Conservation between higher plants and the moss Physcomitrella patens in response to the phytohormone abscisic acid: a proteomics analysis

Background The plant hormone abscisic acid (ABA) is ubiquitous among land plants where it plays an important role in plant growth and development. In seeds, ABA induces embryogenesis and seed maturation as well as seed dormancy and germination. In vegetative tissues, ABA is a necessary mediator in the triggering of many of the physiological and molecular adaptive responses of the plant to adverse environmental conditions, such as desiccation, salt and cold. Results In this study, we investigated the influence of abscisic acid (ABA) on Physcomitrella patens at the level of the proteome using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty-five protein spots showed changes in response to ABA treatment. Among them, thirteen protein spots were down-regulated; fifty-two protein spots were up-regulated including four protein spots which were newly induced. These proteins were involved in various functions, including material and energy metabolism, defense, protein destination and storage, transcription, signal transduction, cell growth/division, transport, and cytoskeleton. Specifically, most of the up-regulated proteins functioned as molecular chaperones, transcriptional regulators, and defense proteins. Detailed analysis of these up-regulated proteins showed that ABA could trigger stress and defense responses and protect plants from oxidative damage. Otherwise, three protein kinases involved in signal pathways were up-regulated suggesting that P. patens is sensitive to exogenous ABA. The down-regulated of the Rubisco small subunit, photosystem II oxygen-evolving complex proteins and photosystem assembly protein ycf3 indicated that photosynthesis of P. patens was inhibited by ABA treatment. Conclusion Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. Sixty-five protein spots showed differences in accumulation levels as a result of treatment with ABA. Detailed analysis these protein functions showed that physiological and molecular responses to the plant hormone ABA appear to be conserved among higher plant species and bryophytes.


Background
Plants undergo continuous exposure to various biotic and abiotic stresses in their natural environment. To survive under such conditions, plants activate signaling cascades that lead to the accumulation of endogenous hormones which, in turn, trigger the induction of defense responses. Abscisic acid (ABA) is a small, lipophilic phytohormone that regulates many important aspects of plant growth and development and plays a crucial role in cellular responses to environmental stresses such as drought, cold, salt, wounding, UV radiation, and pathogen attack [1]. It is ubiquitous in lower and higher plants and has also been found in algae [2], fungi [3], and even mammalian brain tissue [4].
Many studies have been carried out that characterize ABA signaling pathways and physiological responses to ABA in higher plants. Mutational analyses in Arabidopsis have led to the identification of several genes that are involved in ABA signaling pathways. The proteins involved in ABA signaling pathways include G proteins, protein phosphatases, and protein kinases [5]. Target proteins that respond to ABA can be directly involved in cellular protection or can act as transcriptional factors that link extracellular signaling to the regulation of transcription in eukaryotic cells an important part of the physiological response to ABA is achieved through gene expression and protein synthesis. Transcriptome analyses have shown that ABA dramatically alters gene expression. More than 1300 ABA-regulated genes were identified by random mass sequencing of Arabidopsis transcripts [6,7]. Proteome research has also indicated that exogenous ABA can induce the synthesis of many proteins in rice seedlings. These proteins are involved in signaling pathways, transcription, cell growth and division, photosynthesis, protein synthesis and trafficking, and defense/stress-response, among others [8]. To date however, relatively little is known regarding ABA signaling pathways in bryophytes.
Bryophytes were the first land plants and they have been a powerful experimental tool and model for the elucidation of complex biological processes [9][10][11]. Evolutionary studies have indicated that bryophytes may form a sister clade with tracheophytes (vascular plants). Analysis of organisms from these ancient clades can make significant contributions to understanding the development [12], physiology [13], phylogenetics [14] and stress-induced cellular responses among plants [15]. Recently, comparison of the draft genome sequence of P. patens with the genomes of flowering plants revealed evolutionary insights into the conquest of land by plants. Furthermore, bryophyte physiology is regulated by many of the same phytohormones used by higher plants (e.g., auxins, abscisic acid, and gibberellins) and uses similar mechanisms of intracellular messaging (e.g. Ca2+) found in higher plants [13,16,17].
In recent years, the moss P. patens become a new model for studying the phytohormone ABA. ABA is involved in abiotic stress adaptation of P. patens, since exogenously applied ABA increases freezing tolerance [18] and increased endogenous ABA levels are detected upon osmotic stress treatment [19]. Moreover, plants like P. patens comprising both protonemata and gametophores will tolerate complete desiccation following slow drying, if first pretreated with ABA, a process that is associated with a substantial increase in intracellular levels of sucrose [20] as is cold acclimation leading to freezing tolerance [21]. ABA-induced stress tolerance in P. patens is also accompanied by increased expression of stress-related genes [6,7]. However, the role of ABA or the signaling pathways in response to environmental stresses in the moss P. patens has yet to be elucidated.
Here, proteomics has been used to increase understanding of ABA signaling pathways and defense responses in P. patens. We have examined the global changes of proteins in P. patens following ABA treatment using two dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS. Sixty-five proteins have been identified as being up-or down-regulated in response to ABA treatment. These proteins are involved in metabolism and energy, defense responses, protein trafficking and storage, transcription, and signaling pathways, as well as others. Our study reveals the complex signaling pathways and defense responses by which ABA functions in P. patens. It also suggests that molecular responses to ABA may be highly conserved between higher plant species and bryophytes.

Results
Proteome profile changes in P. patens under ABA treatment Changes in the proteome profile between control and ABA treated plants suggested that exogenous ABA could trigger one or more responses in P. patens. In order to ensure statistically results, the experiments were carried out in triplicate. After CBB R-250 staining, more than 1,300 protein spots could be detected for each sample. Representative gels from control and ABAtreated plants and proteins showing altered abundance are presented in Figure 1. Two-dimensional images were analyzed using ImageMaster™ 2D Platinum software. The volume percentage of each spot was estimated and compared across the gels. In the samples following ABA treatment, we repeatedly analyzed 89 protein spots, whose relative abundance was at least 1.5-fold different from the control. The proteins associated with 65 of these protein spots were subsequently identified using LC-MS/MS. Among them, 13 protein spots (D1-D13) were down-regulated, 52 protein spots (U1-U52) were up-regulated by ABA treatment including four protein spots (U22, U24, U40, U43) which were only present in the ABA-treated samples ( Figure 1). Quantitative analysis indicated that the abundance of these proteins changed [see Additional File 1: Supplemental Table S1] in response to ABA treatment ( Figure 2) suggesting that different physiological and biochemical processes are modified following ABA treatment.

Protein identification by LC-MS/MS
The protein spots of interest were excised from preparative 2-D gels. After trypsin autolysis, the digested spots were analyzed by LC-MS/MS. The resulting amino acid sequences were searched against databases using SEQ-EST software. As the P. patens sequence information is limited, there were some samples that did not yield significant matches. For analysis of these samples, the Arabidopsis database was used. Previous transcriptome research has shown that the gene set in P. patens is very similar to that of A. thaliana [22]. The results of the database search are listed in Table 1 and Supplemental  Table S2 [see Additional File 2: Supplemental Table S2].

Discussion
In seed plants, the phytohormone ABA controls many developmental and physiological processes via complicated signaling networks that are composed of receptors, secondary messengers, protein kinase, and transcription factors. Transcription factors can meditate protein synthesis in responses to ABA, which is crucial in plant growth and development as well as in plant responses to various stresses. In this study, a broad spectrum of proteins is synthesized following ABA treatment. Detailed analysis of these proteins indicates that the molecular responses to the plant hormone ABA appears to be conserved among higher plant species and bryophytes, as discussed below.

ABA induced signaling proteins in P. patens
Receptor protein kinases (spot U14) are plasma membrane-bound and play an important role in the perception and transmittance of external signals such as ABA, dehydration, high salt and cold treatments [24][25][26]. Calcium-dependent protein kinases (CDPKs) (spot U4) are implicated as important sensors of Ca 2+ flux in plants in response to a variety of environmental stimuli and hormone signals. Depending upon the calcium signature, the extent and duration of CDPK enzyme activation will vary, but will have a direct effect on the phosphorylation status of its downstream targets [27]. Ser/Thr specific protein kinase (spot U5) catalyzes ATP-dependent phosphorylation of serine and threonine residues on target  Figure 1; B, Proteins down-regulated in response to ABA as identified in Figure 1. The protein abundance is presented as the percentage of the total spot volume associated with each identified spot.  proteins [28]. Phosphorylation by protein kinases is one of the most common and important regulatory mechanisms in signal transduction among all organisms [29]. These results suggested that these same proteins are involved in the ABA signal transduction pathways of P. patens, as reported in higher plants [6,7].

ABA activates the expression of transcriptional regulators
Transcriptional control is a major mechanism whereby cells regulate gene expression. Sequence-specific DNAbinding transcriptional regulators, one class of transcription factors, play an essential role in modulating the rate of transcription of specific target genes [30]. The bZIP transcription factors (spot U26) have been shown to regulate diverse biological processes such as pathogen defense, light and stress signaling, seed maturation and flower development [31]. A group of bZIP proteins have been identified as ABRE-binding factors that activate transcription through this cis element [32]. Members of another large family of plant transcription factors, the MYB proteins (spot U2), have also been linked to plant stress responses, including responses to UV light, wounding, anaerobic stress and pathogens [33,34]. WRKY proteins (spot U22) are a novel family of transcription factors that are unique to plants [35]. The WRKY family contains one or two highly conserved WRKY domains characterized by the hallmark heptapeptide WRKYGOK and a zinc-finger structure distinct from other known zinc-finger motifs. Specific WRKY family members show enhanced expression and/or DNA-binding activity following induction by a range of pathogens, defense signals and wounding. These transcriptional regulators have been shown to induce stress-responsive gene expression and protein synthesis and increase plant tolerance to biotic and abiotic stresses in higher plants [36]. Our results suggested that bryophytes, among the earliest land plants, possessed mechanisms of ABA sensing and signal transduction required for stress responses, similar to higher plants.   [37]. The Hsp70 family (spots U7, U8, U9, U10, U11, U25, U35, U52) represents the most highly conserved of the HSPs, with functional counterparts found in the most primitive of bacteria and the most sophisticated of higher organisms [38]. HSP70 s are involved in almost every step of protein biogenesis [39]. They play essential roles as molecular chaperones assisting the correct folding of nascent polypeptide chains as they emerge from the ribosome, participating in transmembrane protein transport, and limiting cellular damage following stress by their ability to prevent protein aggregation and to restore the function of denatured proteins [40,41]. Luminal binding protein (BiP) (spot U35) is a member of the Hsp70 family that is localized to the endoplasmic reticulum (ER) of eukaryotic cells, where it functions as a chaperone and is believed to support proper protein folding and protein translocation into the ER lumen [42]. HSP is a large protein family that includes HSP90 and HSP60, among others. In our study, different members of Hsp70 subfamily were the major HSPs that were up-regulated by ABA treatment in P. patens.

ABA triggers stress and defense responses
As in higher plants, many defense proteins were upregulated by ABA signal. In this study, we identified twelve proteins related to defense and cellular protection. Intracellular pathogenesis-related proteins (spot U24) are defined as proteins that are induced by pathogens attack as well as abiotic stimuli. They are powerful defense agents that are currently being use to genetically enhance cereals susceptible to endemic disease and insect pests [43]. In order to combat pathogen infection, plants also produce disease resistance protein (spot U29) that specifically detects the appearance of avirulence products. If they lack corresponding disease  Actin U3↑ a The downward arrow represents the down-regulated protein spot on the 2-D gel; b The upward arrow represents the up-regulated protein spot on the 2-D gel; C The * represents the newly induced protein spot on the 2-D gel.
resistance proteins, avirulence products can function as virulence factors, subverting host cellular functions through interactions with plant-encoded pathogenicity targets [44]. Lipoxygenases (LOXs) (spot U30) catalyze the formation of fatty acid hydroperoxides involved in response to stresses such as pathogen attack, wounding, water deficit, and anoxia [45]. N-hydroxycinnamoyl/benzoyl transferase (HCBT) (spot U42) catalyses a reaction in the phytoalexin biosynthetic pathway in carnation [46]. Extensin (spots U18, U19) is a hydroxyproline-rich glycoprotein (HRGP) found in the cell walls of higher plants [47]. It is induced by many stimuli such as jasmonic acid, wounding and pathogen attack and appears to play an important part in the plant defense response [48,49]. Proline-rich proteins (spot U15) are also cell wall proteins that actively contribute to the plant defense response [50]. Enzymes involved in antioxidation include ascorbate peroxidase (spots U33, U45, U46), which catalyses the first reaction between H 2 O 2 and ascorbate in the ascorbate-glutathione cycle, giving rise to monohydroascorbate and H 2 O [51]; and peroxiredoxin (spots. U37, U50), which reduces H 2 O 2 and detoxifies alkyl hydroperoxides and peroxinitrite [52]. Physcomitrin (spots U38, U41) is a novel plant cytolysin but its biological function is not clear. It is significantly induced by ABA treatment, NaCl, and desiccation [53,54]. and is required Further study is required to determine whether physcomitrin makes a significant contribution in response to extreme environmental conditions in P. patens. The identification of a large number of defense proteins implies that ABA could increase the resistance of P. patens towards pathogens and abiotic stresses, protecting the plant from oxidative damage.

ABA inhibits photosynthesis in P. Patens
Proteins involved in photosynthesis and carbon assimilation that responded to ABA treatment are represented by the small subunit of Rubisco (spots D9, D13); photosystem II oxygen-evolving complex proteins (spot D4), that catalyze acid-base reactions or that are involved in proton uptake and release [55]; photosystem assembly protein ycf3 (spot D12), which regulates the assembly of photosystem complex proteins. Down-regulation of these proteins suggests that photosynthesis may be inhibited by ABA in P. patens. These results were in accord with previous data which showed ABA inhibits photosynthesis in higher plant [8]. Together, our results indicated that ABA-responsive bryophytes underwent physiological changes that had features similar to those of higher plants.

Conclusions
Mosses were one of the earliest land plants in evolutionary terms, and have many characteristics in common with seed plants. Therefore, a process common to land plants, such as responding to the plant phytohormone ABA, can be worthwhile to study in the moss species, P. patens. In order to investigate global protein changes after treatment with ABA, we have performed a comparative proteome analysis of P. patens and revealed a complex regulatory network. Upon the application of ABA, P. patens cells can sense ABA and transmit a signal, which, in turn, activates transcriptional factormediated metabolic and defensive genes expression. In higher plants, a broad spectrum of genes is also induced by the ABA treatment, and these genes encode not only proteins involved in the protection of cellular structures or in the biosynthesis of protective metabolites but also key regulatory proteins that induce stress-responsive gene expression and protein synthesis and increase plant tolerance to biotic and abiotic stresses. These responses are similar to those seen in the moss P. patens in response to ABA. Figure 4 illustrates the ABA response process of angiosperm (A. thaliana; from reference 6, 7) and bryophyte (P. patens). Based on these results, it seems likely that P. patens and higher plants share common receptor protein, protein kinases, transcription factors and physiological and biochemical reactions.

Plant materials
P. patens was grown in modified BCD medium containing 0.5% (w/v) glucose and 0.75% (w/v) agar [56]. Gametophores were cultured under the standard conditions (23°C) with a light cycle of 16 h light/8 h darkness and a light intensity of 55 μmol s -1 m -2 . Three-week-old gametophores were treated with 0 μM or 50 μM ABA for 72 h. For ABA treatment, gametophores were transferred to BCD solid medium supplemented with 50 μM (±)-cis-trans ABA. This treatment was carried out three separate times [57]. Gametophores were harvested, frozen in liquid nitrogen and stored at -80°C.

Image and data analysis
The 2-DE gels were scanned at 600 dpi with a UMAX Power Look 2100XL scanner (Maxium Tech Inc., Taiwan, China). The transparency mode was used to obtain a grayscale image. The image analysis was performed with ImageMaster™ 2D Platinum software. To verify the auto detected results, all spots were manually inspected and edited as necessary. After spot detection, quantification, and background subtraction, each analyzed gel was matched individually to the reference gel. To compensate for subtle differences in sample loading, gel staining, and destaining, the volume of each spot (i.e. spot abundance) was normalized as a relative volume. Only those with reproducible changes (quantitative changes more than 1.5-fold in abundance and ttest P < 0.05) among three replicates were used for further analysis.

In-gel digestion and protein identification
Protein spots were manually excised from the gel. In-gel digestion by trypsin and LC-MS/MS were performed according to Wang et al. [54]. All MS/MS samples were analyzed using the TurboSEQUEST program in the Bio-Works 3.1 software suite (Thermo), against a database adapted from P. patens and A. thaliana NCBI protein database, in which each genuine protein sequence was followed by a reversed amino acid sequence. The search parameters included, up to two missed trypsin cleavages with carboxamidomethylation at cysteine. The mass tolerance for precursor ions and fragment ions were considered as 2 amu and 1 amu respectively. Peptides had to be fully tryptically digested and be at least seven amino acids long in the searched databases. The output results were combined together using the software BuildSummary. False positive rate (FPR) was kept less than 5% as the filtration criterion. The FPR was calculated as double of the number of peptides from reversed database divided by the number of peptides from reversed and normal database [59]. An accepted SEQUEST result had a ΔCn score of at least 0.1 (regardless of charge state). Peptides with a +1 charge state were accepted if they were fully tryptic digested and had a cross correlation (Xcorr) of at least 1.9. Peptides with a +2 charge state were accepted if they had an Xcorr ≥2.2. Peptides with a +3 charge state were accepted if they had an Xcorr ≥3.75.

Additional material
Additional file 1: Supplemental Table S1 Quantitative changes in spot intensities. The protein abundance is presented as the percentage of the total spot volume associated with each identified spot. The Vol% value of all identified proteins and the data of statistical analysis among three replicates are listed in this file.
Additional file 2: Supplemental Table S2 Peptides sequence matched of ABA responsive proteins. This file includes the peptides sequence of all identified proteins, total peptide count and unique peptide count which are searched, and charge, XCorr value and score of every peptide.