Differential abundance proteins associated with rapid growth of etiolated coleoptiles in maize

Since Charles Darwin discovered growth movements of the shoot tip (coleoptile) toward light, the grass coleoptiles have long been used as a model system to study plant growth. Rapid growth of the coleoptile is vital for successful seed germination and early seedling establishment. However, the proteome changes underlying the rapid growth of the coleoptiles are not yet clear in the model plant maize ( Zea mays ). In the present study, we investigated proteome changes in vivo occurring in the rapidly growing coleoptiles of maize with two- dimensional gel electrophoresis combined with mass spectrometry. A quantitative comparison of the proteomes at 1.5, 3, and 5 days after germination showed significant changes in protein profiles with coleoptile growth. As a result, 31 differential abundance proteins (DAPs) representing 44 protein spots were identified, of which 21 DAPs with increased abundances were implied in growth- related processes, including translational initiation, transcription regulation, protein and other compound synthesis, H + - transmembrane transport and cytoskeleton organization. The selected DAPs were confirmed by reverse transcrip tion quantitative PCR and immunoblot analysis. We suggested that the rapid growth of the coleoptile is largely due to its ability to quickly enhance relevant cellular pro -cesses, especially the increased synthesis of the growth- related DAPs. The content of indole- 3- acetic acid, salicylic acid, and jasmonic acid decreased significantly, and the content of gibberellins first decreased and then increased during the elongation of coleoptile. This study provides new insight into the significance of proteome changes in coleoptile growth.

In the present study, we investigated proteome changes in vivo occurring in the rapidly growing coleoptiles of maize with two-dimensional gel electrophoresis combined with mass spectrometry. A quantitative comparison of the proteomes at 1.5, 3, and 5 days after germination showed significant changes in protein profiles with coleoptile growth. As a result, 31 differential abundance proteins (DAPs) representing 44 protein spots were identified, of which 21 DAPs with increased abundances were implied in growth-related processes, including translational initiation, transcription regulation, protein and other compound synthesis, H + -transmembrane transport and cytoskeleton organization. The selected DAPs were confirmed by reverse transcription quantitative PCR and immunoblot analysis. We suggested that the rapid growth of the coleoptile is largely due to its ability to quickly enhance relevant cellular processes, especially the increased synthesis of the growth-related DAPs. The content of indole-3-acetic acid, salicylic acid, and jasmonic acid decreased significantly, and the content of gibberellins first decreased and then increased during the elongation of coleoptile. This study provides new insight into the significance of proteome changes in coleoptile growth.

K E Y W O R D S
coleoptile, gel electrophoresis, mass spectrometry, organ growth, proteome change, Zea mays The coleoptile length substantially contributes to deep-sowing tolerance in cereal plants under drought or submerged conditions (Liu et al. 2017;Magneschi & Perata, 2009;Mohan et al. 2013).
Therefore, the coleoptile growth is often targeted for selecting deep-sowing tolerant cultivars.
Several hypotheses have been proposed to explain the dynamics of auxin-induced growth in plants, notably the acid-growth theory (Arsuffi & Braybrook, 2018;Rayle & Cleland, 1992), based on the evidence mostly coming from in vitro experiments using coleoptile/ hypocotyl segments. The mechanisms required for plant growth are not fully clarified yet (Narsai et al. 2015), especially with respect to the proteome changes in vivo in the rapidly growing coleoptiles in model plant maize. The existence of growth-limiting proteins was proposed to explain the regulation of maize coleoptile growth (Kutschera & Wang, 2016), but the candidate proteins are speculative. In Arabidopsis, gene expression or protein synthesis was not suggested to be necessary for the initiation of cell elongation of hypocotyl segments (Schenck et al. 2010). In dark-or light-grown rye seedlings, no significant changes in protein profiles were detected during coleoptile growth (Fröhlich & Kutschera, 1995). By contrast, using in vitro cultures of rye coleoptile segments, proteomic analysis showed that the initiation of coleoptile elongation was associated with at least two auxin-responsive proteins (Deng et al. 2012), while its cessation was related to the degradation of vacuolar H + -ATPase (Kutschera et al. 2010). Using maize coleoptile segments incubated in vitro, 15 proteins with known or predicted roles in cell wall biosynthesis were identified by proteomic analysis (Li et al. 2013).
However, previous proteomic studies were almost performed with in vitro cultures of coleoptile segments, little is known about the significance of proteome changes in vivo in the rapidly growing coleoptiles. Although proteomic studies have showed protein changes during coleoptile elongation in rice under anoxic conditions (Huang et al. 2005;Sadiq et al. 2011), these results are not suitable for interpretation of coleoptile growth in other cereal plants such as maize, wheat, and barley, because their seeds lack the ability to germinate under anoxic conditions (Takahashi et al. 2011). The aim of the present work was to explore the significance of proteome changes in vivo during coleoptile growth. Using two-dimensional gel electrophoresis (2DE) combined with mass spectrometry, we investigated protein changes and identified differential abundance proteins (DAPs) that are associated with rapid growth of maize coleoptiles.

| Plant materials
Seeds of the maize cv. Zhengdan958 were surface sterilized in 1% sodium hypochlorite for 5 min and washed thoroughly with distilled water. After 12-hr soaking, uniformly sized seeds (5 replicates, 30 each) were placed in Petri dishes having three layers of moist Whatman paper and incubated at 28℃ in darkness. Coleoptiles were daily collected over 7 days after germination (DAG). The coleoptiles were excised from the basal 1 mm above the coleoptile node, and the enclosed primary leaves were removed using a pair of forceps.
The coleoptile segments were used for length, fresh weight (FW), and dry weight (DW) assays (Edwards et al. 2012), light microscopy and protein analysis. Analysis of variance was performed using Student's t test.

| Light microscopy
For preparation of paraffin sections, coleoptile segments (1-2 mm long) without the tip region were cut from the same basal region above the coleoptile node at 1.5, 3, and 5 DAG, respectively. The segments were fixed in the solution consisting of 5% formaldehyde, 5% acetic acid, and 45% ethanol for 12 hr and dehydrated in gradient ethanol solutions. The dehydrated tissues were embedded in paraffin and 10μm thick slices were prepared with a microtome (RM-2016, Leica, German). The slices were stained with safranin/fast green (Ruzin, 1999) and recorded under a light microscope equipped with ToupView x 86 software (ToupTek Photonics, China).

| Phytohormone assay
The levels of indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in maize coleoptiles were assayed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) technique as described previously (Niu et al. 2019). Approximately 0.5 g (FW) coleoptile samples were powdered and extracted for 30 min at 4℃ with 10 ml isopropanol/ hydrochloric acid buffer. And then 20 ml dichloromethane was added and shaken at 4℃ for 30 min. After centrifugation at 13,000 g for 5 min at 4℃, the organic phase was extracted and dried in N 2 . The residues were resuspended in 200 μl methanol plus 0.1% methane acid and filtered with a 0.22 μm filter membrane. The filtrate was used for HPLC-MS/MS with a ZORBAXSB-C18 (Agilent Technologies) column (2.1 × 150; 3.5 μm) at 30℃. The sample injection volume was 2 μl.

| Protein extraction and determination
Coleoptile proteins were extracted with trichloroacetic acid/acetone precipitation according to the previous research (Niu, Zhang, et al., 2018). In brief, 10-20 coleoptiles were ground in an SDScontaining buffer (1% SDS, 0.1 M Tris-HCl, pH 6.8, 2 mM EDTA-Na 2 , 20 mM DTT and 2 mM PMSF) and centrifuged at 15,000 g for 5 min (4℃). The supernatant was collected and precipitated with an equal volume of cold 20% trichloroacetic acid/acetone on ice for 5 min and centrifuged as mentioned above. The protein pellets were washed twice with cold acetone and air-dried. Then, the dried pellets were redissolved in Laemmli SDS buffer for SDS-PAGE (Laemmli, 1970) or in 2DE rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer, pH 4-7, GE Healthcare) for isoelectric focusing (IEF). Bradford assay was performed to determine the protein concentration (Bradford, 1976).

| Peroxidase (POD) isozyme activity assay
Crude enzyme was extracted according to the method of Lee and Lin (1995) with some modifications. The coleoptiles (about 1 g FW) were pulverized with liquid N 2 in a mortar and pestle and then homogenized with 1 ml of 10 mM phosphate buffer (pH 6.4). After centrifugation at 16,000 g for 10 min at 4℃, the supernatant was collected for soluble POD activity assay. The precipitate was washed at least five times using an equal volume of extraction buffer until no POD activity was detected in the supernatant. Then the precipitate was extracted in 1 M NaCl for 4 hr at 4℃ with continuous shaking.
After centrifugation, the supernatant was used for cell wall-bound POD activity assay.
The POD activity was measured using the method of López- Kyoto, Japan). One enzyme unit (U) was defined as a change in absorbance of 0.01 in 1 min (Tao et al. 2018).

| 2DE and protein identification
Proteins samples (600 μg in 200 μl) were loaded into IPG strips (11 cm, linear pH 4-7, Bio-Rad) by passive rehydration overnight at room temperature. IEF and subsequent SDS-PAGE were performed as previously described (Wu et al. 2015). After electrophoresis, proteins in gels were visualized with CBB R350, and photographed using a DSLR camera (Nikon D7000) in automatic mode. The digital images of the gels were analyzed using PDQuest software (Bio-Rad, USA).
The relative abundance of protein spots was estimated by taking the average normalized peak area of all spots in 2DE gels. The spots with a significant and consistent change in abundance in the replicate gels of the three biological replicates (Student's t test, p < .05, foldchange ≥2.0) were regarded as the DAPs.
Mass spectrometry was performed as described previously (Niu et al. 2019). The spots of the DAPs were excised and subjected to in-gel trypsin digestion overnight. The digests fragments were subjected to MALDI-TOF/TOF analysis (ABI 5800, Applied Biosystems, CA, USA). The acquired spectra were automatically submitted to Mascot server (Matrix Science, London, UK) for protein identification against the UniProtKB (https://www.unipr ot.org/) database (version 2016_08_16, 210842 sequences of Z. mays). Positive identification required significant scores (p < 0.05) and at least two unique matches. The identified DAPs were grouped by referring to the annotations in the UniProtKB database regarding cellular component, molecular function, and biological process. Subcellular locations were determined according to the annotation in UniProtKB database or predicted by homologous proteins or at Plant-mPLoc server (http://www.csbio.sjtu.edu.cn/bioin f/plant -multi/).

| Total RNA extraction and reverse transcription quantitative PCR (RT-qPCR) analysis
Total RNA of maize coleoptiles was extracted with RNA-Solv® reagent (Omega Bio-Tek, USA) according to the manufacturer's instructions. The purity and concentration of total RNA were determined by a NanoDrop One spectrophotometer (Thermo Scientific, USA).  Table S1). Amplification of the ZmUBI gene was used as an internal reference. The relative expression level of each gene was calculated by the 2 −ΔΔCT method. Each sample was carried out at least three biological replicates. Statistically significant differences in changes in gene expression (|fold-change| ≥ 1.5) due to treatments were accessed by a Student's t test (*p < .05, **p < .01 and ***p < .001).

| Prediction of the phytohormone-related cis-acting motifs
The 1,000-bp promoter sequences of the genes encoding DAPs were retrieved from the maize genomic sequence available at NCBI (https://www.ncbi.nlm.nih.gov/). A search of the extracted promoter sequences for phytohormone-related cis-motifs was conducted using the Plant-CARE (Plant Cis-Acting Regulatory Element, https:// bioin forma tics.psb.ugent.be/webto ols/plant care/html) (Lescot et al. 2002). The distribution of phytohormone-responsive cis-acting motifs was plotted using Microsoft Excel.  (Tetley & Priestley, 1927). The coleoptiles reached the maximal DW and length at 4 and 5 DAG, respectively. Clearly, the coleoptiles elongated rapidly during 2-5 DAG, with an average growth rate of 750 μm/hr. Thus, the coleoptiles at 1.5, 3, and 5 DAG, representing lag phase, exponential phase, stationary phase, individually, were used for further analysis.
Microscopic observation indicated that the coleoptile was surround by an epidermis and within the epidermis, wall-thinned cells (parenchyma) with prominent nuclei were poorly differentiated ( Figure 1c). The coleoptile cells became 5-6 times in length at 5 DAG than at 1.5 DAG and somewhat wider. Two opposite, simple vascular bundles were observed in the cross section of coleoptiles ( Figure 1d). Thus, the rapid growth of the coleoptiles was due to longitudinal extension of coleoptile cells.

| Phytohormone changes with coleoptile growth
To examine the changes in the levels of major phytohormones and phytohormone-responsive DAPs, the contents of IAA, SA, JA, GA3, and ABA were quantified per FW in elongating coleoptiles (Figure 2).
The contents of IAA, SA and JA decreased significantly during the growth of coleoptile by 3.07, 3.21, and 8.06 times, respectively. GA3 first decreased and then increased. In the coleoptile at 3 DAG, the content of GA3 was only 1.11 ng/g FW. ABA showed no significant changes during the growth of the coleoptiles.

| Protein changes with coleoptile growth
Protein content in growing coleoptiles was assayed based on equal FW, DW, and equal number of coleoptile indivuduals. In the growing coleoptiles, protein content decreased per FW and DW, but increased in individual coleoptile (Figure 3a). This difference could be partly explained by growth characteristics of coleoptiles: DW reached the maximum at 4 DAG, whereas the length at 5 DAG ( Figure 1b), that is, the growth of coleoptiles was mainly driven by the increased water input and wall growth.
The quantitative protein changes in the growing coleoptiles were further detected using 2DE at 1.5, 3, and 5 DAG (Figure 3b, Figure S1). PDQuest comparison of 2DE images revealed that among approximately 700 protein spots of each gel, 44 were identified as the DAPs with significant and reproducible changes ( Figure S2; Student's t test, p < .05). These DAPs were grouped as three types (Table 1): I, with steadily increased abundance (24 spots); ΙΙ, with the highest abundance at 3 DAG (6 spots); III, with gradually decreased abundance (14 spots).

| DAPs associated with coleoptile growth
The 44 DAPs were successfully identified by mass spectrometry, belonging to 31 different kinds of proteins (Table 1, Table S2).

F I G U R E 2
Phytohormone content change with coleoptile growth. Statistically significant differences were analyzed by Student's t test (*p < .05, **p < .01 and ***p < .001) According to the annotation in the UniProtKB, these proteins take part in various cellular processes. More importantly, the DAPs of types I and II were involved in growth-related cellular processes, e.g., Therefore, the cellular processes, especially growth-and stressrelated processes, were selectively enhanced to support the needs of the rapidly growing coleoptiles.

| Verification of the DAPs
The activities of soluble POD and wall-bound POD increased significantly with the elongation of coleoptile, and the activity of wall-bound POD was higher than that of soluble POD (Figure 4a).
Soluble POD activity staining showed that isozyme patterns significantly changed with coleoptile elongation, especially two major POD activities emerged (Figure 4b). These results were largely consistent with POD abundance changes in 2DE images ( Figure S2, spots 19, 31 and 32) during germination.
Immunoblot analysis showed that on an equal protein basis, the accumulation of actin, tubulin, and expansin strongly increased with coleoptile growth, with the highest abundance at 5 DAG ( Figure S3a,b); nevertheless, for a single coleoptile, they accumulated to the highest abundance at 3 DAG ( Figure S3c,d). These results were consistent with the results of 2DE.
RT-qPCR showed that the expression patterns ( Figure 5) of 11 selected genes in the growing coleoptiles were partly consistent with the abundance changes (Table 1) of the corresponding DAPs.

F I G U R E 3 Protein changes in the growing coleoptiles of maize. (a) Changes of protein content per g (DW) coleoptile (left), per g (FW)
coleoptile (middle) and as per coleoptile (right). Statistically significant differences were analyzed by Student's t test (**p < .01). (b) Proteome changes in the growing coleoptiles on an equal protein basis. Representative 2DE images from three biological replicates were shown with the marked DAPs

TA B L E 1 (Continued)
For example, the expression of O-Methyltransferase ZRP4 steadily increased with coleoptile growth, as its product O-methyltransferase ZRP4 substantially accumulated; the levels of 16.9 kDa class I heat shock protein 1 and its gene sharply decreased with coleoptile growth, and so did Auxin-binding protein 1. However, several genes were inconsistent between gene expression and protein accumulation, such as Actin-7, Tubulin α-1 chain, Profilin-5.

| D ISCUSS I ON
The coleoptile is a short-lived organ with low differentiation; its growth is highly dependent upon the elongation of the cells preformed in the embryo (Jones & Rost, 1989). In the present study, we aimed to characterize proteome changes and the DAPs in the rapidly growing coleoptiles of maize. Due to space limitation, the discussion below focused on a few DAPs.
Our work showed the significant changes in protein content and 1D profiles with maize coleoptile growth, especially protein content per coleoptile steadily increased. 2DE analysis further revealed quantitative the proteome changes with 31 DAPs (44 spots) associated with rapid growth of the coleoptile. Therefore, the synthesis and degradation of specific proteins were selectively enhanced during coleoptile growth. Importantly, 21 DAPs with increased abundance (type I and II) are mainly involved in growth-related, and thus are necessary for rapid growth of the coleoptile. This implied that protein synthesis plays a vital role in rapid growth of maize coleoptile, as found in rice (Edwards et al. 2012;Lasanthi-Kudahettige et al. 2007;Narsai et al. 2009). For the first time, β-D-glucoside glucohydrolase, nucleoside diphosphate kinase1, ferritin-1, kiwellin-1, and ABA stress ripening 2 were implied to be involved in coleoptile growth.
Cell wall and cuticle play a key role in plant growth and form an outermost barrier against pathogen infection (Keegstra, 2010). Using maize coleoptile segments incubated in vitro, two proteins involved in cutin or cell wall biosynthesis were identified (Li et al. 2013). In the present study, the DAPs involved in lignin biosynthesis, aromatic compound biosynthesis, and strengthening of walls were highly accumulated with coleoptile growth. To grow, plants must loosen rigid walls in a pH-dependent manner (Arsuffi & Braybrook, 2018) by expansins (Choi et al. 2003;Cosgrove, 2000). Though expansins were not found as DAPs here possibly due to their low abundance, immunoblotting and RT-qPCR showed their change with coleoptile growth ( Figure 5, Figure S3). POD plays a key role in H 2 O 2 -mediated cell wall stiffening in maize coleoptiles (Schopfer, 1996). In the present study, the increase in POD abundance and activity with coleoptile growth was consistent with the presence of POD in the growth-limiting outer epidermal wall (Schopfer, 1996), implying their role in cell wall stiffening and stress response.
Actin and tubulin are essential components of cell cytoskeleton, involved in cytoskeleton-based processes, e.g., cell division and elongation, vesicle transport, signal transduction, and cell wall deposition (Hashimoto, 2015;Kandasamy et al. 2001). The accumulation of α-tubulin, especially acetylated ones, gradually increased during F I G U R E 4 POD activity assay in maize coleoptiles. (a) Determination of soluble POD and wall-bound POD activity during coleoptile elongation. Error bars indicate the SE values of five biological replicates (n = 5). Asterisks show significant differences assessed by a Student's t test (**p < .01). (b) In-gel soluble POD activity assay on an equal protein basis. Arrows indicate two emerging major POD activities in the growing coleoptiles F I G U R E 5 Verification of transcript abundance of the genes encoding the selected DAPs. RT-qPCR was repeated in three biological replicates, with ZmUBI as a reference gene. Gene name in red indicates an inconsistency between gene expression and protein accumulation. Asterisks show significant differences in expression changes as assessed by a Student's t test (|fold-change| ≥ 1.5; *p < .05, **p < .01, ***p < .001) TA B L E 2 Analysis of hormone-responsive cis-acting element in the promoter regions in the genes encoding DAPs the early growth stages of Brassica rapa seedlings and correlated to the maturation of plant organ (Nakagawa et al. 2013). A similar situation existed with actin (Nakagawa et al. 2013). We here confirmed the increased expression of tubulin and actin by 2DE, immunoblotting and RT-qPCR, strongly showing their important roles in coleoptile growth.
In the present work, the mRNA levels of eleven DAPs were partly correlated with protein abundance ( Figure 5). The discordance of transcript/protein level has been well documented in maize (Ponnala et al. 2014), possibly because mRNA is not a direct indication of protein level. The protein abundance can not only be regulated at transcription level but also translation level and turnover level.
The roles of auxin in rapid growth of maize coleoptile have been extensively studied before (Kutschera et al. 1987;Kutschera & Khanna, 2020). In the present study, 2DE analysis failed to identify any auxin-related proteins as the DAPs, this was mainly due to the possibility that 2-fold changes in abundance were set to filter DAPs.
In the growing coleoptiles, the abundance changes of auxin-related proteins were possibly lower than twofolds based on equal protein amounts, or auxin-related proteins accumulated in low abundance, which could not be detected by 2DE combining CBB R350 staining due to the poor resolution. In a previous study, auxin-associated proteins were also not identified in IAA-induced rapidly growing maize coleoptiles by 2DE and mass spectrometry (Li et al. 2013). Thus, we performed experiments to assay auxin content and detect the gene expression of auxin-related proteins in growing maize coleoptile.
Peroxidases are a group of enzymes that catalyze the oxidation of a substrate by hydrogen peroxide or an organic peroxide. As discussed previously, antioxidant enzymes (e.g., peroxidases and superoxide dismutase) and their activities did not always match the extent of alteration in their abundance (Niu, Xu, et al. 2018). It is difficult to correlate specific enzyme activities to protein abundance. Soluble POD activity staining showed that isozyme patterns significantly changed with coleoptile elongation, especially two major POD activities emerged (Figure 4b). These results were largely consistent with POD abundance changes in 2DE images ( Figure S2, spots 19, 31 and 32). These POD (spots 19, 31, and 32) were localized within cell internals rather in cell walls (Table 1). Thus, we inferred that the two major POD activities in Figure 4b possibly corresponded to spots 19, 31, and 32. However, the verification of POD identities in Figure 4b should cut the gel bands with POD activities and then subjected to mass spectrometry.
Our results showed that to respond to stress conditions during germination, coleoptiles substantially accumulated stress-responsive DAPs, especially herbicide SBP, ABA stress ripening 2, major latex protein 22, kiwellin, and NAD(P)-binding Rossmann-fold superfamily (Han et al. 2019;Hatzios, 1983;Yamauchi et al. 2011). Herbicide safeners have long been used to protect crops from herbicide injury (Hatzios, 1983). Cereal coleoptile is an important site for the action of herbicides and safeners (Scott-Craig et al. 1998). We found here that the abundance of SBP (spots 40 and 41) increased at least tenthousand times at 3-5 DAG and became one of the most abundant proteins in the growing coleoptiles, suggesting that the coleoptiles have prepared themselves for confronting possible herbicide injury during germination.
It is worth noting that several enzymes identified here, especially H + -ATPase subunit B and β-glucosidase and fructokinase-2 were highly accumulated in the rapidly growing coleoptiles. The expression of H + -ATPase genes was found to positively correlate with cellular growth (Viereck et al. 1996). β-Glucosidase was found to be most abundant in the coleoptiles (Esen, 1992) and involved in carbohydrate metabolic process and cytokinin-activated signaling pathway. Fructokinase-2 takes part in starch biosynthesis and may also be involved in a sugar-sensing pathway (Zhang et al. 2003). The high abundance of these enzymes revealed here suggested their important roles in rapid growth of maize coleoptiles.
In addition, our work provides complementary results compared with the previous study (Li et al. 2013) that used SDS-PAGE and mass spectrometry to identify the proteins of the cultures in vitro of excised maize coleoptiles. Both studies indicated that eukaryotic initiation factor 4a (eIF4A), S-adenosylmethionine synthase, tubulin, SBP, 14-3-3-like protein, POD and profilin-5 increased in abundance either in vivo or in vitro growth of maize coleoptiles.

| CON CLUS ION
Our results showed that the rapid growth of maize coleoptile is largely due to its ability to quickly enhance relevant cellular processes, especially the selective synthesis of the DAPs that are involved in growth-related cellular processes. This study will contribute to the understanding of the mechanisms underlying coleoptile growth in maize and other cereal plants. The DAPs identified here may be useful for regulating plant growth and for increased biomass in agriculture.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.