Stolon anatomy
Anatomical observation showed that the first node of the stolon (10 cm length) in cultivated strawberry (F. × ananassa Duch.) was extremely small and could be easily missed. When the bract of the first node was peeled off, a tiny bud (Fig. 1A) was observed. In contrast, upon observation of the second node, two different types of buds were observed underneath the bract—one was a plump bud (Fig. 1B), and the other was a leaf cluster mixed with several developing leaf buds (Fig. 1C). Slice observation showed that the buds in the first node stopped growing and stepped into dormancy at an early stage (Fig. 2A). The dormancy of the bud located at the first node could be released only under favorable environmental conditions, and it continued to develop into a new stolon branch (Fig. 2B). The leaf buds, located inside of the bract of the second node, showed a distinct trifoliolate structure (Fig. 2C), and the vascular bundles of the newly formed stolon, which is laterally located on the leaf buds, were connected inward with the primary stolon (Fig. 2D). The structure of the strawberry stolon was observed by cross-sectional anatomy. The tissues, from outside to inside, of the stolon included the epidermal hair and epidermis, thick cortex, cambium, phloem, xylem, and the pith, which are composed of a large number of parenchymatous cells (Fig. 2E). These two lateral buds on the first and second nodes of stolon were inwardly connected with the primary stolon in a similar pattern (Figs. 2F-G). At the base of the second node, which is connected to the terminal strawberry stolon buds, there are numerous adventitious root primordia (Figs. 2H-I). Each adventitious root primordium originated from the cambium tissue, which consists of meristematic cells containing dense cytoplasm and swollen nuclei (Figs. 2E, 2I).
Stereoscopic and SEM observation of developing stolon buds
Dormancy bud in the first node––In order to acquire more details on the developmental characteristics of the first and the second node buds of the strawberry stolon, stolon buds in different developmental phases were observed under the stereomicroscope and scanning electron microscope (SEM), respectively. In the early stage of stolon elongation (when the stolon length was 4–10 cm), the buds on the first node of the stolon grew with the subsequent development of the stolon at the early stage (Figs. 3 A–C). For example, when the stolon was 4 cm in length, a tiny growing point located in the center of a trifoliolate bud could be seen after the outermost bract was peeled off from the first node (Fig. 3A). Continually, along with the growth of stolon (when the stolon length was 6–7 cm), the top trifoliolate leaf bud on the first node developed further, and the growth point at the central base of the buds also grew (Figs. 3B–C). With the further elongation of the primary stolon, when the primary stolon reached a length of 8–9 cm, the original trifoliolate bud gradually developed into a young trifoliolate bract and was densely covered with trichomes (Figs. 3D–E). When this young trifoliolate was peeled off sequentially, another tightly closed thin trifoliolate bud could be seen protecting the underlying growing point (Fig. 3F). This is a landmark at which the bud of the first node in the strawberry stolon ceases to develop and enters into dormancy; under continual observation, when the primary stolon elongated further, this thin trifoliolate bud structure showed no change. Our conclusion was further confirmed under the magnified observation of SEM; the first trifoliolate bud under the bract of the first node continuously develops into a young trifoliate, with the development of the primary stolon occuring at an early stage of stolon development or elongation (Figs. 3G, 3J). Similarly, when this young trifoliolate leaf was peeled off, the structure of the thin, tightly closed trifoliolate bract was visible (Fig. 3J). With all this, the outermost new trifoliolate leaf bud and the inner growing point ceased to develop, and showed no further development while the primary stolon elongated continually, indicating its stepping into a state of dormancy.
Activity shoot bud in the second node––The active stolon bud (ASB) under the outermost bract of the second node of a strawberry stolon is quite different from the DSB located in the first node, showing an active development state throughout the whole process of primary stolon development (Fig. 4). The process of ASB developmental inside the second node also showed that the trifoliolate leaf bud developed first (Figs. 4A–D). When this trifoliolate leaf bud developed into a young trifoliolate (Fig. 4E), the new inner trifoliolate could be seen (Fig. 4F) and, at the same time, a new growth point started to develop near the trifoliolate cluster (Fig. 4F). SEM observation revealed further details of the development of trifoliolate leaf buds, which exhibited a high developmental activity (Figs. 4G–I).
Ramet buds in the second node––Unlike the axillary development of DSBs and ASBs, the developmental process of the strawberry ramet leaf bud (RLB) is relatively simple and rapid (Fig. 5). The apical region of a strawberry stolon contains multiple leaf bud primordia; when one leaf primordium gradually develops into a young trifoliolate, the next leaf primordium is initiated out as a visible developing trifoliolate (Figs. 5A, 5B). Subsequently, each leaf primordium develops into a young leaf, orderly, to form a young leaf cluster (Figs. 5C–F). The stolon branch developing activity is located laterally to these leaf clusters (Figs. 5E, 5F). SEM observation showed that the trifoliolate bract first to grow out is located at the top of each trifoliolate primordium to protect the inner part (Fig. 5G). Each trifoliolate bract was tightly connected with the others in a complementary manner (Fig. 5G) to save the limited growth space. As a result, each connected young trifoliolate in the ramet of the second node is well positioned, and the active stolon bud located adjacent to them (Figs. 5I, 5J).
Protein profiles among different strawberry stolon buds
For searching the regulatory factors involved in modulating the heterogeneity of strawberry stolon buds at the proteomic level, DEPs among different types of buds were assessed by profiling the proteome using the TMT labeling system. The expression profiles of the proteins extracted from DSBs (labeled with 126, 127N, 127C), ASBs (labeled with 128N, 128C, 129N), and RLBs (labeled with 129C, 130N,130C) on a 10-cm long strawberry stolon were analyzed.
LC-MS/MS––A total of 42,737 peptide fragments, of which 26,135 were unique peptides corresponding to a total of 7,271 proteins (Fig. 6A), were assessed by TMT-based LC-MS/MS mass spectrometry identification and a search against the P20180400239_hebing_76764 database (detailed information in the Materials and Mathods) employing MASCOT engine integrated with Proteome Discoverer 1.4 software (supplemental Table S1, S2). A 1.2-fold-change cut-off with P-value<0.05 was used to indicate significant changes in the abundance of DEPs among different strawberry stolon buds (supplemental Fig. S2). By analyzing the quality control data, we found that the TMT results that were achieved by using a high-quality Q Exactive mass spectrometer was reliable. The accuracy and high resolution achieved in our experiment can maintain good quality deviation during the process of data acquisition, and produce high-quality MS1 and MS2 spectrograms. The quality deviation of all identified peptides was mainly within 10 ppm (supplemental Fig. S3), indicating that the identification results were accurate and reliable. When the rigid analyzing tool of MASCOT (FDR<0.01) was used for judging each MS2 spectrogram, we obtained an ideal score with a median of 34.06, and more than 86.21% peptides scored higher than 20 (supplemental Fig. S4). The protein ratio (approximately 1.0) distribution of the three groups (ASB/DSB, RLB/DSB, and RLB/ASB) are shown in supplemental Fig. S5.
Features of identified proteins––The distribution of unique peptides defining each protein is shown in Figure 6B;over 61% of proteins, includied at least two unique peptides (supplemental Table S1). The average molecular mass of the identified gene products ranged from 10 to 70 kDa (Fig. 6C). The t (PI) distribution of the identified proteins was mainly in the range of 5.0–10.0, with most PIs ranging from 6.0 to 7.0 (supplemental Fig. S6). Comparisons between the DSB, ASB, and RLB groups led to the identification of 1,307 ASB/DSB (including 691 up-regulated and 616 down-regulated), 363 RLB/DSB (168 up-regulated and 195 down- regulated) and 626 RLB/ASB (256 up-regulated and 370 down-regulated) DEPs (supplemental Table S3). The variation among the three biological replicates of each group (DSB, ASB, and RLB) was calculated according to their quantitative data, with most proteins exhibiting less than 20% variation among replicates (supplemental Fig. S7), indicating the high quality and repeatability of the data.
For exhibiting the DEPs among each group in detail, we identified top 10 up-regulated and top 10 down-regulated DEPs according to their fold changes (supplemental Table S3) [in total of 1,307 in ASB/DSB, 691 up-, 616 down-; 363 in RLB/DSB, 168 up- and 195 down-; and 626 in RLB/ASB, 256 up- and 370 down-regulated DEPs]. These top DEPs were selected out and listed in table 1, and by further analyzing, there could be found 5, 1and 6 common top DEPs among (ASB/DSB and RLB/DSB), (RLB/DSB and RLB/ASB) and (ASB/DSB and RLB/ASB), respectively, and additionally with 3 common top DEPs were found in all above three groups (supplemental Table S4). All these 5 common top DEPs between the groups of ASB/DSB and RLB/DSB were down-regulated DEPs, they were [cucumisin-like], [serine carboxypeptidase-like 27], [ornithine decarboxylase-like], [36.4 kDa proline-rich protein], and [ribulose bisphosphate carboxylase small chain, chloroplastic-like protein]. Indicating that all these 5 common top DEPs are highly expressed in DSB when compared with ASB and RLB, and we could speculate that they are mainly involved in stimulating the developmental phases or promoting the dormancy process of the first node of DSB in a strawberry stolon. However, between the groups of RLB/DSB and RLB/ASB, only 1 up-regulated common top DEP of [pentatricopeptide repeat-containing protein] was found.
Between ASB/DSB and RLB/ASB, all 6 common top DEPs showed consistent up-regulated expression in ASB/DSB and down-regulated expression in RLB/ASB. They were [fasciclin-like arabinogalactan protein 12] (double), [glucuronoxylan 4-O-methyltransferase 3-like], [aquaporin TIP2-1], [putative 4-hydroxy-4-methyl-2-oxoglutarate aldolase 2], and [fruit protein pKIWI501-like]. Thus, all these six common DEPs were highly expressed in ASBs, when compared with DSBs and RLBs, suggesting they are highly involved in the ASB developmental process. At last, these 3 common top DEPs in all three groups were [hypothetical protein CARUB_v10021660mg], [blue copper protein-like] and [glycine-rich cell wall structural protein], and also showed the consistent expression modes of ASB/DSB-up, RLB/DSB-up, and RLB/ASB-down. This implied that these 3 common top DEPs have the ability to promote the entry of DSBs into dormancy and to stimulate the development of ASBs at the same time. The other DEPs among the top 10 up- or down-regulated DEPs are specifically expressed in each group (supplemental Table S5).
Table 1. Top 10 of up-regulated and down-regulated differentially expressed proteins between groups
Groups
|
No.
|
Protein ID
|
Description
|
Coverage
|
Fold change
|
P-value
|
ASB/DSB
|
up-1
|
XP_004304418.1
|
Fasciclin-like arabinogalactan protein 12
|
20.73
|
6.23
|
0.00186255
|
up-2
|
FANhyb_rscf00000173.1.g00014.1
|
hypothetical protein CARUB_v10021660mg
|
22.06
|
5.29
|
0.02442000
|
up-3
|
FANhyb_rscf00000386.1.g00003.1
|
fasciclin-like arabinogalactan protein 12
|
20.73
|
4.78
|
1.3548E-07
|
up-4
|
XP_011462288.1
|
glycine-rich cell wall structural protein
|
50.36
|
4.64
|
0.015453
|
up-5
|
XP_004299681.1
|
blue copper protein-like
|
14.21
|
4.52
|
0.004586
|
up-6
|
FANhyb_rscf00004026.1.g00001.1
|
glucuronoxylan 4-O-methyltransferase 3-like
|
7.360
|
3.80
|
0.00520136
|
up-7
|
FANhyb_icon00011962_a.1.g00001.1
|
fructokinase-5
|
38.28
|
3.61
|
0.00072744
|
up-8
|
XP_011458504.1
|
anthocyanidin 3-O-glucosyltransferase 7-like
|
19.91
|
3.53
|
0.00100593
|
up-9
|
FANhyb_rscf00000027.1.g00021.1
|
zinc finger protein
|
2.580
|
3.47
|
0.00693375
|
up-10
|
FANhyb_rscf00000295.1.g00005.1
|
histone H2A.1
|
37.84
|
3.43
|
0.00117361
|
down-1
|
XP_004307317.1
|
cucumisin-like
|
7.830
|
0.34
|
0.01617526
|
down-2
|
XP_004307401.1
|
serine carboxypeptidase-like 27
|
11.79
|
0.35
|
0.01705444
|
down-3
|
FANhyb_rscf00000020.1.g00007.1
|
pectinesterase
|
4.170
|
0.37
|
0.03259767
|
down-4
|
XP_004297134.2
|
ornithine decarboxylase-like
|
4.660
|
0.38
|
0.00237870
|
down-5
|
FANhyb_rscf00000011.1.g00007.1
|
36.4 kDa proline-rich protein
|
9.320
|
0.38
|
0.00620498
|
down-6
|
FANhyb_icon00014184_a.1.g00001.1
|
putative laccase-9
|
2.760
|
0.41
|
0.01597461
|
down-7
|
XP_004303137.1
|
ribulose bisphosphate carboxylase small chain, chloroplastic-like
|
31.87
|
0.42
|
0.01480696
|
down-8
|
FANhyb_rscf00001104.1.g00003.1
|
aquaporin TIP2-1
|
7.260
|
0.43
|
0.00599268
|
down-9
|
FANhyb_icon00031506_a.1.g00001.1
|
putative 4-hydroxy-4-methyl-2-oxoglutarate aldolase 2
|
47.83
|
0.45
|
0.00430290
|
down-10
|
XP_004293363.1
|
fruit protein pKIWI501-like
|
50.00
|
0.45
|
0.02295962
|
RLB/DSB
|
up-1
|
XP_004303183.1
|
protein LIGHT-DEPENDENT SHORT HYPOCOTYLS 10-like
|
13.88
|
3.24
|
0.01882948
|
up-2
|
FANhyb_rscf00005475.1.g00002.1
|
pentatricopeptide repeat-containing protein At4g38150-like
|
10.04
|
2.29
|
0.01396803
|
up-3
|
XP_004307632.1
|
flocculation protein FLO11 isoform X3
|
2.470
|
1.76
|
0.00227661
|
up-4
|
FANhyb_rscf00000173.1.g00014.1
|
hypothetical protein CARUB_v10021660mg
|
22.06
|
1.76
|
0.00017183
|
up-5
|
FANhyb_icon00004730_a.1.g00001.1
|
squamosa promoter-binding-like protein 9
|
7.090
|
1.69
|
0.00267638
|
up-6
|
FANhyb_rscf00000264.1.g00010.1
|
uncharacterized protein LOC101290827
|
12.68
|
1.61
|
0.03225374
|
up-7
|
XP_011462288.1
|
glycine-rich cell wall structural protein
|
50.36
|
1.58
|
0.00426962
|
up-8
|
XP_004299681.1
|
blue copper protein-like
|
14.21
|
1.58
|
0.00418736
|
up-9
|
XP_004310149.1
|
mediator-associated protein 2
|
10.27
|
1.57
|
0.04857720
|
up-10
|
FANhyb_rscf00005241.1.g00001.1
|
zinc-finger homeodomain protein 6
|
16.35
|
1.56
|
0.02325629
|
down-1
|
XP_004307317.1
|
cucumisin-like
|
7.830
|
0.41
|
0.02484890
|
down-2
|
XP_004297134.2
|
ornithine decarboxylase-like
|
4.660
|
0.41
|
0.00581982
|
down-3
|
XP_004307401.1
|
serine carboxypeptidase-like 27
|
11.79
|
0.41
|
0.02026585
|
down-4
|
FANhyb_rscf00001190.1.g00001.1
|
telomere repeat-binding factor 1-like
|
9.040
|
0.50
|
0.00752576
|
down-5
|
FANhyb_rscf00000011.1.g00007.1
|
36.4 kDa proline-rich protein
|
9.320
|
0.51
|
0.01674637
|
down-6
|
FANhyb_rscf00000159.1.g00006.1
|
glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic
|
14.16
|
0.54
|
0.01030551
|
down-7
|
FANhyb_rscf00005433.1.g00001.1
|
fasciclin-like arabinogalactan protein 13
|
8.540
|
0.55
|
0.00275099
|
down-8
|
XP_004303137.1
|
ribulose bisphosphate carboxylase small chain, chloroplastic-like
|
31.87
|
0.56
|
0.03678190
|
down-9
|
FANhyb_icon00037348_a.1.g00001.1
|
ribulose bisphosphate carboxylase small chain, chloroplastic-like
|
54.00
|
0.56
|
0.03512944
|
down-10
|
XP_004297630.1
|
carbonic anhydrase 2 isoform X2
|
19.37
|
0.57
|
0.00032382
|
RLB/ASB
|
up-1
|
FANhyb_rscf00000329.1.g00005.1
|
1-aminocyclopropane-1-carboxylate oxidase 1-like
|
16.07
|
2.16
|
0.00162178
|
up-2
|
FANhyb_rscf00001394.1.g00002.1
|
glutamate dehydrogenase 1
|
20.87
|
2.05
|
0.04362181
|
up-3
|
FANhyb_icon00031506_a.1.g00001.1
|
putative 4-hydroxy-4-methyl-2-oxoglutarate aldolase 2
|
47.83
|
2.05
|
0.00751062
|
up-4
|
XP_004293441.1
|
major latex allergen Hev b 5-like
|
68.04
|
2.04
|
0.00379106
|
up-5
|
XP_004293363.1
|
fruit protein pKIWI501-like
|
50.00
|
2.00
|
0.00846049
|
up-6
|
FANhyb_rscf00001104.1.g00003.1
|
aquaporin TIP2-1
|
7.260
|
1.98
|
0.00734669
|
up-7
|
FANhyb_rscf00000704.1.g00001.1
|
putative formamidase C869.04 isoform X1
|
3.690
|
1.98
|
0.01632577
|
up-8
|
FANhyb_rscf00005475.1.g00002.1
|
pentatricopeptide repeat-containing protein At4g38150-like
|
10.04
|
1.89
|
0.02607894
|
up-9
|
FANhyb_rscf00000980.1.g00003.1
|
glutathione S-transferase F13-like
|
43.45
|
1.81
|
0.00588200
|
up-10
|
XP_004291285.2
|
probable sarcosine oxidase
|
8.690
|
1.80
|
0.00202212
|
down-1
|
XP_004304418.1
|
fasciclin-like arabinogalactan protein 12
|
20.73
|
0.23
|
0.00070797
|
down-2
|
FANhyb_rscf00000386.1.g00003.1
|
fasciclin-like arabinogalactan protein 12
|
20.73
|
0.25
|
0.00001714
|
down-3
|
FANhyb_rscf00004026.1.g00001.1
|
glucuronoxylan 4-O-methyltransferase 3-like
|
7.360
|
0.29
|
0.00706772
|
down-4
|
XP_011457444.1
|
F-box protein SKIP14
|
2.380
|
0.30
|
0.00827485
|
down-5
|
FANhyb_icon00004976_a.1.g00001.1
|
cytochrome P450 84A1
|
28.68
|
0.31
|
7.5325E-07
|
down-6
|
XP_004294339.1
|
probable galacturonosyltransferase 12
|
3.000
|
0.31
|
0.01158193
|
down-7
|
FANhyb_rscf00000173.1.g00014.1
|
|hypothetical protein CARUB_v10021660mg
|
22.06
|
0.33
|
0.04426256
|
down-8
|
XP_011462288.1
|
glycine-rich cell wall structural protein
|
50.36
|
0.34
|
0.02679911
|
down-9
|
XP_004299681.1
|
blue copper protein-like
|
14.21
|
0.35
|
0.03378082
|
down-10
|
FANhyb_rscf00002637.1.g00001.1
|
L-type lectin-domain containing receptor kinase IX.1-like
|
2.040
|
0.35
|
0.03378082
|
Additional large-scale analysis of DEPs between groups showing co-up regulation and co-down regulation was also carried out, as shown in the Venn diagram in Figure 7 (detailed information in supplemental Table S6, S7, S8). We found that no DEP showed co-up regulation; only one DEP of GDSL esterase/lipase showed co-down regulation among all three groups (Fig. 7A, 7B). When the total number of co-up regulated and co-down regulated proteins was counted, 45 DEPs common to all three groups were found (Fig. 7C). Among all statistics, one group of data showed special performance, that is, ASB/DSB and RLB/ASB have almost no co-up or co-down proteins (Fig. 7A, 7B) separately, but when we calculated the total co-up and co-down DEPs, 407 common DEPs could be found between groups of ASB/DSB and RLB/ASB, simultaneously (Fig. 7C). These results indicated that each of the 407 DEPs shared common down-regulated or up-regulated expression patterns in the groups of DSB and RLB when compared with ASB. This suggests that all these proteins have dual functions in stimulating the RLB formation while promoting the DSBs stepping into dormancy; however, they also have an antagonistic effect on the ASB development in strawberry stolons, simultaneously. Thus, exploring the regulatory mechanisms of these 407 DEPs is of great significance to clarify the dormancy of first node DSBs and the formation of the second node RLBs.
Bioinformatics analysis
All DEPs detected by MS were subjected to a bioinformatics analysis for further classification.
Cluster Analysis––The hierarchical clustering results were expressed as a respective heat map (Fig. 8). The X- and Y-coordinates represented sample and differentially expressed proteins, respectively. As determined by a horizontal comparison, the samples could be classified into three categories: DSB, ASB, and RLB. Such a classification was associated with high accuracy, suggesting that the selected DEPs could effectively distinguish between samples. Furthermore, a vertical comparison indicated that the selected proteins could be classified into two categories with opposite directional variation, which displayed the expression patterns of DEPs in three groups (supplemental Fig. S8), demonstrating the rationality of the selected DEPs. The cluster analysis, thus, supported that the DEPs screened through this experiment were accurate.
GO Functional Annotation and Analysis––The DEPs (1,307, 363, and 626) between ASB and DSB, RLB and DSB, and RLB and ASB groups corresponded to 1,931, 1,194, and 1,276 functional annotations, respectively (supplemental Table S9-11). The DEPs were individually analyzed against the Gene Ontology (GO) database using three sets of ontologies: biological process, molecular function, and cellular component (Fig. 9). The analysis showed that among ASB/DSB, RLB/DSB, and RLB/ASB, numerous DEPs could be classified in the same GO category (supplemental Table S9-11). The top two common biological process categories were metabolic process (over 35%) and cellular process (over 25%). The top four common molecular function categories were catalytic activity (over 35%), binding (over 25%), transporter activity, and structural molecule activity. The top four common cellular component categories were cell (over 25%), cell part (over 25%), organelle (over 15%), and membrane proteins (over 15%). A small number of other DEPs existed in cellular component categories, including membrane part, organelle part, and macromolecular complex, with the ratio of approximately 10%. For further exhibition of the top 20 enriched GO terms (supplemental Fig. S9), we know detailed information of functional proteins in biological process (BP) of ASB/DSB were oxidation-reduction process (~100 DEPs) and regulation of RNA metabolic process (supplemental Fig. S9A), in RLB/DSB, they were DNA metabolic process and photosynthesis, with the same number of DEPs (14), as well as DNA conformation change and replication (supplemental Fig. S9B), whereas for RLB/ASB (supplemental Fig. S9C), there were small numbers of DEPs, which were classified into the secondary metabolic process, one-carbon metabolic process, secondary metabolite biosynthetic process, and phenylpropanoid metabolic process.
Molecular function (MF) analysis showed that the GO terms in ASB/DSB were catalytic activity (~450 DEPs) and oxidoreductase activity (over 100 DEPs), and DNA binding (supplemental Fig. S9A). In RLB/DSB, the MF proteins were DNA binding and helicase activity (supplemental Fig. S9B). In the RLB/ASB MF GO terms, a large numbers of DEPs belonged to catalytic activity (~250), oxidoreductase activity (~60), and protein dimerization activity (supplemental Fig. S9C).
The cellular component (CC) terms in ASB/DSB were thylakoid and thylakoid part and chromatin. Combining the GO terms identified in the MF and BP analysis above, we found that the differences existed mainly at the RNA level (supplemental Fig. S9A). Between the groups of RLB and DSB, the GO terms were thylakoid (22), thylakoid part (16), plastid thylakoid (14), chloroplast thylakoid (14), photosynthetic membrane (14), and thylakoid membrane (12) (supplemental Fig. S9B). This suggested that the differences between RLB and DSB mainly occurred in terms of their capacity for photosynthesis. In RLB/ASB, all DEPs of CC were functional compartments of chromosome- or DNA-related proteins, and showed a low coincidence trend (supplemental Fig. S9C).
KEGG Pathway Analysis—By searching the major biological pathways and relevant regulatory processes involved in the Kyoto Encyclopedia of Genes and Genomes (KEGG), we analyzed all DEPs among groups (Fig. 10). The results indicated that the spliceosome (43 DEPs, as shown below) and ribosome (29) had high enrichment between ASB and DSB (Fig. 10A, supplemental Table S12). This suggested that the differences in transcription or translation are the fundamental reason for the difference between ASB and DSB. As for RLB/DSB (Fig. 10B, supplemental Table S13), the photosynthesis (13) pathway had the highest enrichment of DEPs. This is an additional proof for the fact that the main RLB function is photosynthesis for the next clonal generation of ramets. In addition, two highly enriched pathways (DNA replication and spliceosome) were still present indicating that both genetic and transcriptional differences exist between RLB and DSB. The participation of DEPs in the phenylpropanoid biosynthesis pathway (18), as well as in carbon metabolism related pathways, such as starch and sucrose metabolism (15), amino sugar and nucleotide sugar metabolism (14), and glycolysis/gluconeogenesis (11), showed high enrichment between the groups RLB and ASB (Fig. 10C, supplemental Table S14). This indicated that phenylpropaniod biosynthesis caused differences differentiation between RLBs and ASBs in the second node of strawberry stolon, especially with respect to the formation of vessels during the ASB developmental processes, as discussed below.
Protein-Protein Interaction (PPI) Analysis––We used the PPI database and relevant literature to confirm the interactions of the identified proteins or DEPs, as well as of other proteins that interacted directly with them. The PPI network (supplemental Table S15), expressed as nodes and links, contributed to extracting effective protein information from various points of view and obtaining comprehensive information that could not be obtained through the analysis of only a single protein (Fig. 11). According to the analysis, 20 high-connectivity degree DEPs, with a degree value of more than 30, were identified between the groups ASB and DSB (Table 2). 6 and 11 DEPs, with connectivity degrees higher than 10, were identified from the RLB/DSB and RLB/ASB groups, respectively (Table 2). The results obtained were highly consistent with those obtained using KEGG, indicating that the difference between ASB and DSB was mainly due to the differences at the transcriptional level, while the difference between RLB and DSB was mainly due to the differences at the genetic level. For further validating direct protein-protein interactions, we selected four typical DEPs, namely NADH-GOGAT & GDH, PK, MCM 2–4, and 6–7, as the PPI core (Fig. 11A–C). In additional, we drew the PPIs in the phenylpropanoid biosynthesis pathway (Fig. 11D) to further clarify the key protein-protein interactions.
Table 2 DEPs with high connectivity degree in PPI analysis between groups
Group
|
Protein accession
|
Degree
|
Description
|
Unique peptide
|
Fold Change
|
ASB/DSB
|
XP_004304484.1
|
42
|
splicing factor 3A subunit 2 isoform X1
|
5
|
1.29
|
FANhyb_rscf00000157.1.g00010.1
|
37
|
60S ribosomal protein L7a-2
|
6
|
1.24
|
FANhyb_rscf00000522.1.g00007.1
|
37
|
small nuclear ribonucleoprotein-associated protein B & apos
|
3
|
1.31
|
XP_004290666.1
|
36
|
small nuclear ribonucleoprotein Sm D1-like
|
4
|
1.28
|
XP_004303828.1
|
35
|
60S ribosomal protein L7-2
|
2
|
1.53
|
XP_004287907.1
|
34
|
U1 small nuclear ribonucleoprotein 70 kDa
|
10
|
1.36
|
XP_004308170.1
|
33
|
40S ribosomal protein S20-2
|
6
|
1.28
|
XP_004299660.1
|
33
|
40S ribosomal protein S6
|
8
|
1.25
|
FANhyb_rscf00005750.1.g00001.1
|
33
|
30S ribosomal protein S17, chloroplastic-like
|
2
|
1.27
|
XP_004307020.1
|
33
|
pyruvate kinase, cytosolic isozyme-like
|
16
|
0.83
|
XP_004304484.1
|
42
|
splicing factor 3A subunit 2 isoform X1
|
5
|
1.29
|
XP_004297699.1
|
33
|
pyruvate kinase, cytosolic isozyme-like
|
18
|
0.82
|
FANhyb_icon14422311_s.1.g00001.1
|
32
|
60S ribosomal protein L27a-3
|
2
|
1.28
|
XP_004296676.1
|
32
|
60S ribosomal protein L27a-3
|
2
|
1.24
|
XP_004306930.1
|
32
|
40S ribosomal protein S13
|
1
|
1.62
|
FANhyb_rscf00000738.1.g00007.1
|
32
|
serine/arginine-rich splicing factor RS31-like isoform X1
|
7
|
1.50
|
XP_004306844.1
|
32
|
serine/arginine-rich splicing factor RS41-like isoform X1
|
5
|
1.50
|
XP_011470401.1
|
32
|
serine/arginine-rich splicing factor RS41 isoform X1
|
5
|
1.29
|
XP_004300041.1
|
31
|
uncharacterized RNA-binding protein C1827.05c
|
3
|
1.26
|
XP_004299114.1
|
31
|
SART-1 family protein DOT2
|
12
|
1.26
|
XP_004297699.1
|
33
|
pyruvate kinase, cytosolic isozyme-like
|
18
|
0.82
|
RLB/DSB
|
FANhyb_icon00002169_a.1.g00001.1
|
19
|
LOW QUALITY PROTEIN: replication factor C subunit 1
|
1
|
1.37
|
XP_011458268.1
|
12
|
DNA replication licensing factor MCM4
|
18
|
1.22
|
XP_004299199.1
|
12
|
protein LIGHT-DEPENDENT SHORT HYPOCOTYLS 10-like
|
1
|
3.24
|
FANhyb_icon00004233_a.1.g00001.1
|
12
|
structural maintenance of chromosomes protein 2-1-like
|
1
|
1.46
|
XP_011460181.1
|
11
|
DNA replication licensing factor MCM3
|
2
|
1.26
|
XP_004300454.1
|
11
|
DNA replication licensing factor MCM7
|
18
|
1.27
|
RLB/ASB
|
FANhyb_rscf00000086.1.g00005.1
|
16
|
glutamate synthase 1 [NADH], chloroplastic isoform X1
|
20
|
0.74
|
XP_004299600.1
|
15
|
acetyl-CoA carboxylase 1-like isoform X1
|
29
|
0.65
|
XP_004297699.1
|
14
|
pyruvate kinase, cytosolic isozyme-like
|
14
|
1.23
|
FANhyb_icon00042639_a.1.g00001.1
|
12
|
hypothetical protein B456_008G262000
|
1
|
0.75
|
FANhyb_rscf00005750.1.g00001.1
|
12
|
30S ribosomal protein S17, chloroplastic-like
|
2
|
0.77
|
XP_004308170.1
|
12
|
40S ribosomal protein S20-2
|
6
|
0.81
|
FANhyb_rscf00001394.1.g00002.1
|
12
|
glutamate dehydrogenase 1
|
5
|
2.05
|
XP_004287377.1
|
12
|
probable histone H2A variant 3
|
2
|
0.54
|
XP_004309949.1
|
12
|
4-coumarate-CoA ligase 2-like
|
4
|
0.50
|
FANhyb_rscf00001010.1.g00003.1
|
11
|
40S ribosomal protein S24-1-like
|
1
|
0.80
|
FANhyb_rscf00000697.1.g00007.1
|
11
|
40S ribosomal protein S24-1
|
1
|
0.69
|
Parallel Reaction Monitoring (PRM) verification
To further verify the results of MS, three DEPs (Pyruvate Kinase (PK), MCM2, and PAL1) were selected for PRM analysis (Fig. 12). The screening criteria were formulated based on the following two principles: 1) proteins with potential biological functions and peptide fragments greater than 1, as identified by LC-MS/MS; and 2) proteins that were specifically expressed in one group of buds when compared with the other two groups of buds and have not been reported yet.
The results of the LC-PRM/MS analysis performed on 12 peptide fragments of three target proteins from three groups of strawberry samples showed that the quantitative information of target peptide fragments could be obtained for all nine samples. Subsequently, the relative quantitative analysis was carried out on target peptide fragments and proteins through the incorporation of heavy isotope-labeled peptide fragments. The results indicated that of the three target proteins, the expression quantities of PK and PAL1 in the ASB group was markedly up-regulated compared with that in the DSB and RLB groups; whereas the expression quantity of MCM2 in the RLB group was significantly up-regulated compared with that in the DSB and ASB groups; these results verified the accuracy of the TMT method for this study.