Abstract
Background
Pod size is an important yield target trait for peanut breeding.
However, the molecular mechanism underlying the determination of peanut
pod size still remains unclear.
Results
In this study, two peanut varieties with contrasting pod sizes were
used for comparison of differences on the transcriptomic and endogenous
hormonal levels. Developing peanut pods were sampled at 10, 15, 20, 25
and 30 days after pegging (DAP). Our results showed that the process of
peanut pod-expansion could be divided into three stages: the
gradual-growth stage, the rapid-growth stage and the slow-growth stage.
Cytological analysis confirmed that the faster increase of cell-number
during the rapid-growth stage was the main reason for the formation of
larger pod size in Lps. Transcriptomic analyses showed that the
expression of key genes related to the auxin, the cytokinin (CK) and
the gibberellin (GA) were mostly up-regulated during the rapid-growth
stage. Meanwhile, the cell division-related differentially expressed
genes (DEGs) were mostly up-regulated at 10DAP which was consistent
with the cytological-observation. Additionally, the absolute
quantification of phytohormones were carried out by
liquid-chromatography coupled with the tandem-mass-spectrometry
(LC–MS/MS), and results supported the findings from comparative
transcriptomic studies.
Conclusions
It was speculated that the differential expression levels of TAA1 and
ARF (auxin-related), IPT and B-ARR (CK-related), KAO, GA20ox and GA3ox
(GA-related), and certain cell division-related genes
(gene-LOC112747313 and gene-LOC112754661) were important participating
factors of the determination-mechanism of peanut pod sizes. These
results were informative for the elucidation of the underlying
regulatory network in peanut pod-growth and would facilitate further
identification of valuable target genes.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12870-023-04382-w.
Keywords: Peanut (Arachis hypogaea L.), Pod size, Pod growth,
Cytological analysis, Transcriptome, Phytohormone
Background
Cultivated peanut (Arachis hypogaea L.) can provide humans with
nutrients such as protein and essential fatty acids [[43]1], and is
widely cultivated worldwide as an important oilseed crop and cash crop.
In recent years, peanut has become one of the three major oilseed crops
in China, which plays an important role in ensuring the safety of
edible oil in China [[44]2]. Yield potential has always been a vital
target of plant breeding in peanut, and the pod size directly
influences the final yield and quality of peanut. The peanut pod is
composed of shell and seed. Swelling of the shell can affect potential
yield [[45]3]. Larger shells provide more room for development and are
more likely to obtain larger seeds [[46]4]. Nevertheless, the sizes of
seeds and organs are not always positively correlated because they have
separate regulatory pathways [[47]5]. For peanut, previous studies have
shown that pod-growth can be divided into two stages of pod expansion
and seed filling [[48]6]. Pod expansion is mainly performed at the
early-growth stage (10DAP-30DAP), during which the pod reaches its
final size. Therefore, studies on the peanut pod-growth during this
critical period might be helpful to understand the
determination-mechanism of peanut pod sizes.
Plant fruit development is mainly controlled by cell division and cell
expansion [[49]7]. And fruit size is determined by the number and size
of cells [[50]8]. Rice OsSPL16 encodes a protein that is a positive
regulator of cell proliferation. The high expression of this gene
promotes cell division and generates wider grain [[51]9]. On the
contrary, restriction of cell proliferation produces smaller organs and
seeds [[52]5, [53]10]. Moreover, studies suggest that the grain size of
rice could be modulated by increasing cell expansion in spikelet hulls
[[54]11, [55]12]. However, the regulation of cell-number and cell-size
are often controlled and coordinated by the mechanism of regulating
plant and organ size. Changing either determinants does not necessarily
change the final organ size [[56]13]. At present, it is still unclear
whether the difference in pod size of peanut is caused by the
difference in cell number or cell size.
Phytohormones play essential roles in the regulation of pod-growth, pod
size and crop yield. Auxin is a critical phytohormone that plays
crucial roles in embryogenesis, organogenesis, cell determination and
division, flower and fruit development [[57]14]. The auxin biosynthesis
mutation of garden pea resulted in small seeds, and the phenotypic
effect of the mutation was partially reversed by auxin application
[[58]15]. The growth and development of peanut pegs and pods are
regulated by auxin [[59]16]. The pod weight and yield per pod treated
by indole-3-acetic acid (IAA) and auxin polar transportinhibitor
2,3,5-triiodobenzoic acid (TIBA) were significantly increased,
indicating that auxin may increase yield by promoting pod development
[[60]17]. Cytokinin (CK) regulates plant growth and development and
plays a key role in regulating cell proliferation [[61]18]. The AtENO2
mutant reduces seed size by decreasing the content of cytokinin
[[62]19]. Furthermore, the triple cytokinin receptor mutant produces
larger seeds [[63]20]. Studies have shown that cytokinin and auxin can
synergistically promote cell division and thus influence fruit size
[[64]21, [65]22]. Gibberellin (GA) is a phytohormone that promotes cell
division and elongation and participate in many developmental processes
[[66]23]. DELLA proteins (aspartic acid–glutamic
acid–leucine–leucine–alanine) inhibit plant growth by reducing both
cell proliferation and expansion rates [[67]24]. GA promotes cell
division and expansion by inhibiting the activity of DELLA proteins
[[68]25, [69]26]. In rice, miR396ef mutation promotes the increase in
the level of GA precursor, thereby promoting the biosynthesis of GA,
and improving grain yield by increasing grain size [[70]27]. In peanut,
AhGRF5a and AhGRF5b are response factors to GA3 and express higher
levels in pod. These two genes may play key roles in peanut pod-growth
[[71]6].
Currently, several studies on transcriptome research related to peanut
pod-growth have been reported [[72]3, [73]28–[74]31]. However, few
studies have focused on phytohormones regulation pod-growth during
early-stage. In the present study, cytological observation, RNA-Seq and
LC–MS/MS for absolute phytohormones quantification were performed to
explore the physiological and molecular changes of peanut pod during
this period. The results will facilitate understanding the decisive
role of a series of changes during early-growth stage on the formation
of peanut pod size, and be helpful for cloning of candidate genes and
molecular breeding.
Results
Pod differences between Tif and Lps during early-growth stage
In general, peanut pods begin to expand at 10DAP and reach the final
size during 20DAP to 30DAP. In this study, the pod length and width of
Tif and Lps were measured at 10, 15, 20, 25, and 30 days after pegging
(DAP), respectively. The results showed that pods of both Tif and Lps
developed rapidly from 10 to 15DAP. Subsequently, pod reticulation
appeared at 25DAP and reached the final size in about 25DAP to 30DAP
(Fig. [75]1a). Notably, the pod length and width of Lps still increased
significantly during DAP25-DAP30 compared to Tif. The growth curve of
the peanut pods was close to the S type, and the pod length and width
could be well fitted by a logistic growth curve (Fig. [76]1b, c).
Calculations showed that the pod length and width of both Tif and Lps
increased fastest during about 8DAP to 16DAP (t[1]-t[2]). The time of
maximum rates (Tm) of both pod length and width of Tif was about 12DAP,
and the Tm of pod length and width of Lps was about 12DAP and 11DAP,
respectively (Table S[77]2). In addition, the pod length and width of
Lps were significantly greater (p < 0.01) than those of Tif at each
growth stage (Fig. [78]1d, e).
Fig. 1.
[79]Fig. 1
[80]Open in a new tab
Phenotypes of Tif and Lps during the early-growth stage. a The
phenotypic characteristics of Tif and Lps pods at five different growth
stages. b Observed and fitted the pod length of Tif and Lps using
logistic growth function. c Observed and fitted the pod width of Tif
and Lps using logistic growth function. d Pod length of Tif and Lps
during early-growth stage. e Pod width of Tif and Lps during
early-growth stage. Scale bar = 2 cm in (a). Error bar is SD. **
p < 0.01
Distinct cell number and cell size between Tif and Lps
Since the pod size of Tif and Lps changed significantly from 10 to
20DAP, we performed histological analysis in longitudinal and
transverse sections of the shell at 10DAP, 15DAP, and 20DAP,
respectively. Cell number was calculated along the black lines in one
single-cell line. Cells calculated in this way were regarded as cell
numbers in longitudinal or transverse sections. Cell areas in the red
box were calculated as the cell areas of the longitudinal or transverse
section (Fig. [81]2a, b). The cell number of Tif and Lps increased
rapidly during 10DAP to 15DAP. In the longitudinal section of each
stage, the cell-number of Lps was 32.0%, 38.1% and 18.4% more than that
of Tif, respectively (Fig. [82]2e). For the transverse section, the
cell-number in Lps was significantly more (p < 0.05) than that of Tif
at 10DAP, 15DAP and 20DAP by 48.19%, 11.69% and 10.32%, respectively
(Fig. [83]2e). The cell-area of Tif and Lps increased rapidly during
15DAP to 20DAP. The cell-area of Lps was significantly greater
(p < 0.05) than that of Tif only on the longitudinal of 10DAP and
transverse sections of 20DAP (Fig. [84]2f).
Fig. 2.
[85]Fig. 2
[86]Open in a new tab
Differences in cell development between Tif and Lps lines. a The
longitudinal sections of shell of Tif and Lps at three growth stages. b
The transverse sections of shell of Tif and Lps at three growth stages.
c The longitudinal sections of Tif and Lps at three growth stages. d
The transverse sections of Tif and Lps at three growth stages. e Cell
number of longitudinal sections and transverse sections in Tif and Lps
at three growth stages. f Cell area of longitudinal sections and
transverse sections in Tif and Lps at three growth stages. g
Correlation between pod length and cell number. h Correlation between
pod transversal diameter and cell number. Error bar is SD. Scale
bar = 5000 μm in (a); scale bar = 2000 μm in (b); scale bars = 200 μm
in (c) and (d)
Based on the investigation, we found that both cell number and cell
size of Lps were greater than that of Tif during the early-growth
stage. Herein, we analyzed the relationship between pod length and pod
transversal diameter with cell number and cell area, respectively. In
both longitudinal and transverse sections, pod length and transversal
diameter were significantly correlated with cell number and cell area
(p < 0.01), indicating that the growth of pod was promoted by the
increase of cell number and area. For the longitudinal section, pod
length showed a strong linear relationship (r = 0.9357) with cell
number (Fig. [87]2g). However, compared with the number of cells, the
linear relationship between pod length and cell area is
weak(r = 0.6725) (Fig. S[88]1a). For the transverse section, a strong
linear relationship (r = 0.8448) was found in the analysis of pod
transversal diameter vs. cell number (Fig. [89]2h), not in pod
transversal diameter vs. cell area (r = 0.6834) (Fig. S[90]1b).
Therefore, the pod length and width were mainly determined by the cell
number.
Transcriptome sequencing
The transcriptome sequencing was performed for peanut shell at 10DAP,
15DAP and 20DAP. After removing the low-quality reads, a total of
853,569,042 clean reads were obtained. The percentages of Q30 and GC
were 93.38–94.28% and 44.64–45.23%, respectively, indicating that the
quality of transcriptome sequencing data is high. Gene expression of
Tif and Lps pods during early-growth stage was compared. There were
6888 (Tif10DAP vs. Tif15DAP), 5992 (Tif15DAP vs. Tif20DAP), 11,129
(Lps10DAP vs. Lps15DAP) and 8683 (Lps15DAP vs. Lps20DAP) genes
identified as DEGs (Fig. [91]3a). The results showed that during 10DAP
to 15DAP, there were more DEGs in Tif (Tif10DAP vs. Tif15DAP) and Lps
(Lps10DAP vs. Lps15DAP), and after that, the number of DEGs decreased.
During 10DAP-15DAP, GO enrichment analysis (Table S[92]3) revealed that
DEGs of Tif were mainly enriched in cell wall polysaccharide metabolic
process (GO: 0010383), cell wall macromolecule metabolic process (GO:
0044036) and hemicellulose metabolic process (GO: 0010410). For Lps,
DNA packaging complex (GO: 0044815), protein-DNA complex (GO: 0032993)
and nucleosome (GO: 0000786) were the main enriched terms. During
15DAP-20DAP, the term response to chitin (GO: 0010200) was the most
enriched in Tif, followed by microtubule binding (GO: 0008017) and
tubulin binding (GO: 0015631). Meanwhile, DEGs of Lps were mainly
enriched in response to chitin (GO: 0010200), trihydroxystilbene
synthase activity (GO: 0050350) and plant-type cell wall (GO: 0009505).
The enrichment analysis of KEGG pathways showed that the DEGs of both
Tif and Lps were significantly enriched in several major metabolic
pathways at each comparison group, including plant hormone signal
transduction, mitogen-activated protein kinase (MAPK) signaling
pathway-plant, starch and sucrose metabolism and phenylpropanoid
biosynthesis (Fig. [93]3b; Table S[94]4).
Fig. 3.
[95]Fig. 3
[96]Open in a new tab
DEGs of Tif and Lps at 10DAP, 15DAP and 20DAP. a Venn diagram of genes
in Tif and Lps in four comparison groups. b KEGG enrichment analysis of
Tif and Lps in four comparison groups. c Venn diagram of genes in Tif
and Lps at three growth stages. d KEGG enrichment analysis of Tif and
Lps at three growth stages
Further analysis showed that there were 5628, 7969 and 7378 genes
identified as DEGs between Tif and Lps at 10DAP, 15DAP and 20DAP,
respectively (Fig. [97]3c). Among them, 1096 genes were overlapped
DEGs. The annotation of overlapping genes showed that DNA replication
was the most significantly enriched pathway (ko03030) and biological
process (GO: 0006260) (Table S[98]4). GO enrichment analysis of all
DEGs in three comparison groups showed that DEGs were mainly enriched
in DNA replication initiation (GO: 0006270), DNA packaging complex (GO:
0044815), protein-DNA complex (GO: 0032993), MCM complex (GO: 0042555),
mitotic cell cycle process (GO: 1,903,047), phenylpropanoid metabolic
process (GO: 0009698), nucleosome (GO: 0000786), trihydroxystilbene
synthase activity (GO: 0050350), microtubule binding (GO: 0008017) and
tubulin binding (GO: 0015631) (Fig. S[99]2a-c). KEGG pathway analysis
divided DEGs into 135, 138, and 136 pathways, respectively (Table
S[100]5). Notably, plant hormone signal transduction was significantly
enriched at three comparison groups, indicating that plant hormones
played an important role in the regulation of peanut pod-growth.
Moreover, MAPK signaling pathway-plant, starch and sucrose metabolism,
and phenylpropanoid biosynthesis were also found to be enriched in
three comparison groups (Fig. [101]3d).
Key DEGs related to phytohormones biosynthesis and signaling pathways during
the rapid -growth stage
Previous studies have shown that the biosynthesis process and
signal-mediated transduction of auxin, CK and GA are related to cell
division and cell expansion [[102]32], thus affecting the size of plant
fruit. The KEGG analysis presented in this study provides evidence for
a significant change in the gene expression of auxin, CK and GA both in
biosynthesis process and signal-mediated transduction pathway.
Therefore, we focused on the DEGs involved in biosynthesis and signal
transduction of these phytohormones.
Auxin is a well-known phytohormone that has a strong effect on cell
enlargement and plant growth. There were 46 (25 up- and 21
down-regulated), 76 (35 up- and 41 down-regulated) and 63 (32 up- and
31 down-regulated) DEGs between Tif and Lps at 10DAP, 15DAP and 20DAP,
respectively, participating in the auxin biosynthetic process, auxin
-mediated signalling pathway and response to auxin (Table S[103]6).
Both TAA1 and TDC had positive regulatory effects on auxin
biosynthesis. Gene-LOC112801162 (TAA1) and gene-LOC112727890 (TDC) were
found to be up-regulated at 10DAP in Lps. The genes that participated
in auxin-mediated signalling, namely, GH3 (auxin responsive GH3 gene
family: gene-LOC112737581) and ARF (auxin response factor:
gene-LOC112743715) were found to be differentially expressed at all
stages. In Lps, the expression of gene-LOC112737581 (GH3) was
up-regulated at 10DAP and down-regulated at 15DAP and 20DAP, and
gene-LOC112743715 (ARF) was up-regulated at all stages. In addition,
two ARFs (gene-LOC112712603, gene-LOC112728970) were found to be
significantly up-regulated at 10DAP and gene-LOC112803082 (ARF) was
up-regulated at 15DAP (Fig. [104]4a).
Fig. 4.
[105]Fig. 4
[106]Open in a new tab
Expression profiles of the DEGs involved in biosynthesis and signaling
transduction pathway of auxin, CK and GA. a Heatmap of DEGs related to
the auxin biosynthesis and signaling pathway. b Heatmap of DEGs related
to the CK biosynthesis and signaling pathway. c Heatmap of DEGs related
to the GA biosynthesis and signaling pathway
There were 16 (9 up- and 7 down-regulated), 33 (14 up- and 19
down-regulated) and 30 (9 up- and 21 down-regulated) DEGs participating
in the biosynthesis and signal transduction of CK, respectively (Table
S[107]6). Isopentenyltransferases (IPTs) are responsible for the bulk
of CK biosynthesis [[108]33]. Gene-LOC112741152 and gene-LOC112795845
were two DEGs encoding IPTs, both of which were significantly
up-regulated in Lps at 10DAP. Another DEG: gene-LOC112705443 (IPT) was
significantly up-regulated at 15DAP. There are two types of response
regulators (ARRs) in CK signaling pathway. Positive regulation of CK
response by type B ARR protein. Gene-LOC112771796 (B-ARR) and
gene-LOC112712721 (B-ARR) were up-regulated in Lps compared with Tif at
10DAP (Fig. [109]4b).
GA plays an important role in promoting cell division and elongation
[[110]34]. For the biosynthesis and signal transduction of GA, 43(25
up- and 18 down-regulated), 59(22 up- and 37 down-regulated) and 46(24
up- and 22 down-regulated) DEGs were involved in (Table S[111]6).
ent-kaurenoic acid oxidase (KAO), GA3-oxidase (GA3ox), GA 20-oxidase
(GA20ox) and GA 2-oxidase (GA2ox) are key enzymes in GA biosynthesis.
The results showed that gene-LOC112756333 (KAO), gene-LOC112695926
(KAO), gene-LOC112706011 (GA20ox), gene-LOC112711704 (GA20ox),
gene-LOC112709954 (GA3ox) and gene-LOC112727565 (GA3ox) were all
up-regulated in Lps at 10DAP (Fig. [112]4c).
Quantitative analysis of phytohormones during early-growth stage of pods.
The LC–MS/MS absolute quantification analysis of these phytohormones
was performed since there were many DEGs in the biosynthesis of auxin,
CK and GA. The contents of 26 auxins, 36 CKs and 12 GAs were detected
during the early-growth stage (Table S[113]7). Results suggested that
the phytohormones contents of Tif and Lps were higher during the
rapid-growth stage of peanut pods, and then decreased rapidly, which
was consistent with the expression trend of related genes (Fig. [114]4,
Table S[115]8). We further compared the differences in the contents of
IAA, tZ (Trans zeatin) and GA3 between Tif and Lps. At 10DAP, the
content of IAA in Lps was significantly increased with a fold change
(Lps/Tif) of 1.61 and a P-value of 0.0035. No significant difference in
IAA contents between Lps and Tif at 15DAP (Fig. [116]5a). However, the
content of IAA in Lps was significantly decreased with a fold change
(Lps/Tif) of 0.61 and a P-value of 0.0017 at 20DAP. The tZ contents of
Lps was significantly higher than that of Tif during the early -growth
stage. The fold changes (Lps/Tif) of 10DAP, 15DAP and 20DAP were 1.22,
2.01 and 1.21, respectively (Fig. [117]5b). The contents of GA3 in Lps
increased significantly during rapid-growth stage of pod with fold
changes (Lps/Tif) of 2.48 and 2.04, respectively. At 20DAP, compared
with Tif, the GA3 content of Lps was decreased, but no significant
difference (Fig. [118]5c).
Fig. 5.
[119]Fig. 5
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Auxin, CK and GA contents of Tif and Lps pods at 10DAP, 15DAP and
20DAP. a Indole-3-acetic acid (IAA) content. b trans-zeatin (tZ)
content. c GA3 content. Error bar is SD. ** p < 0.01.* p < 0.05
Analysis of DEGs participating in cell division
According to the cytological observation results, we assumed that the
larger pod size of Lps was associated with cell division. Hence, the
DEGs related to cell division were investigated. The results showed
that there were 57 (45 up- and 12 down-regulated), 146 (8 up- and 138
down-regulated) and 118 (9 up- and 109 down-regulated) DEGs at 10DAP,
15DAP and 20DAP, respectively (Table S[121]9). Since cell division
mainly occurs during the rapid-growth stage, we focus on the DEGs
significantly up-regulated in Lps during this period. D-type cyclins
are conserved in plants, known as sensors for growth conditions and
trigger the G1/ S transition [[122]35]. In the present study,
gene-LOC112747313 (log2FoldChange = 2.6617) encoding cyclin-D4 was
significantly up-regulated at 10DAP. At 15DAP, gene-LOC112754661 was
most significantly up-regulated. Likewise, gene-LOC112716260 (Cell
division control protein) and three genes encoding dynamin-related
protein (gene-LOC112730699, gene-LOC112776526 and gene-LOC112733381)
were found to be up-regulated significantly (Table [123]1).
Table 1.
The top 5 significantly up-regulated DEGs in Lps at rapid-growth stage
Group Gene ID Log2FC p-value Description
Tif10DAP vs. Lps10DAP gene-LOC112747313 2.66 1.50E-03 Cyclin-D4
gene-LOC112695821 1.98 2.16E-07 Wee1-like protein kinase
gene-LOC112712603 1.83 2.42E-04 Auxin response factor
gene-LOC112721292 1.80 1.92E-08 Targeting protein for Xklp2
gene-LOC112766595 1.67 2.54E-04 Cell division control protein 6
Tif15DAP vs. Lps15DAP gene-LOC112754661 1.21 5.26E-12 Signal
recognition particle receptor
gene-LOC112716260 1.11 2.20E-07 Cell division control protein 48
gene-LOC112730699 1.26 2.78E-11 Dynamin-related protein
gene-LOC112776526 1.26 2.28E-09 Dynamin-related protein
gene-LOC112733381 1.06 2.30E-06 Dynamin-related protein
[124]Open in a new tab
Validation of candidate DEGs by qRT-PCR analysis
Five, five, and six genes involved in the biosynthesis and signal
transduction of auxin, CK and GA and three genes related to cell
division were selected, respectively, and qRT-PCR was used to analyze
their expression to verify the transcriptome data sets from RNA-Seq.
The qRT-PCR results for these 19 genes were in close agreement with the
corresponding relative transcript abundances obtained from RNA-Seq
(Fig. [125]6), validating the reliability of RNA-seq results.
Fig. 6.
[126]Fig. 6
[127]Open in a new tab
qRT-PCR verification of DEGs between Tif and Lps. The left y-axis shows
the relative expression levels analyzed by qRT-PCR and the right y-axis
shows the RPKM value analyzed by RNA-seq. Data represent the mean of
three replicates ± SD
Discussion
The development of peanut pods affects the final yield. In this
process, the peanut shell develops first and acts as a protective and
perceived organ to ensure the normal development of seeds [[128]36].
Larger pods (shells) are the basis for obtaining larger seeds. However,
the development of pods and seeds is not synchronized, and large pods
do not necessarily obtain large seeds. For example, Ca^2+ deficiency
can cause empty pods at the seed-filling stage [[129]37]. Thus, the
relationship between pod size and shelling percentage should be
synthetically considered in breeding efforts. Peanut forms pegs after
fertilization and the pod-development process is triggered only if
elongating pegs penetrate into the soil [[130]38]. In order to explore
the developmental pattern of peanut pods, we analyzed the length and
the width of Tif and Lps pods during five stages after pegging.
According to the logistic growth-function, the pod expansion could be
divided into three stages. The first stage was the gradual-growth stage
of the pod. During this period, the sizes of pods increased slowly.
Subsequently, the pod size increased rapidly and the pod entered the
rapid-growth stage (10DAP-15DAP). The next is the slow-growth stage,
when the pod size increased slowly and reached its final size. Previous
studies showed that the final size of plant fruit is determined during
early growth stage [[131]39, [132]40]. In this study, our findings
suggested that the final size of peanut pod may be determined during
rapid-growth stage.
The fruit size is mostly determined by cell number rather than cell
size [[133]41, [134]42]. The anatomical structure of Tif and Lps pods
was compared first in our study. In agreement with previous studies,
our results suggested that the cell-number was the critical factor
causing the difference in pod sizes between Tif and Lps. However, some
previous studies on the peanut pod size showed that the cell-area was
the main factor affecting pod size [[135]3, [136]29]. On one hand, this
could be due to the fact that the cellular basis of pods differs
amongst peanut varieties. On the other hand, the development-pattern of
pod cells during rapid-growth stage (especially 15DAP) was not
investigated in the studies mentioned above. During the fruit
development process in plant, the cell division occurs first. Once the
cell division is completed, the cell expansion begins [[137]43]. The
cell-number of Tif and Lps pods increased rapidly from 10 to 15DAP and
slowed down after 15DAP. In the aspect of cell-area, there was no
significant increase until 15DAP, but increased significantly
thereafter 15DAP. All these results suggested that the cell-number
increase was the main developmental events during the rapid-growth
stage of peanut pods. After the rapid-growth stage the cell number no
longer increased, but the cell area began expanding. Therefore, the
peanut pod-size was determined by the cell number during the
rapid-growth stage.
To probe the molecular mechanisms underlying the determination of
peanut pod sizes, we performed transcriptional profiling of peanut pods
during early-growth stage. The result showed that the number of DEGs
between Tif and Lps was increased significantly during the rapid-growth
stage. KEGG pathway enrichment analysis revealed that many of these
DEGs were enriched in starch and sucrose metabolism and plant hormone
signal transduction, etc. Starch and sucrose metabolism are energy
sources for plant-growth [[138]44, [139]45]. SUS catalyzes reversible
reaction to decompose sucrose into UDPG [[140]46], thus providing
substrate for cellulose biosynthesis [[141]47]. Moreover, GAUT is an
enzyme promoting pectin synthesis [[142]48, [143]49]. In this study,
most of SUSs and GAUTs in Lps exhibited higher gene expression levels
at 10DAP (Fig. [144]6). This could lead to accumulate more of cellulose
and pectin during the rapid-growth stage in Lps. Plant cell division
requires coordinated synthesis and deposition of new walls between two
daughter cells [[145]50]. It is well known that cellulose and pectin
are the major components of plant cell walls. Hence, the up-regulation
of these genes was related to more cells in Lps during rapid-growth
stage.
Phytohormones, including auxin, CK and GA, play an important role
during the early-growth stage of peanut pods [[146]28]. These
phytohormones affect pod size mainly by regulating cell division and
expansion. In this study, we found that the cell-number in the
rapid-growth stage was the main factor determining peanut pod sizes.
Meanwhile, this critical period was also the stage where the
phytohormones content of pods was the highest. Therefore, we speculated
that the difference in phytohormones content during this period was the
main reason for the difference in the cell-number of Tif and Lps.
Tryptophan is an important precursor for auxin biosynthesis [[147]51].
Tryptophan is first converted by the TAA family of amino transferases
to indole-3-pyruvic acid (IPA), and then IAA is produced from the IPA
by the YUC family of flavin monooxygenases [[148]52–[149]54]. In
Arabidopsis, the research has shown that auxin levels can be regulated
by modulation of TAA1 gene transcription [[150]55]. In this study, the
expression pattern of gene-LOC112801162 (TAA1) was consistent with the
changing trend of IAA content, indicating that the differentially
expressed of TAA1 resulted in the different content of IAA between Tif
and Lps. ARF and GH3 play downstream roles in IAA signaling pathway and
are responsible for plant growth [[151]34]. Previous studies suggested
that auxin regulates seed size mainly through auxin response factors
(ARFs) [[152]56]. Meanwhile, GH3 is regulated by ARF [[153]57] and
participates in tissue or organ development in leguminous plants
[[154]58]. Luo et al. identified 63 AhARF genes from an allotetraploid
peanut cultivar, of which AhARF14/26/45 were significantly associated
with root development [[155]59]. In Lps, two ARFs (gene-LOC112712603
and gene-LOC112728970) were significantly up-regulated at 10DAP, and
gene-LOC112743715 (ARF) was up-regulated at all stages. Furthermore,
gene-LOC112737581 (GH3) was up-regulated at 10DAP (Fig. [156]7). These
DEGs were predicted to be important genes involved in peanut
pod-growth.
Fig. 7.
[157]Fig. 7
[158]Open in a new tab
Phytohormones biosynthesis and signal transduction pathways.
Biosynthesis and signal transduction are represented by blue and green
lines, respectively. The solid arrow indicates a direct step, and the
broken arrow indicates an indirect step. FPKM values of the genes were
Z-score standardized. For genes, the key is located at right side with
FPKM values increasing from skyblue to red. For phytohormones, the
color scale indicates low (darkblue) to high (yellow) content
The ability of CK to promote cell division was first discovered more
than sixty years ago [[159]60]. Trans-zeatin (tZ) is the main active
form of CK in most plants [[160]61]. Isopentenyl transferases (IPTs)
are involved in the first step in CK biosynthesis by catalyzing
isopentenyl transfer from dimethylallyl diphosphate to adenine
nucleotides [[161]62]. In peanut, overexpression of the IPT gene
improves drought tolerance and increases yield [[162]63]. In this
study, two IPTs (gene-LOC112741152, gene-LOC112795845) were
significantly up-regulated in Lps at 10DAP, which led to more
accumulation of CK in Lps. There are two types of type-A Arabidopsis
response regulators (ARRs) involved in CK signaling: type-A ARRs and
type-B ARRs [[163]64]. The type-B ARR proteins are activated by changes
in their phosphorylation state, which positively regulates CK response
by activating transcription of their downstream targets [[164]65]. At
present, the genome-wide identification of type-B ARR family members
has not been reported in peanut. In this study, gene-LOC112771796
(B-ARR) and gene-LOC112712721 (B-ARR) were significantly up-regulated
in Lps at 10DAP (Fig. [165]7).The up-regulation of these DEGs
associated with CK was speculated to be responsible for the difference
in the cell-number between Tif and Lps.
GA is involved in various aspects of plant growth and development.
ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox) and GA
3-oxidase (GA3ox) are key enzymes in GA biosynthesis [[166]66]. It has
been reported that the gene NA encode KAO in pea, and the na mutant
showed a GA-deficient dwarf phenotype [[167]67]. In Arabidopsis, GA20ox
regulates plant growth and development by modulating GA levels
[[168]68]. Overexpression of GA20ox can enhance seed size [[169]69].
PsGA3ox1 transgenic plants were reported to have longer pea fruits
[[170]70]. In this study, gene-LOC112756333 (KAO), gene-LOC112706011
(GA20ox), gene-LOC112711704 (GA20ox), gene-LOC112709954 (GA3ox) and
gene-LOC112727565 (GA3ox) were significantly up-regulated in Lps at
10DAP (Fig. [171]7). In a recent study, Wang et al. [[172]29] reported
that GA20ox genes were significantly down-regulated in a peanut mutant
with a small pod, which was consistent with our results. This suggests
that further study of GA20ox genes in peanut is necessary. Overall,
these key DEGs may positively regulate pod size through modulation of
GA biosynthesis.
In the present study, the auxin, CK, and GA contents measured by
LC–MS/MS were consistent with the RNA-Seq analysis results. The
contents of these phytohormones were higher during the rapid -growth
stage and decreased significantly after 15DAP, indicating that the
regulation of phytohormones on peanut pod-growth was mainly during the
rapid-growth stage. In addition, we found that only the tZ contents
were extremely significant different between Tif and Lps at both 10DAP
and 15DAP, which suggested that CK might be a decisive factor
contributing to the difference in peanut pod sizes. In summary, we
proposed a simple model for peanut pod-growth during early stage
regulated by phytohormones (Fig. [173]8). In this model, the difference
in phytohormones levels is due to DEGs associated with phytohormones
biosynthesis. Subsequently, changes in phytohormones levels and
phytohormones signal transduction related DEGs lead to differences in
cell division of peanut pod. Finally, the cell number during the
rapid-growth stage determines the pod size. However, if we want to
construct a comprehensive development-network in peanut pods, we may
also need more studies such as the changes in proteomic and
metabolomic, and genome-wide identification of key gene families (such
as type-B ARR), which is one of our future works.
Fig. 8.
[174]Fig. 8
[175]Open in a new tab
Schematic representation of early-growth stage. Cell division was
mainly carried out from 10 to 15 DAP, and cell expansion was mainly
carried out from 15 to 20DAP. The changes of auxin, CK and GA content
in Lps compared to Tif showed in the figure.↑↑and ↑indicate the
significant increase (p < 0.01) and (p < 0.05), respectively.—indicates
no significant difference between Tif and Lps
Methods
Plant materials
Two peanut varieties with contrasting pod size were used in this study.
The larger pod size line Lps is the backbone parent used for breeding
high yield peanut germplasm in Northern China, showing exciting
potential for breeding purposes in the long-term breeding work. The
smaller pod size variety is Tifrunner (the reference genome of peanut,
hereafter referred as Tif.). Both Tif and Lps were planted in the same
areas (Laixi, Shandong, China). The peg that had not penetrated the
soil of Tif and Lps were tied with colored tags (cotton thread),
respectively. Peg penetrated into the soil on different days were
marked with different colored tags. After marking, the soil was covered
to ensure that the pegs were buried. Subterranean pods were collected
from plants grown in the field at 10, 15, 20, 25 and 30DAP.
Trait measurements and calculations
Pod length and width were measured by Vernier caliper (five biological
replicates for each material). The development pattern of pod length
and width fitted by Logistic growth model [[176]71, [177]72] with
CurveExpert 1.4 software, the formula is:
[MATH: Y=K/(1+ae-bt) :MATH]
(1). In the formula (1): Y is the length or width at any time (cm •
pod^−1); K is the maximum length or width (cm • pod^−1); t is the
number of days after pegging; a and b are undetermined coefficients.
The velocity function of Logistic growth process can be obtained by
calculating the first derivative of formula (1):
[MATH: Vt=dy/dt=Kabe-bt/(1+ae-bt)2 :MATH]
(2). In the formula (2): V (t) is the rate of development; the
following formulas are obtained by the first-order derivation and the
second-order derivation of formula (2) and making it equal to 0:
[MATH: tmax=lna/b :MATH]
;
[MATH: t1=lna-1.317/b :MATH]
;
[MATH: t2=lna+1.317/b :MATH]
. In these formulas: t[max] is the occurrence time of maximum growth
rate; t[1] and t[2] are the start and end time of rapid-growth stage.
t[1] and t[2] divided the process of peanut pods expansion into
gradual-growth stage, rapid-growth stage and slow-growth stage of
length (or width).
Cytological observation and analysis
Peanut pods were collected at 10DAP, 15DAP and 20DAP, and immediately
fixed in formalin-aceto-alcohol (FAA), dehydrated with a graded series
of ethanol (75%, 85%, 90%, 95%, and 2 × 100%), infiltrated with 100%
xylene and embedded in paraffin. Serial 6-μm sections were cut with an
RM2016 microtome (Leica, Shanghai, China), stained with toluidine blue,
and visualized with a Nikon ECLIPSE E100 microscope (Nikon Instruments,
Japan). Image J software was explored to measure cell number and size
of the parenchymal cell of exocarp.
RNA-Seq
We selected 10 representative pods from Tif and Lps for each biological
library construction (three biological replicates for each time point).
RNA-Seq was performed using RNA extracted from peanut pods using
RNAprep Pure Plant Plus Kit (TIANGEN BIOTECH, Beijing, China). To meet
the requirements of RNA library construction, the RNA concentration and
RNA integrity were detected by RNA Nano 6000 Assay Kit of the
Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and Qubit® RNA
Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA),
respectively. Library quality was assessed on the Agilent Bioanalyzer
2100 system (Agilent Technologies, Palo Alto, California, USA). The
cDNA libraries were sequenced on the Illumina sequencing platform by
Metware Biotechnology Co., Ltd. (Wuhan, China). Use fastp (version
0.19.3) to filter the original data, mainly to remove reads with
adapters clean reads were mapped to the reference genome sequence
(Arachis hypogaea cv. Tifrunner)
([178]https://www.peanutbase.org/data/public/Arachis_hypogaea/). All
subsequent analyses are based on clean reads. Use feature Counts v1.6.2
to calculate the gene alignment, and then calculate the FPKM of each
gene based on the gene length. DESeq2 v1.22.1 was used to analyze the
differential expression between the two groups, and the P value was
corrected using the Benjamini & Hochberg method. The corrected P value
and fold change |log2| are used as the threshold for significant
differential expression. To identify differentially expressed genes
(DEGs), a stringent value of |fold change|> 2 and corrected
p-value < 0.05 were used as thresholds. The enrichment analysis is
performed based on the hypergeometric test. For KEGG, the
hypergeometric distribution test is performed with the unit of pathway;
For GO, it is performed based on the GO term.
Quantitative analysis of phytohormones by LC–MS/MS
Peanut pods collected at 10DAP, 15DAP and 20DAP were used to detect the
content of phytohormones. Phytohormones including auxin, CK and GA were
detected by MetWare ([179]http://www.metware.cn/) based on the AB Sciex
QTRAP 6500 LC–MS/MS platform. Each treatment contained three
replicates. Determination by the method described previously [[180]73].
Quantitative real-time PCR (qRT-PCR)
The samples used for qRT-PCR analysis were the same as RNA-seq.
Gene-specific primers for qRT-PCR were shown in Table S[181]1. The
qRT-PCR was conducted using a SYBR Premix Ex Taq™ kit (TaKaRa, Dalian,
China) following the manufacturer’s instructions. The amplification
conditions were as follows: predenaturation at 95 °C for 10 min,
denaturation at 95 °C for 15 s, and annealing and extension at 60 °C
for 30 s. Fluorescence signals were collected during annealing and
extension and the whole process was repeated for 40 cycles. To
determine the relative expression of each gene among different samples,
the 2^−△△Ct method was used along with the internal reference actin
gene to normalize the results.
Statistical analysis
Data was analyzed using Microsoft Excel and plotted using GraphPad
Prism 8.0.2 and OriginPro 2021b software. Statistical analyses were
performed using SPSS 17.0 software (SPSS, Inc.). Cell statistics using
CaseViewer 2.4 and ImageJ software. Circle diagrams and bubble plots
were prepared using OmicShare tools ([182]www.omicshare.com/tools).
Heatmaps were created by R software using “ComplexHeatmap” and
“circlize” package.
Supplementary Information
[183]Additional file 1: Fig. s1.^ (347.4KB, tif)
[184]Additional file 2: Fig. s2.^ (2.7MB, jpg)
[185]12870_2023_4382_MOESM3_ESM.xlsx^ (8.9KB, xlsx)
Additional file 3: Table S1. Gene-specific primers for qRT-PCR.
[186]12870_2023_4382_MOESM4_ESM.xlsx^ (9.1KB, xlsx)
Additional file 4: Table S2. Logistic growth function.
[187]12870_2023_4382_MOESM5_ESM.xlsx^ (2.5MB, xlsx)
Additional file 5: Table S3. GO enrichment analysis.
[188]12870_2023_4382_MOESM6_ESM.xlsx^ (175.9KB, xlsx)
Additional file 6: Table S4. Enrichment analysis of overlapping DEGs.
[189]12870_2023_4382_MOESM7_ESM.xlsx^ (264.2KB, xlsx)
Additional file 7: Table S5. KEGG enrichment analysis.
[190]12870_2023_4382_MOESM8_ESM.xlsx^ (57.6KB, xlsx)
Additional file 8: Table S6. DEGs related to phytohormones.
[191]12870_2023_4382_MOESM9_ESM.xlsx^ (19.8KB, xlsx)
Additional file 9: Table S7. Quantitative analysis of phytohormones.
[192]12870_2023_4382_MOESM10_ESM.xlsx^ (15.9KB, xlsx)
Additional file 10: Table S8. DEGs consistent with the change of
phytohormones.
[193]12870_2023_4382_MOESM11_ESM.xlsx^ (107.4KB, xlsx)
Additional file 11: Table S9. DEGs participating in cell division.
Acknowledgements