70
(2010) Global transcriptome profiling of wild
10:
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S,
Tokimatsu T
36:
Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ
13:
soybean (Glycine soja) roots under NaHCO3 treatment. BMC Plant Biol.
153
(2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res
D480-D484
(2001)
Gene expression profiles during the initial phase of salt stress in rice. The Plant Cell Online
889-905
Kobayashi S, Abe N, Yoshida KT, Liu S, Takano T
plasma
membrane-and
vacuolar-type
(2012) Molecular cloning and characterization of
Na+/H+
125:
Li R, Yu C, Li Y, Lam T-W, Yiu S-M, Kristiansen K, Wang J
25:
monocot, Puccinellia tenuiflora. J. plant res
short read alignment. Bioinformatics
of
an
alkaline-salt-tolerant
(2009) SOAP2: an improved ultrafast tool for
1966-1967
19
antiporters
587-594
Lindsay WL
Makarevitch I, Thompson A, Muehlbauer GJ, Springer NM
7:
Miller G, Suzuki N, Ciftic-Yilmaz S, Mittler R
(1979) Chemical equilibria in soils. John Wiley and Sons Ltd.
brassinosteroid C-6 oxidase. PLoS ONE
(2012) Brd1 gene in maize encodes a
e30798
(2010)
Reactive
oxygen
signalling during drought and salinity stresses. Plant, Cell Environ.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B
5:
Munns R, Termaat A
Parida AK, Das AB
60:
Qing D-J, Lu H-F, Li N, Dong H-T, Dong D-F, Li Y-Z
transcriptomes by RNA-Seq. Nat. Methods
species
33:
homeostasis
and
453-467
(2008) Mapping and quantifying mammalian
621-628
(1986) Whole-plant responses to salinity. Funct. Plant Biol.
13:
143-160
(2005) Salt tolerance and salinity effects on plants: a review. Ecotoxicol. Environ. Saf
324-349
(2009) Comparative profiles of gene expression in
leaves and roots of maize seedlings under conditions of salt stress and the removal of salt
50:
Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA
326:
Sheng Y-m, Shi D-c, Xao H-x, Xu Y
4:
Shi D-C, Yin L-J
35:
Shi D, Sheng Y
54:
Shi D, Wang D
271:
Shi L-R
1:
Sun Gr, Peng Yz, Shao Hb, Chu Ly, Zhao Xn, Min Hy, Cao W, Wei C
46:
Sun W, Xu X, Zhu H, Liu A, Liu L, Li J, Hua X
51:
stress. Plant Cell Physiol.
889-903
(2009) The B73 maize genome: complexity, diversity, and dynamics. Science
1112-1115
(1999) The effect on growth of sunflower to mixed salts with various
natural and alkaline. Journal of Northeast Normal University (Natural science edition)
014
(1993) Difference Between Salt (Nacl) and Alkaline (Na2CO3) Stresses on Pucinellia
tenuiflora (Griseb.) Scribn. et Merr. Plants. Acta. Bot. Sin.
144-144
(2005) Effect of various salt–alkaline mixed stress conditions on sunflower seedlings
and analysis of their stress factors. Environ. Exp. Bot.
8-21
(2005) Effects of various salt-alkaline mixed stresses on Aneurolepidium chinense (Trin.)
Kitag. Plant Soil
15-26
(2007) Effects of Complex Saline-Alkali Stress On The Seed Germination of Zea mays L. J.
Hengshui University
003
(2005) Does Puccinelia tenuiflora
have the ability of salt exudation? Colloids Surf. B Biointerfaces
197-203
(2010) Comparative transcriptomic profiling of a
salt-tolerant wild tomato species and a salt-sensitive tomato cultivar. Plant Cell Physiol.
997-1006
Wang H, Miyazaki S, Kawai K, Deyholos M, Galbraith DW, Bohnert HJ
(2003) Temporal progression of
gene expression responses to salt shock in maize roots. Plant Mol. Biol.
Wang Z, Gerstein M, Snyder M
10:
Wu L, Chen X, Ren H, Zhang Z, Zhang H, Wang J, Wang X-C, Huang R
52:
873-891
(2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev.
Genet.
57-63
transcriptionally
modulates
the
expression
of
abscisic
(2007) ERF protein JERF1 that
acid
enhances the tolerance under salinity and cold in tobacco. Planta
Yang C, Guo W, Shi D
(2010)
Physiological
Roles
of
Organic
Acids
biosynthesis-related
226:
in
102:
Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L
34:
Yu J, Chen S, Wang T, Sun G, Dai S
14:
Zörb C, Schmitt S, Mühling KH
Alkali-Tolerant Halophyte. Agron J.
gene
815-825
Alkali-Tolerance
of
the
1081-1089
for plotting GO annotations. Nucleic Acids Res
(2006) WEGO: a web tool
W293-W297
(2013) Comparative Proteomic Analysis of
Leaves under Na2CO3 Stress. Int. J. Mol. Sci
Puccinellia tenuiflora
1740-1762
(2010) Proteomic changes in maize roots after short-term adjustment
20
10:
Zörb C, Schmitt S, Neeb A, Karl S, Linder M, Schubert S
to saline growth conditions. Proteomics
4441-4449
(2004) The biochemical reaction of maize ( Zea
mays L.) to salt stress is characterized by a mitigation of symptoms and not by a specific
adaptation. Plant Sci.
Zhu J-K
167:
91-100
(2002) Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol.
21
53:
247
Figure legends
Figure 1. The design and photos showing representative seedlings used for the
experiments.
A. A diagram showing the design of the experiments. The stress imposed by Na2CO3
to plants is a combination of Na+, high pH and CO32-. In the diagram, the stress of
50mM Na2CO3 is compared with that of 100mM NaCl and 1.28x10-4mM NaOH.
B. Photos showing representative 14-d-old seedlings following treatments for five
hours from left to right in double distilled water, 25mM, 50mM and 100mM
Na2CO3, respectively. A concentration of 50mM Na2CO3 was used for the
subsequent experiments.
C. Photos showing representative 14-d-old seedlings following treatments for five
hours in double distilled water, NaOH, NaCl and Na2CO3, respectively.
Figure 2. Summary of RNA-Seq data generated for comparison of genes
differentially expressed under Na2CO3, NaCl and NaOH stresses.
A. A pie chart showing the classification of raw RNA-Seq reads. Clean reads amount
to 98% of the total reads obtained.
B. Genes mapped by clean reads, as indicated when the clean reads are over 3000k,
the gene coverage reached 80% of the genes identified in maize genome.
C. A diagram showing the number of reads in relation to their relative positions in the
gene with a window size of 100bp.
D. A diagram showing the coverage of the clean reads mapped to the genes on
chromosome 7 in relation to the gene coverage and consistence of the two
biological repeats.
Figure 3. Summary of the numbers of total and shared genes differentially
expressed upon treatment by Na2CO3, NaCl and NaOH, respectively.
A. A Venn diagram showing the genes up-regulated by Na2CO3, NaCl and NaOH
stress, respectively. The numbers of genes shared and distinctive to each treatment
22
were shown.
B. A Venn diagram showing the genes down-regulated by Na2CO3, NaCl and NaOH
stress, respectively. The numbers of genes shared and distinctive to each treatment
were shown.
C. Analysis of gene ontology (GO) categories in the genes up-regulated by Na2CO3,
NaCl and NaOH stress, respectively.
D. Analysis of gene ontology (GO) categories in the genes down-regulated by
Na2CO3, NaCl and NaOH stress, respectively.
Figure 4. Diagrams showing the quantitative PCR results confirming the results
of RNA-Seq.
Seven genes whose expression was up-regulated by Na2CO3 but down-regulated by
NaCl and NaOH were selected for validation and the results of quantitative PCR and
RNA-Seq were consistent.
Figure 5. A diagram from KEGG pathway analysis for phenylpropanoid
biosynthesis under Na2CO3 stress.
Enzymes framed by red squares indicate that the corresponding genes were
up-regulated, whereas those framed by green squares indicate that the corresponding
genes were down-regulated, and the enzymes framed half by red and half by green
indicate that multiple genes in the maize genome encode for the designated enzymes
and some of them showed up-regulation and some down-regulation.
Figure 6. A diagram from KEGG pathway analysis for brassinosteriod
biosynthesis under Na2CO3 stress.
Enzymes framed by red squares indicate that the corresponding genes were
up-regulated, whereas those framed half by red and half by green indicate that
multiple genes in the maize genome encode for the designated enzymes and some of
them showed up-regulation and some down-regulation.
23
Figure 7. A diagram from KEGG pathway analysis for fatty acid biosynthesis
under Na2CO3 stress.
Enzymes framed by red squares indicate that the corresponding genes were
up-regulated, whereas those framed by green squares indicate that the corresponding
genes were down-regulated, and the enzymes framed half by red and half by green
indicate that multiple genes in the maize genome encode for the designated enzymes
and some of them showed up-regulation and some down-regulation.
Figure S1. Experimental repeatability analysis of RNA-Seq.
Correlation analysis of two parallel experiments provides the evaluation of the
reliability of experimental results as well as operational stability. The closer the value
of correlation gets to 1, the better the repeatability between two parallel experiments.
Figure S2. Digital expression genes profiling after treatment with different stress
group in comparison to control.
Figure S3. A diagram from KEGG pathway analysis for Nitrogen metabolism
under Na2CO3 stress.
Enzymes framed by red squares indicate that the corresponding genes were
up-regulated, whereas those framed by green squares indicate that the corresponding
genes were down-regulated, and the enzymes framed half by red and half by green
indicate that multiple genes in the maize genome encode for the designated enzymes
and some of them showed up-regulation and some down-regulation.
Figure S4. A diagram from KEGG pathway analysis for Protein processing in
endoplasmic reticulum under Na2CO3 stress.
Enzymes framed by red squares indicate that the corresponding genes were
up-regulated, whereas those framed by green squares indicate that the corresponding
genes were down-regulated, and the enzymes framed half by red and half by green
indicate that multiple genes in the maize genome encode for the designated enzymes
24
and some of them showed up-regulation and some down-regulation.
Figure S5. A diagram from KEGG pathway analysis for Ascorbate and aldarate
metabolism under Na2CO3 stress.
Enzymes framed by red squares indicate that the corresponding genes were
up-regulated, whereas those framed by green squares indicate that the corresponding
genes were down-regulated, and the enzymes framed half by red and half by green
indicate that multiple genes in the maize genome encode for the designated enzymes
and some of them showed up-regulation and some down-regulation.
25
Table 1. Statistical data of RNA-Seq reads mapped to the maize reference B73 genome
RNA-Seq
ddH2O (CK)
Na2CO3
NaCl
NaOH
Total reads
6137869
(100.00%)
5929349
(100.00%)
6070077
(100.00%)
5912892
(100.00%)
Total mapped reads
3950604
4997515
4612105
4294205
(64.36%)
(84.28%)
(75.98%)
(72.62%)
3479443
(56.69%)
4252399
(71.72%)
4049630
(66.71%)
3764200
(63.66%)
Unique match
26
Table 2. Comparison of GO categories significantly (p < 0.05) enriched in up-regulated genes
under Na2CO3, NaCl and NaOH stresses
CMb
GO Information
GO term
Ontoa Description
FDRc
1 2 3 Na2CO3 NaCl
NaOH
Regulation of macromolecule biosynthetic process
5.7e-14
8e-11
---
1e-10
GO:0010556
P
GO:0019219
P
Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
8.8e-14
GO:0051171
P
Regulation of nitrogen compound metabolic process
1.6e-13 1.4e-10
---
GO:0080090
P
Regulation of primary metabolic process
6.3e-12 1.6e-09
---
GO:0060255
P
Regulation of macromolecule metabolic process
1.1e-11 1.8e-09
---
GO:0043687
P
Post-translational protein modification
5.2e-11 5.3e-07
---
GO:0006468
P
Protein amino acid phosphorylation
1.6e-10 9.3e-07
---
GO:0006464
P
Protein modification process
1.6e-08 9.9e-06
---
GO:0006793
P
Phosphorus metabolic process
3.1e-08 8.4e-05
---
GO:0034641
P
Cellular nitrogen compound metabolic process
GO:0043412
P
Macromolecule modification
8.7e-08 2.7e-05
GO:0042493
P
Response to drug
8.9e-06
GO:0019439
P
Aromatic compound catabolic process
4.3e-05 0.0007
GO:0006725
P
Cellular aromatic compound metabolic process
GO:0009072
P
GO:0004713
F
GO:0004674
5e-08
7e-05
---
3.1e-10 0.00014
---
--0.0089
---
2.6e-05
---
Aromatic amino acid family metabolic process
0.00024 1.9e-06
---
Protein tyrosine kinase activity
4.4e-14 5.6e-09
---
F
Protein serine/threonine kinase activity
2.2e-11 1.9e-07
---
GO:0016773
F
Phosphotransferase activity, alcohol group as acceptor
5.5e-10 1.4e-07
---
GO:0003700
F
Transcription factor activity
5.5e-10 4.4e-09
---
GO:0016301
F
Kinase activity
1.3e-08 2.4e-06
---
GO:0016757
F
Transferase activity, transferring glycosyl groups
GO:0015291
F
Secondary active transmembrane transporter activity
4.5e-06
GO:0005506
F
Iron ion binding
8.1e-06 4.4e-09
---
GO:0016758
F
Transferase activity, transferring hexosyl groups
1.4e-05 0.00036
---
GO:0016491
F
Oxidoreductase activity
2.2e-05 0.00016 0.015
GO:0009055
F
Electron carrier activity
3.6e-05 0.00016
---
GO:0004497
F
Monooxygenase activity
7.8e-05 9.3e-07
---
GO:0008194
F
UDP-glycosyltransferase activity
0.00022
---
---
GO:0048037
F
Cofactor binding
0.00074
---
---
GO:0005529
F
Sugar binding
0.0031 0.00096
GO:0030145
F
Manganese ion binding
---
0.00016
---
GO:0046906
F
Tetrapyrrole binding
---
0.00022
---
GO:0016747
F
Transferase activity, transferring acyl groups other than amino-acyl groups
---
0.00077
---
GO:0048046
C
Apoplast
0.00032 0.00025
---
2e-08 0.00022
---
--0.0019
---
a
Gene Ontology (GO) Categories: P: Biological processes, F: Molecular function, C: Cellular
components;
b
1: Na2CO3, 2: NaCl, 3: NaOH; The colour-coded blocks represent the levels of gene enrichment for
each term. The gray blocks represent no gene enrichment, the darker (the more red) the colour, the
higher the degree of the gene enrichment. The adjusted P-value of the term determines the degree of
27
color saturation of the corresponding box.
c
FDR correction methods: Benjamini and Hochberg (1995).
28
Table 3. Comparison of GO categories significantly (p < 0.05) enriched in the down-regulated
genes under Na2CO3, NaCl and NaOH stresses
CMb
GO Information
GO term
Ontoa
GO:0034641
P
GO:0033036
Description
1
Na2CO3
NaCl
NaOH
Cellular nitrogen compound metabolic process
2e-07
5.4e-07
---
P
Macromolecule localization
2e-07
---
---
GO:0034613
P
Cellular protein localization
0.00013
---
---
GO:0007264
P
Small GTPase mediated signal transduction
0.00013
---
---
GO:0007242
P
Intracellular signaling cascade
0.00013
---
---
GO:0045184
P
Establishment of protein localization
0.00016
---
---
GO:0051604
P
Protein maturation
0.00034
---
---
GO:0046907
P
Intracellular transport
0.00034
---
---
GO:0019725
P
Cellular homeostasis
0.00071
---
---
GO:0051246
P
Regulation of protein metabolic process
0.00079
---
---
GO:0019538
P
Protein metabolic process
0.0015
1.5e-10
---
GO:0044267
P
Cellular protein metabolic process
0.0028
4.1e-14
---
GO:0018130
P
Heterocycle biosynthetic process
0.021
5.7e-05
---
GO:0006412
P
Translation
---
3.2e-53
---
GO:0010467
P
Gene expression
---
2.6e-13
---
GO:0034645
P
Cellular macromolecule biosynthetic process
---
3.1e-12
---
GO:0044249
P
Cellular biosynthetic process
---
2.4e-10
---
GO:0044260
P
Cellular macromolecule metabolic process
---
0.0026
---
GO:0004298
F
Threonine-type endopeptidase activity
2.4e-05
---
---
GO:0003735
F
Structural constituent of ribosome
---
3.2e-62
---
GO:0016762
F
Xyloglucan: xyloglucosyltransferase activity
---
0.00015
---
GO:0044444
C
Cytoplasmic part
1.7e-05
3.8e-42
---
GO:0005839
C
Proteasome core complex
2.5e-05
---
---
GO:0044464
C
Cell part
0.00019
4.5e-05
---
GO:0032991
C
Macromolecular complex
0.00029
6.5e-24
---
GO:0048475
C
coated membrane
0.00039
---
---
GO:0005622
C
Intracellular
0.0027
7.3e-08
---
GO:0030529
C
Ribonucleoprotein complex
0.031
1e-57
---
GO:0043232
C
Intracellular non-membrane-bounded organelle
---
3.5e-34
---
GO:0043229
C
Intracellular organelle
---
2.1e-12
---
GO:0015934
C
Large ribosomal subunit
---
7.6e-06
---
GO:0031970
C
Organelle envelope lumen
---
2.4e-05
---
a
2
FDRc
3
Gene Ontology (GO) Categories: P: Biological processes, F: Molecular function, C: Cellular
components;
29
b
1: Na2CO3, 2: NaCl, 3: NaOH; The color-coded blocks represent the levels of gene enrichment for
each term. The gray blocks represent no gene enrichment, the darker (the more red) the color, the
higher the degree of the gene enrichment. The adjusted P-value of the term determines the degree of
color saturation of the corresponding box.
c
FDR correction methods: Benjamini and Hochberg (1995).
30
Table 4. Comparison of common KEGG pathways significantly (p < 0.05) enriched under
Na2CO3, NaCl and NaOH stresses
Na2CO3
Response style
NaCl
NaOH
Pathways
Regulation
Regulation
P value
Regulation
P value
P value
Metabolism of xenobiotics by cytochrome P450
Up
1.14E-05
Up
1.29E-04
Up
1.12E-05
α-Linolenic acid metabolism
Up
1.39E-04
Up
3.18E-06
Up/down
2.70E-04
ABC transporters
Up
1.99E-03
Up
2.30E-03
Up
1.92E-04
Phenylpropanoid biosynthesis
Up/down
2.14E-06
Up
3.43E-08
Up/down
1.94E-06
Phenylalanine metabolism
Up/down
1.62E-03
Up/down
2.55E-04
Up/down
4.82E-07
Linoleic acid metabolism
Up/down
5.05E-03
Up
3.56E-04
Up/down
1.01E-02
Down
9.00E-04
Up
2.15E-02
Up/down
3.19E-07
—
—
—
—
—
—
—
—
Up/down
7.12E-04
Down
4.99E-02
Common to three
stresses
Nitrogen metabolism
Benzoxazinoid biosynthesis
Up
5.59E-03
Up
4.89E-05
Common to Na2CO3
Flavone and flavonol biosynthesis
Up
1.19E-02
Up
7.43E-05
and NaCl stresses
Plant-pathogen interaction
Up/down
6.12E-08
Up
7.82E-10
Retinol metabolism
Up/down
3.35E-02
Up
4.45E-02
—
—
—
—
Up
3.74E-07
Up
5.55E-05
Common to Na2CO3
Methane metabolism
Down
1.19E-03
and NaOH stresses
3-Chloroacrylic acid degradation
Down
2.55E-02
—
—
—
—
—
—
—
—
Stilbenoid, diarylheptanoid and gingerol biosynthesis
Common to NaCl
Cysteine and methionine metabolism
and NaOH stresses
Flavonoid biosynthesis
Biosynthesis of secondary metabolites
31
Up
1.18E-03
Up/down
1.45E-03
Up/down
1.95E-05
Up/down
2.48E-06
Up/down
3.89E-03
Up/down
1.53E-04
Table 5. Comparison of key amino acid metabolic processes under Na2CO3, NaCl and
NaOH stresses
Na2CO3
NaCl
NaOH
Pathways
Number
Regulation
Number
Regulation
Number
Regulation
Cysteine and methionine metabolism
63 (1.51%)
Up
38*(2.24%)
Up
12*(3.72%)
Up /down
Phenylalanine, tyrosine and tryptophan biosynthesis
17 (0.41%)
Up
14* (0.82%)
Up
5* (1.55%)
Up /down
Valine, leucine and isoleucine biosynthesis
15 (0.36%)
Up/down
7 (0.41%)
Up
—
—
21 (0.5%)
Up/down
5* (1.55%)
Up/down
12* (0.71%)
Up /down
Alanine, aspartate and glutamate metabolism
22 (0.53%)
Down
9 (0.53%)
Up
7 (2.17%)
Up
Glycine, serine and threonine metabolism
24 (0.58%)
Down
8 (0.47%)
Up
2 (0.62%)
Up
Valine, leucine and isoleucine degradation
20 (0.48%)
Down
3 (0.18%)
Up
2 (0.62%)
Up
Lysine biosynthesis
10 (0.24%)
Down
4 (0.24%)
Down
1 (0.31%)
Up
Lysine degradation
24* (0.58%)
Down
3 (0.18%)
Up
2 (0.62%)
Up
29 (0.7%)
Down
10 (0.59%)
Up/down
9* (2.79%)
Up /down
13 (0.31%)
Down
4 (0.24%)
Up
1 (0.31%)
Down
84* (2.02%)
Down
44* (2.59%)
Up
19* (5.88%)
Up
14 (0.34%)
Down
6 (0.35%)
Up
2 (0.62%)
Up /down
Tyrosine metabolism
Arginine and proline metabolism
Histidine metabolism
Phenylalanine metabolism
β-Alanine metabolism
*
p value < 0.05
Table 6. KEGG significantly enrichment (p < 0.05) of up/down regulation expression genes only
with each stress
Response style
Pathway
Brassinosteroid biosynthesis
Regulation
Number (Ratio)
P value
Q value
Pathway ID
Up
9 (0.22%)
0.036484
2.49E-01
ko00905
Ascorbate and aldarate metabolism
Down
35 (0.84%)
0.001395
3.91E-02
ko00053
Protein processing in endoplasmic reticulum
Down
147 (3.53%)
0.004177
6.19E-02
ko04141
Only in Na2CO3 stress
Only in NaCl stress
N-Glycan biosynthesis
Down
29 (0.7%)
0.004583
6.37E-02
ko00510
Fatty acid biosynthesis
Down
27 (0.65%)
0.006335
7.14E-02
ko00061
Circadian rhythm - plant
Down
58 (1.39%)
0.020697
1.91E-01
ko04712
χ-Hexachlorocyclohexane degradation
Up
42 (2.47%)
1.22E-05
4.77E-04
ko00361
Naphthalene and anthracene degradation
Up
32 (1.88%)
0.00051
9.29E-03
ko00626
Limonene and pinene degradation
Up
32 (1.88%)
0.00124
1.83E-02
ko00903
Systemic lupus erythematosus
Up
20 (1.18%)
0.00249
3.25E-02
ko05322
Diterpenoid biosynthesis
Up
16 (0.94%)
0.00464
5.45E-02
ko00904
Phosphatidylinositol signaling system
Ribosome
Glutathione metabolism
Only in pH stress
Up
19 (1.12%)
0.03442
3.00E-01
ko04070
Down
162 (9.53%)
7.76E-26
1.82E-23
ko03010
Up
10 (3.1%)
0.00064
9.43E-03
ko00480
Arginine and proline metabolism
Up/down
9 (2.79%)
0.00035
5.69E-03
ko00330
Metabolic pathways
Up/down
104 (32.2%)
0.00481
4.51E-02
ko01100
32
B
A
C
Cont
r
ol
(
ddH2O) 25mM Na2CO3
50mM Na2CO3 100mM Na2CO3
Cont
r
ol
(
ddH2O)NaOH(
pH11.
39)NaCl
(
100mM)Na2CO3(
50mM)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
map00940
Fig. 5
file:///F|/郝老师转录组信息/转录组数据/转录组数据_58455/转录组数据/华大基因分析结果/GeneDiffExp/Pathway/0-VS-2_map/map00940.html2013/4/6 15:12:11
map00905
Fig. 6
file:///F|/郝老师转录组信息/转录组数据/转录组数据_58455/转录组数据/华大基因分析结果/GeneDiffExp/Pathway/0-VS-2_map/map00905.html2013/4/6 15:01:38
Fig. 7