Protein Expression Changes in Maize Roots in Response to Humic Substances

  • Paolo Carletti
  • Antonio Masi
  • Barbara Spolaore
  • Patrizia Polverino De Laureto
  • Mariangela De Zorzi
  • Loris Turetta
  • Massimo Ferretti
  • Serenella Nardi
Article

Abstract

Humic substances are known to affect plant metabolism at different levels. We characterized humic substances extracted from earthworm feces by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and used them to treat corn, Zea mays L., seedlings to investigate changes in patterns of root protein expression. After root plasma membrane extraction and purification, proteins were separated by two-dimensional gel electrophoresis, and differential spot intensities were evaluated by image analysis. Finally, 42 differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The majority of them were downregulated by the treatment with humic substances. The proteins identified included malate dehydrogenase, ATPases, cytoskeleton proteins, and different enzymes belonging to the glycolytic/gluconeogenic pathways and sucrose metabolism. The identification of factors involved in plant responses to humic substances may improve our understanding of plant–soil cross-talk, and enable a better management of soil resources.

Keywords

DRIFT spectroscopy Humic substances LC-MS-MS Proteomics Sucrose metabolism Two-dimensional gel electrophoresis Zea mays L. 

Introduction

Humic substances (HS) consist of a mixture of different organic compounds resulting from the decomposition of plant and animal residues (MacCarthy 2001). They are found in all terrestrial and aquatic environments and constitute one of the most abundant forms of organic matter on the surface of the Earth (MacCarthy 2001). HS interact with the organic component of soil and the root apparatus of plants in the soil matrix, where HS can have a fundamental influence not only on overall soil fertility and conservation, but also on plant physiology (Nardi et al. 2002). Under laboratory and field conditions, HS enhance plant growth, as measured in terms of an increase in length or in the fresh and dry weights of shoots and roots. HS also result in the production of higher leaf chlorophyll concentrations, more lateral root initials, an improved micro- and macronutrient uptake, and many other biological effects (Nardi et al. 2002). These effects have been attributed partly to the complexing properties of HS, which increase the availability of micronutrients from scarcely soluble hydroxides, and particularly to the maintenance of sufficient levels of Fe and Zn in solution (Cesco et al. 2000).

Other targets of HS on plant roots are the enzymes associated with the plasma membrane (PM) (Canellas et al. 2002). At the molecular level, the effects of HS have been demonstrated on the expression of specific genes, such as the two H+-ATPase isoforms Mha1 and Mha2 (Quaggiotti et al. 2004). Muscolo et al. (2007b) showed that HS, and particularly those with a low molecular mass, are taken up by plant cells and are able to affect plant metabolism. In this sense, a hormone-like activity has been suggested for several humic fractions (Zandonadi et al. 2007) whose biological action appears to mimic the responses induced by gibberellic and indole-3-acetic acid (IAA). The metabolic pathways involved in these responses have been only partially described, and the whole mechanism implicated is far from being elucidated.

The PM represents the site for the exchange of information and substances between the cell and its environment. Proteins associated with root cell PMs can thus be reasonably assumed to be a target for HS, and changes in their expression may be seen as the primary reactions leading to the biological responses reported so far. Since protein analysis is the most direct approach for studying the dynamics of plant cell metabolism, valuable information on plant cell and HS interactions can be obtained from a proteomic assay.

We extracted HS from earthworm feces, and used the extract to treat 11-d-old seedlings of corn, Zea mays. Next, we used a proteomics approach to assess changes in protein expression in PM-enriched root extracts in response to HS treatment. The biochemical reaction of Z. mays to HS at the level of root proteins is shown here for the first time. The main aim of this study was to establish which proteins are differentially regulated after exposure to HS, and to provide new insight on the molecular basis of the response of Z. mays PM to these compounds.

Methods and Materials

Preparation of Humic Extract

The feces of Nicodrilus [=Allolobophora (Eisen)=Aporrectodea (Oerley)] caliginosus (Savigny) and Allolobophora rosea (Savigny) (Minelli et al. 1995) were collected from the Ah horizon of an uncultivated couchgrass, Agropyron repens L., growing in soils classified as Calcaric Cambisol (CMc-F.A.O. classification) (FAO-UNESCO 1990). Earthworm culture conditions, HS extraction, and extract purification were conducted as reported in Quaggiotti et al. (2004). HS extraction and purification were performed with 0.1 N KOH. The extract was desalted by using 14 kDa cut-off dialysis Visking (Medicell, London, UK) tubing against distilled water. Subsequently, the extract was desalted on ion exchange Amberlite IR-120 (H+ form), assessed for organic carbon content, and lyophilized before conducting the following analyses.

Diffuse Reflectance Infrared Fourier Transform Spectroscopy

DRIFT spectroscopy was performed with a Nicolet Impact 400 FT-IR Spectrophotometer (Nicolet Instruments, Madison, WI, USA) fitted with an apparatus for diffuse reflectance (Spectra-Tech. Inc., Stamford, CT, USA). SiC disks (320-grid-Carb paper obtained from Spectra-Tech) were used to collect 5 mg of lyophilized sample. Spectra were obtained by combining at least 200 scans at a resolution of 4 cm−1. The background was obtained on the abrasive SiC disk. Spectral data were analyzed with Grams 386 spectral software (Galactic Industries, Salem, NH, USA).

Plant Material and Growth Conditions

Maize (Zea mays L. cv. DKc 5783, Dekalb, Monsanto Agricoltura SpA, Lodi, Italy) seeds were soaked for one night in running water and germinated in the dark at 27°C on filter paper wetted with 1 mM CaSO4 for 96 hr (Nardi et al. 2000b). Seedlings were grown in pools containing 40 l of Hoagland solution for 11 d as follows: 16 hr of light at 25°C and 60% relative humidity, 8 hr of dark at 18°C and 80% relative humidity. For the treatment, a batch of plants was moved for 16 hr to a nutrient solution with HS added at a concentration of 1 mg/l carbon (C), while controls were moved in fresh medium without HS. The HS concentration was chosen according to Nardi et al. (2000b) and in light of preliminary experiments (data not published). At the end of the treatment time, about 120 g of plant roots were collected, snap frozen in liquid nitrogen, and immediately treated for extraction.

Protein Extraction from Root Tissues and Two-Dimensional Polyacrylamide Gel Electrophoresis

A total of eight independent samples, i.e., biological replicates (four control and four HS-treated), were extracted. Plasma membrane enriched fractions were obtained by two-phase partitioning (Sandelius and Morrè 1990; Ephritikhine et al. 2004). The sensitivity of the H-ATPase (Mg-dependent) activity to vanadate was used as a marker of PM purity. The activity that was sensitive to vanadate amounted to 83.3% (N = 8) of the total ATPase activity, which was similar to the purity reported in other studies (Santoni et al. 1998). PM-enriched samples were submitted to phenol extraction followed by methanolic ammonium acetate precipitation, according to the method of Hurkman and Tanaka (1986). Proteins were then solubilized in 7 M urea, 2 M thiourea, 2% CHAPS, 2% DTT, and a protease inhibitor cocktail. The amount of protein in the PM fraction was assayed with the Biorad (Hercules, CA, USA) protein quantitation kit following the manufacturer’s instructions.

After solubilization, isoelectric focusing (IEF) was performed with IPG strips (11 cm, pH range 3–10) by using IPGphor instrumentation following the manufacturer’s instructions (GE Healthcare Bio-Science AB, Uppsala, Sweden). IPG strips were loaded with 100 μg protein, 2% ampholytes (pH range 3–10, GE Healthcare), 60 mM DTT, and bromophenol 121 blue (as a tracer) in 210 μl final volume. Rehydration was done overnight. Next day, a pre-focalization program was run at low voltage (300 V) for 2 hr to enable salt removal, then focalization was performed according to the manufacturer’s instructions (GE Healthcare), followed by reduction and alkylation with iodoacetoamide and equilibration in a solution containing 2% SDS, 15 mM DTT, 62 mM TRIS–HCl pH 6.8, and 10% glycerol. Strips were then placed on top of precast gels and sealed with agarose. Separation on the second dimension was done on polyacrylamide gradient precast gels (8–16%, Biorad) in electrophoretic chambers accommodating up to 12 gels at once (Criterion DodecaCel, Biorad). Running conditions: buffer 25 mM Tris-Base, 192 mM glycine, and 0.1% SDS; 20 mA per gel. Proteins were fixed at least 30 min in 10% methanol + 7% acetic acid. The fixing solution was changed at least twice to wash the gel. Gels were then incubated in undiluted Sypro Ruby (Biorad) stain overnight and rinsed with water. Each of the eight biological replicates was analyzed through three separate gels (technical replicates) for a total of 24 gels. Gel images were acquired with the Chemidoc system (Biorad).

Image and Data Analysis

Gel images were analyzed with the Imagemaster 2D Platinum 6.0 software (GE Healthcare Amersham Biosciences AB, Uppsala, Sweden,) To select only high-quality protein spots for expression profiling, the following threshold criteria were applied: Each protein spot had to be present in at least three biological replicate gels per class (treated or untreated) and detected in at least six gels per class. Only spots with over 1.5-fold changes in volume after normalization between the two classes were defined as altered and further validated with the nonparametric Wilcoxon–Mann–Whitney test (Imagemaster 2D Platinum 6.0 software with either P < 0.05 or P < 0.01) as recommended in Wilkins et al. (2006). Spots appearing in only one class on visual inspection were also defined as newly induced or suppressed by the treatment.

In-Gel Digestion, Protein Identification and Database Search

Excised from the gel spots were washed with 50% v/v acetonitrile (ACN) in 0.1 M NH4HCO3, and vacuum-dried. The gel fragments were reduced for 45 min at 55°C in 10 mM DTT in 0.1 M NH4HCO3. After cooling, the DTT solution was immediately replaced with 55 mM iodoacetamide in 0.1 M NH4HCO3. After washing with 50% ACN in 0.1 M NH4HCO3, the dried gel pieces were swollen in a minimum volume of 10 μl digestion buffer containing 25 mM NH4HCO3 and 12.5 ng/l trypsin (Promega, Madison, WI, USA) and incubated overnight at 37°C. Tryptic-digested peptides were extracted according to the protocol described by Kim et al. (2004).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses were performed on a Micromass CapLC unit (Waters, Milford, MA, USA) interfaced with a Micromass Q-TOF Micro mass spectrometer (Waters) equipped with a nanospray source. For each 2D gel spot, 6.4 μl of tryptic digest were injected at a flow rate of 20 μl/min into an Atlantis dC18 Trap Column. After valve switching, the sample was separated in an Atlantis dC18 NanoEase column (Waters, 0.075 × 150 mm, 3-μm particle size) at a flow rate 200 nl/min with a gradient from 10% B to 55% B in 35 min (solvent A: 95% H2O, 5% ACN, 0.1% formic acid; solvent B: 5% H2O, 95% ACN, 0.1% formic acid). Instrument control, data acquisition, and processing were performed with MassLynx V4.0 software (Waters, Milford, MA, USA). Database searching was done with the on-line version of the Mascot program (www.matrixscience.com) by using the following parameters: one missed cleavage; 0.8 and 0.6 Da mass accuracy allowed for parent and fragment ions, respectively; carbamidomethyl as fixed modification; oxidized methionine as variable modification; 100 hits allowed. Proteins identified with at least two peptides, both showing a molecular weight search (MOWSE) score (Perkins et al. 1999) higher than 40, were considered as matched. For proteins identified by only one peptide with a MOWSE score higher than 40, the peptide sequence was routinely checked manually. All database searches were performed in both the National Center for Biotechnology Information (NCBI) complete database with no species specified, and in the NCBI Viridiplantae-specified database.

Results and Discussion

DRIFT Spectroscopy

The DRIFT spectrum of the HS considered in this study (Fig. 1) shows an intense and broad band centered at 3,282 cm−1 attributed to water bridging of OH stretching of the phenolic hydroxyl groups or to stretching of the hydrogen-bonded OH and NH groups (Niemeyer et al. 1992); the shoulder at 3,089 cm−1 might be attributable to aromatic CH stretching, whose weak relative intensity may be due to the extensive substitution of aromatic rings in HS macromolecules and/or to masking from the broad band of the OH stretching. The peak at 2,930 cm−1 and the weaker shoulder at 2,850 cm−1 are attributable to CH asymmetric and symmetric stretching in aliphatic groups, respectively (Montecchio et al. 2006). The broad band at 2,626 cm−1 was attributed to the formation of intermolecular hydrogen bonding between OH groups in oxygenated compounds (Niemeyer et al. 1992). The sharp, strong band at 1,717 cm−1 is caused by C=O stretching vibration, mainly of COOH. This band assigned to C—O stretching and OH deformation and the band at 1,231 cm−1 are the most clearly distinct bands of the COOH group vibrations (Ding et al. 2002). In addition, the band at 1,231 cm−1 might also be due to C—OH stretching of the phenolic groups (Francioso et al. 2002). The strong band at 1,655 cm−1 might denote the presence of C=O stretching in quinonic and ketone (Gressel et al. 1995) or amide groups (Niemeyer et al. 1992). The peak at 1,542 cm−1 is due to secondary amide deformation (Gressel et al. 1995) and to C=C stretching vibrations of aromatic moieties. The region between 1,400–1,300 cm−1 is assigned to CH2 and CH3 bending, C—OH deformation of COOH, and COO- symmetric stretching (Ding et al. 2002). The intense peak at around 1,040 cm−1 is attributed to C—O stretching of polysaccharide-like components. Taken together, these findings show that this humic fraction is endowed with the characteristic structural network described for most HS isolated from different sources of organic matter (Clapp and Hayes 1999).
Fig. 1

Diffuse reflectance infrared Fourier Transform (DRIFT) spectroscopy spectrum for Humic Subtances (HS) extracted from earthworm feces

Proteomic Analysis

In many cases, plant gene expression modifications are induced by changes in nutrient availability. In particular, Ca++ affects the activity of many actin-binding proteins, and HS are able to chelate cations. In our case study, the Ca++ was provided in the nutrient solution as 0.2 mmol/L of Ca(NO3)2. Both treated and untreated plants were grown in the same medium and HS ash content was very low (<1%). Thus, the reported effects in the proteome could be regarded neither as Ca-dependent nor as related to improved nutrient contents following HS addition.

Image analysis of the 24 sypro-stained gels enabled the detection of approximately 480–550 spots on each gel. By using the threshold criteria (see Methods and Materials), a total of 325 protein spot groups were matched, manually validated, and quantified. Sixty-three spots were found affected by HS treatment, representing more then 10% of the total proteins detected on each gel. Twenty-two spots were upregulated by the HS treatment, whereas 38 were downregulated (Fig. 2). The most upregulated spot (ID 370) represented a 2.92-fold enhancement, whereas the most downregulated protein (ID 1272) resulted in a 2.53-fold diminishment (Table 1). Moreover, two spots appeared to be suppressed by HS treatment, whereas one spot was detected only in gels from treated plants. These 63 spots (Fig. 3) were excised from the gels, digested with trypsin, and the peptides were analyzed by LC-MS-MS, thus enabling the identification of 42 protein species (some of which are illustrated in Fig. 4). In most cases, the database search revealed that the experimental pI and Mr values of the identified proteins matched their theoretical values (Table 1). In a few cases, a discrepancy was observed, but this happens frequently in proteomic analyses and is probably a consequence of posttranslational modification, proteolytic cleavage, or matching proteins from different species. In this expression study, proteins were isolated from plasma membrane-enriched fractions. Although the purity was good and in line with other studies (Santoni et al. 1998), some contamination from other cell components certainly occurred. Even if some of the identified protein species have been previously described from other subcellular compartments, the reproducibility and statistical analysis of the expression data indicate that the differences depended on HS treatment.
Fig. 2

Relative intensities, spot ID, and P values (Wilcoxon–Mann–Whitney test) for the 60 differentially expressed protein spots

Fig. 3

Two-dimensional polyacrylamide gel electrophoresis (2-DE) analysis of maize, Zea mays, root plasma membrane proteins after HS-treatment. One hundred micrograms of protein were extracted and separated by 2-DE, as described in “Methods and Materials” and visualized by Sypro-Ruby staining. A and B show 2-DE gel patterns of protein samples prepared from control and HS-treated roots, respectively. Numbers indicate differentially expressed proteins subjected to LC-MS-MS analysis. Numbers marked with asterisk indicate identified proteins

Fig. 4

Magnified views of some differentially expressed proteins in maize roots plasma membrane in response to HS treatment. Plants were controls (column A) or treated with HS for 16 hr (column B) (see “Methods and Materials”)

Table 1

HS-induced differentially expressed maize root PM proteins identified by LC-MS/MS analysis

Spot ID

Protein fold changea

Accession Numberb

Identified Proteinc

SC (%)d

PMe

Theoretical Mr/pI

Experimental Mr/pI

Protein Classification

Peptide sequencesf

370

+2.92

YP_588408

ATPase subunit 1

10

3

55.4/5.8

53.0/5.8

Cellular transport, transport facilitation and transport routes

VVDALGVPIDGK

AAELTTLLESR

NILSTINPELLK

545

+1.83

CAA66901

Annexin p35

13

1

35.5/6.8

36.5/6.5

Cellular transport, transport facilitation and transport routes

TPAQLFAVK

(m/z = 487.7368; z = 2; error (Da) = −0.1006)i

1012

+1.79

CAA66901

Annexin p35

17

5

35.5/6.8

35.5/6.8

Cellular transport, transport facilitation and transport routes

TPAQLFAVK

LLVPLVSAYR

AQLLATFNSYK

AVILWTLDPAER

DYEDIMLALLGAE

224

+1.76

P49608

Aconitate hydratase, cytoplasmic (ACO) (Cucurbita maxima)

3

2

98.6/5.7

99.8/5.8

Energy and metabolism

SDETVSMIEAYLR

SNLVGMGIIPLCFK

1209

+1.69

P12863

Triosephosphate isomerase, cytosolic (TIM)

24

5

27.2/5.5

27.0/5.5

Energy and metabolism

VAYALSQGLK

TNASPEVAESTR

VIACVGETLEQR

ALLGESNEFVGDK

EAGSTMDVVAAQTK

1131

+1.67

CAN68719

Hypothetical protein (Vitis vinifera)

13

2

34.5/6.3

35.1/6.1

Unclassified proteins

AVQEEDELPPEK

LINPQTMMLGQEPR

341

+1.58

P49087

Vacuolar ATP synthase catalytic subunit A

27

10

62.2/5.9

59.8/5.7

Cellular transport, transport facilitation and transport routes

SGDVYIPR

FDPDFIDIR

LYDDLTTGFR

LAADTPLLTGQR

KLYDDLTTGFR

EDDLNEIVQLVGK

TTLVANTSNMPVAAR

EASIYTGITIAEYFR

LAEMPADSGYPAYLAAR

ISYIAPAGQYNLQDTVLELEFQGIK

290

+1.56

CAA31663

Heat shock protein (HSP) 70 (Petunia x hybrida)

4

2

71.1/5.1

71.1/4.6

Interaction with the environment, signalling, defense, cell rescue

VEIIANDQGNR

NQVAMNPINTVFDAK

488

+1.01

Q09054

Glyceraldehyde-3-phosphate dehydrogenase, cytosolic 2 (GAPD)

31

3

36.6/6.4

41.3/6.4

Energy and metabolism

VVISAPSK

AASFNIIPSSTGAAK

VPTVDVSVVDLTVR

286

−1.50

AAL40137

Phenylalanine ammonia-lyase (PAL)

20

11

75.3/6.5

72.2/5.9

Interaction with the environment, signalling, defense, cell rescue

ELISAIDR

VFVGISQGK

KVDAAEAFK

VGQVAAVASAK

NPSLDYGFK

DGPALQVELLR

VNELDPLLKPK

TKDGPALQVELLR

EVNSVNDNPVIDVHR

DASGVAVELDEEARPR

EAVFTYAEDAASASLPLMQK

1301

−1.50

P49101

Calcium-dependent protein kinase 2 (CDPK)

2

1

58.3/6.1

54.8/5.7

Interaction with the environment, signalling, defense, cell rescue

DIVGSAYYVAPEVLR

(m/z = 826.4362; z = 2; error (Da) = −0.0039)i

1293

−1.50

P30792

Phosphoglycerate mutase (PGAM)

5

2

60.7/5.3

72.2/5.6

Energy and metabolism

LDQLQLLLK

YLVSPPEIDR

1215

−1.51

AAL57038

UDP-glucosyltransferase BX9

22

6

50.5/5.2

47.2/5.2

Unclassified proteins

LSALLAAEGR

TDLTDLVDLIK

ALSVPVFAVAPLNK

GFESGALPDGVEDEVR

VDTSDLEEFAELLAR

LLASEDIAAIVTTLNASCDAPFR

312

−1.51

P49087

Vacuolar ATP synthase catalytic subunit A (V-ATPase)

33

10

62.2/5.9

65.9/5.4

Cellular transport, transport facilitation and transport routes

LASFYER

SGDVYIPR

TVISQALSK

FDPDFIDIR

LYDDLTTGFR

LAADTPLLTGQR

EDDLNEIVQLVGK

TTLVANTSNMPVAAR

DMGYNVSMMADSTSR

EASIYTGITIAEYFR

NIIHFNTLANQAVER

LVSQKFEDPAEGEEALVGK

EVLQREDDLNEIVQLVGK

1325

−1.51

P08440

Fructose-bisphosphate aldolase, cytoplasmic isozyme (FBA)

17

3

39.0/7.5

43.7/6.2

Energy and metabolism

NAAYIGTPGK

VTPEVIAEYTVR

IGPNEPSQLAIDLNAQGLAR

566

−1.51

AAF71261

Beta-glucosidase aggregating factor (BGAF)

6

1

31.9/6.1

33.6/5.9

Unclassified proteins

AGVYLDALGVYVR

(m/z = 698.3304; z = 2; error (Da) = −0.1095)i

404

−1.51

Q02245

Tubulin alpha chain (Alpha-tubulin)

18

4

50.5/4.9

49.9/5.2

Cellular transport, transport facilitation and transport routes

DVNAAVATIK

EIVDLCLDR

LISQIISSLTTSLR

AIFVDLEPTVIDEVR

P18025

Tubulin beta-chain (Beta tubulin)

11

3

50.5/4.8

49.9/5.2

YLTASAMFR

FPGQLNSDLR

EVDEQMLNVQNK

1251

−1.53

P49036

Sucrose synthase 2 (SUS)

22

9

93.5/6.0

87.1/6.1

Energy and metabolism

SLSALQGALR

AMENEMLLR

ASALLVDFFDK

NLTGLVELYGR

LKDGAFEDVLR

VNVSELAVEELR

LLPDATGTTCGQR

TMASTVPLAVEGEPSSK

IGDSLSAHPNELVAVFTR

P04712

Sucrose synthase 1 (SUS) (Shrunken-1)

17

5

92.1/6.0

87.1/6.1

ALENEMLLR

MYSLIDEYK

YIEMFYALK

AADILVNFFDK

VIGTEHTDIIR

405

−1.54

Q02245

Tubulin alpha-5 chain (Alpha-5 tubulin)

 

5

50.2/4.9

48.9/5.2

Cellular transport, transport facilitation and transport routes

EDAANNFAR

EIVDLCLDR

LISQIISSLTTSLR

AIFVDLEPTVIDEVR

AVCMISNNTAVAEVFSR

P33627

Tubulin alpha-6 chain (Alpha-6 tubulin)

5

50.2/4.9

48.9/5.2

EDAANNFAR

EIVDLCLDR

LISQIISSLTTSLR

AIFVDLEPTVIDEVR

AVCMISNNTAVAEVFSR

P14640

Tubulin alpha-1 chain (Alpha-1 tubulin)

4

50.2/4.9

48.9/5.2

EDAANNFAR

LISQIISSLTTSLR

AIFVDLEPTVIDEVR

AVCMISNNTAVAEVFSR

1048

−1.55

P49106

14-3-3-like protein GF14-6

49

9

29.7/4.7

27.4/4.8

Interaction with the environment, signalling, defense, cell rescue

NLLSVAYK

DSTLIMQLLR

DAAENTMVAYK

KDAAENTMVAYK

TVDSEELTVEER

LLETHLVPSSTAPESK

AAQDIALAELAPTHPIR

QAFDEAISELDTLSEESYK

DNLTLWTSDISEDPAEEIR

1276

−1.55

Q08062

Malate dehydrogenase, cytoplasmic (MDH)

13

3

35.9/5.8

39.9/5.7

Energy and metabolism

LNVQVSDVK

IVQGLPIDEFSR

VLVVANPANTNALILK

466

−1.55

P26301

Enolase 1 (ENO) (2-phosphoglycerate dehydratase 1)

3

1

48.2/5.2

43.0/5.7

Energy and metabolism

VQIVGDDLLVTNPTR

(m/z - 820.4318; z - 2; error (Da) = −0.0451)i

546

−1.55

Q6XZ79

Fructokinase-1 (FRK)

8

1

34.8/4.9

36.5/5.0

Energy and metabolism

APGGAPANVAIAVSR

(m/z = 675.8791; z = 2; error (Da) = 0.0021)i

1033

−1.56

P49076

ADP-ribosylation factor (ARF)

16

2

20.7/6.3

17.7/5.9

Interaction with the environment, signalling, defense, cell rescue

DAVLLVFANK

ILMVGLDAAGK

1298

−1.58

P93804

Phosphoglucomutase, cytoplasmic 1 (Glucose phosphomutase 1) (PGM)

22

8

63.3/5.4

66.6/5.6

Energy and metabolism

GATIVVSGDGR

LVTVEDIVR

LSGTGSVGATIR

SMPTSAALDVVAK

YDYENVDAGAAK

DSQDALAPLVDVALK

ATTPFDGQKPGTSGLR

YNMGNGGPAPESVTDK

P93805

Phosphoglucomutase, cytoplasmic 2 (Glucose phosphomutase 2) (PGM)

20

7

63.2/5.4

66.6/5.6

GATIVVSGDGR

LVTVEDIVR

LSGTGSVGATIR

SMPTSAALDVVAK

YDYENVDAGAAK

DSQEALAPLVDVALK

YNMGNGGPAPESVTDK

422

−1.59

AAL57038

UDP-glucosyltransferase BX9

24

6

50.5/5.2

46.9/5.3

Unclassified proteins

LSALLAAEGR

FVPVTVEADPK

TDLTDLVDLIK

VGTELVGEQLER

GFESGALPDGVEDEVR

VDTSDLEEFAELLAR

1262

−1.64

ABC59687

Lipoxygenase (LOX)

20

10

100.5/6.2

100.5/6.2

Interaction with the environment, signalling, defense, cell rescue

EADAVFIR

MPAALVPYR

DTLNINALAR

MSDFLGYSLK

ETLAGVNPVLIK

AAHLEEAVVSLK

QTLINADGIFER

VFFANDTYLPSK

GVAVPDQSSPYGVR

INELEGNFIYASR

1274

−1.65

AAO32643

Cytosolic 3-phosphoglycerate kinase (PGK)

33

5

31.6/5.0

46.2/5.7

Energy and metabolism

IGVIESLLAK

ELDYLVGAVANPK

GVSLLLPTDIVVADK

GVTTIIGGGDSVAAVEK

LAAALPEGGVLLLENVR

1031

−1.66

P80639

Eukaryotic translation initiation factor 5A (eIF-5A)

10

2

17.7/5.6

19.0/5.7

Unclassified proteins

LPTDETLVAQIK

DDLRLPTDETLVAQIK

1078

−1.69

AAT42176

Putative 3-glucanase

10

4

52.4/4.8

52.5/4.8

Unclassified proteins

SVAPGNFER

YAMDLSGQGR

ALNDAGFGDTIK

ATVPLNADVYNSPK

275

−1.72

AAL33589

Methionine synthase (MS)

39

20

84.7/5.7

80.2/5.7

Energy and metabolism

YLFAGVVDGR

IPSAEEIADR

FALESFWDGK

ISEEEYVTAIK

FETCYQIALAIK

IPSAEEIADRIDK

DEAYFAANAAAQASR

GTQTLGLVTSAGFPAGK

YGAGIGPGVYDIHSPR

YTEVKPALTNMVSAAK

GMLTGPVTILNWSFVR

TLTSLSSVTAYGFDLVR

KYTEVKPALTNMVSAAK

ISEEEYVTAIKEEINK

LQEELDIDVLVHGEPER

ALAGQKDEAYFAANAAAQASR

LNLPILPTTTIGSFPQTVELR

LVVSTSCSLMHTAVDLVNETK

KLNLPILPTTTIGSFPQTVELR

YIPSNTFSYYDQVLDTTAMLGAVPER

1155

−1.72

AAR33048

Allene oxide synthase (AOS)

9

3

53.2/6.5

51.3/6.2

Interaction with the environment, signalling, defense, cell rescue

VTLAAVER

QALDTAEGLGLSR

VFGDTAGDFVPDR

230

−1.77

P49608

Aconitate hydratase, cytoplasmic (ACO) [Cucurbita maxima]

5

3

98.5/5.7

99.0/5.9

Energy and metabolism

TSLAPGSGVVTK

SDETVSMIEAYLR

SNLVGMGIIPLCFK

308

−1.78

P49087

Vacuolar ATP synthase catalytic subunit A (V-ATPase)

23

5

62.2/5.9

66.6/5.4

Cellular transport, transport facilitation and transport routes

FDPDFIDIR

LYDDLTTGFR

LAADTPLLTGQR

TTLVANTSNMPVAAR

LAEMPADSGYPAYLAAR

1083

−1.84

P49106

14-3-3-like protein GF14-6

17

4

29.7/4.7

29.8/4.8

Interaction with the environment, signalling, defense, cell rescue

NLLSVAYK

DSTLIMQLLR

DAAENTMVAYK

AAQDIALAELAPTHPIR

1253

−1.87

P04712

Sucrose synthase 1 (SUS) (Shrunken-1)

5

2

92.1/6.0

87.1/6.1

Energy and metabolism

ALENEMLLR

AADILVNFFDK

498

−2.13

Q08062

Malate dehydrogenase, cytoplasmic (MDH)

19

2

35.9/5.8

41.1/5.7

Energy and metabolism

LNVQVSDVK

MELVDAAFPLLK

1263

−2.16

ABC59687

Lipoxygenase (LOX)

10

7

100.5/6.2

100.5/6.1

Interaction with the environment, signalling, defense, cell rescue

DTLNINALAR

STTDGETVYR

ETLAGVNPVLIK

QTLINADGIFER

VFFANDTYLPSK

GVAVPDQSSPYGVR

INELEGNFIYASR

1254

−2.28

P49036

Sucrose synthase 2 (SUS)

10

3

93.5/6.0

87.7/6.0

Energy and metabolism

AMENEMLLR

VNVSELAVEELR

NMTGLVEMYGK

544

Ng

CAA66900

Annexin p33

19

3

35.7/7.13

36.6/6.2

Cellular transport, transport facilitation and transport routes

SITDEISGDFER

AYAEAYGEELLR

QAIAGLGTDENSLTR

CAA66901

Annexin p35

18

2

35.5/6.8

36.6/6.2

TPAQLFAVK

LLVPLVSAYR

1356

STh

CAN75785

Hypothetical protein (Vitis vinifera)

10

3

48.4/6.5

52.4/6.2

Unclassified proteins

NPLDLLPPSK

YNDENTVSFVTLNK

VPVLETPDGPVFESNAIAR

1369

STh

AAB40105

Actin

4

1

37.2/5.5

55.6/4.7

Cellular transport, transport facilitation and transport routes

SYELPDGQVITIAADR

(m/z = 874.3623; z = 2; error (Da) = −0.1688)i

aIncreased or decreased (±) compared to the control.

bNCBI accession number.

cProteins have been identified in Zea mays L. protein database when no species is reported.

dSequence coverage. Peptides with individual ion MOWSE scores lower then the threshold indicating identity or extensive homology (P<0.05) have not been reported, but have been considered in sequence coverage calculation.

eNumber of peptide matches.

fUnderscored methionine in sequences reported indicate oxidation.

gNewly induced.

hSuppressed by the treatment.

iIn instances where proteins were identified based on a single peptide, precursor mass, charge, and mass error observed are provided.

According to the FunCat functional classification (Ruepp et al. 2004), the proteins identified were grouped into the following categories: energy and metabolism; cellular transport, transport facilitation and transport routes; interaction with the environment, signaling, defense, cell rescue; and unclassified proteins (Table 1). Below, we briefly discuss the possible significance of these protein groups and the interpretation of changes in abundance of the protein species.

Energy and Metabolism

Glycolytic enzymes have long been considered to exist as soluble proteins in the cytosolic compartment, but in recent years it has been suggested that the cytoskeleton and/or membranes contribute to this compartmentalization (Holtgrawe et al. 2005). Protein–protein interactions exist between actin or tubulin and a number of glycolytic enzymes and enzymes of sucrose metabolism (Holtgrawe et al. 2005) that have also been found to co-localize (Anderson and Carol 2005), so it is hardly surprising to find these enzymes in PM-enriched fractions (Santoni et al. 1998, 1999b). In the present study, the enzymes identified were triosophosphate isomerase (TIM; EC 5.3.1.1), glyceraldehyde-3-P dehydrogenase (GAPD; EC 1.2.1.12), phosphoglycerate mutase (PGAM; EC 5.4.2.1), fructose biphosphate aldolase (FBA; EC 4.1.2.13), 2-phosphoglycerate dehydratase (enolase, ENO; EC 4.2.1.11), phosphoglucomutase (PGM; EC 5.4.2.2), and 3-phosphoglycerate kinase (PGK; EC 2.7.2.3). In our experimental conditions, all of the above-mentioned enzymes appear to be downregulated after HS treatment except for TIM, which showed a 69% increase, and GAPD, which maintained the same level of expression in treated and untreated samples. An effect of HS on enzymes involved in the glycolytic/gluconeogenic pathways has already been reported (Muscolo et al. 2007a), and it was thought to impair these pathways. This impairment may affect starch and sucrose metabolism. Three spots (IDs 1251; 1253; 1254) match with sucrose synthase (SUS; EC 2.4.1.13). Spot 546 has been identified as fructokinase-1 (FRK; EC 2.7.1.4) and spot 1298 as PGM, representing the link between glycolysis and starch synthesis pathways. These two proteins were downregulated by HS treatment. SUS can associate with actin, the cytoskeleton, and the plasma membrane, where it is thought to funnel uridine diphosphoglucose (UDP-glucose) to cellulose synthase to produce cellulose (Amor et al. 1995). Merlo et al. (1991) reported that SUS activity appeared to be lower in HS-treated maize leaves. The depletion of these enzymes after HS treatment in our experimental conditions may indicate a slackening of the energy machinery.

Malate has a central role in the energetics of the plant cell, and malate dehydrogenase (MDH; EC 1.1.1.37) is one of the key enzymes in malate metabolism. Sukalovic et al. (1999) found that purified PMs isolated from maize roots contain a closely associated, deeply buried, NAD+-dependent MDH, and this enzyme was also identified in Arabidopsis PM-enriched fractions (Santoni et al. 1998). This enzyme is involved in the plasma membrane redox system (Berczi and Moller 2000). Spots 498 and 1276 identified as MDH, showed downregulation after HS treatment in both cases. An in vitro inhibition of this enzyme has already been reported after treatment with HS (Pflug and Ziechmann 1981), whereas activity was reportedly stimulated in carrot cell cultures and, more recently, in maize leaves (Nardi et al. 2007).

Aconitase (ACO; EC 4.2.1.3) catalyzes the reversible isomerization of citrate to isocitrate via the intermediate product cis-aconitate. There are two isoforms of ACO; the mitochondrial isoform is a component of the citric acid cycle, whereas the cytosolic isoform participates in the glyoxylate cycle (Moeder et al. 2007). We identified two adjacent protein spots as cytosolic ACO from Cucurbita maxima, one showing downregulation (spot 230, −77%) and the other upregulation (spot 224, +79%). This result is suggestive of a posttranslational modification of this enzyme. Citric acid is a component of root exudates and plays an important role in disaggregating HS (Nardi et al. 2000a). ACO expression may induce changes in root excretion participating in the rhizospheric cross-talk between plant and soil. Methionine synthase (MS, EC 2.1.1.14) was found as a downregulated protein: the function of this enzyme is the de novo synthesis of methionine on one hand and, on the other, the regeneration of the methyl group of S-adenosyl methionine from S-adenosyl homocysteine after methylation reactions (Hesse et al. 2004). Although cytosolic and plastid isoforms have been reported, this enzyme has also been identified in PM extracts (Santoni et al. 1999b). Muscolo and Nardi (1997) found that HS-treated plants showed no change in methionine concentrations.

Cellular Transport, Transport Facilitation and Transport Routes

Annexin, actin and tubulin isoforms were identified in our samples. These proteins have been reported already in studies on the Arabidopsis plasma membrane proteome (Santoni et al. 1999a, 2000; Marmagne et al. 2004). Actin and tubulin are major components of the cytoskeleton, forming microfilaments and microtubules, respectively. The cytoskeleton coordinates all aspects of growth in plant cells, including exocytosis of the membrane and wall components during cell expansion. Both proteins are reported to be associated with plasma membranes and also an interaction between the two proteins has been demonstrated by Wasteneys and Galway (2003). It is generally assumed that annexins are implicated in several processes related to membranes, including the regulation of membrane organization, membrane trafficking, interactions with the cytoskeleton, and secretion (Konopka-Postupolska 2007), but the primary physiological function of annexins has yet to be elucidated. Certain proteins from this family were identified as actin binding, making them ideal mediators in cell membrane and cytoskeleton interactions (Konopka-Postupolska 2007). In this study, the protein spots 545 and 1012, identified as annexin, were upregulated by about 80%. Spot 544, also identified as annexin, was detectable in 10 of the 12 gels generated from treated roots and in none of the gels obtained from control samples. The fact that multiple spots in the same gel area are recognized as annexin may be due to posttranslational modifications, e.g., phosphorylation has been reported for maize annexin (Delmer and Potikha 1997). Due to the role of annexins in cell exocytosis and cell expansion, their upregulation may be correlated with modifications in plant metabolism leading to humic substances-induced stimulation of plant growth (Canellas et al. 2002). Spot 1369, identified as actin, was detectable in seven of the gels obtained from control samples and in none of the HS-treated ones: this indicates that PM-associated actin is low in untreated plants and drops to below the detection limits in treated plants. Spots identified as tubulin showed a depletion of about 53% in the gels obtained from treated plants. Alpha tubulin expression was reported to be modulated by cell sugar content (Datta and Chourey 2001), so this result may correlate with the previously shown modifications in the enzymes of the sucrose pathway. These effects point to an involvement of the cytoskeleton proteins in the response to HS. The responsiveness of the cytoskeleton to environmental cues has been widely documented (for a review, see Wasteneys and Galway 2003).

ATPases are membrane-bound ion transporters that couple ion movement through a membrane with the synthesis or hydrolysis of ATP. Different forms of membrane-associated ATPases have evolved over time to meet specific cell demands. V-ATPases hydrolyze ATP to drive a proton pump but cannot work in reverse to synthesize ATP. They are involved in a variety of vital intra- and inter-cellular processes such as receptor-mediated endocytosis, protein trafficking, and active transportation of metabolites. The F-ATPases (F(0)F(1)-ATPases) can synthesize ATP by using a H+ gradient, and they work in reverse to create a H+ gradient by using the energy gained from the hydrolysis of ATP. Subunits belonging to either the V1 vacuolar sector or the F1 mitochondrial complex of ATPase were found among the differentially expressed proteins in this research: these proteins are classically thought to reside in the tonoplast (Nelson and Harvey 1999) and in the mitochondrial membrane, respectively, although they were found in the PM in other organisms (Nelson and Harvey 1999). The presence of these two proteins either reveals a true contamination caused by technical problems or it reflects functional interactions between the PM and these particular structures (Marmagne et al. 2004). Moreover, V-ATPase subunits have been identified already in highly purified plasma membrane extracts (Santoni et al. 1998; Marmagne et al. 2004). In the case of the regulatory alpha subunit of mitochondrial ATP synthase that shows an almost three-fold relative increase in the treated samples, this effect may be caused by an enhanced metabolic energy demand in response to HS. In the case of vacuolar ATPase, the result is apparently contradictory, given the presence of two downregulated spots (308 and 312, −78% and −51%, respectively) and one upregulated spot (341, +58%). This variation may result from posttranslational modifications of the protein that could not be resolved in this work. In most of the previous cases, the results were attributed to the plasma membrane E1-E2-type ATPase, but changes in vacuolar (Pinton et al. 1992) and mitochondrial (Visser 1987) ATPases have also been found.

Others

A number of the differentially expressed proteins are involved in the plant’s interaction with the environment, while the functions of others have yet to be clarified. The interpretation of the changes in their abundance after the HS treatment is more challenging because of the complex network of signaling pathways. In some cases, the changes observed might reflect interesting aspects of plant metabolism that deserve further study. For example, beta-glucosidase-aggregating factor (BGAF) (spot 566, lowered by the treatment) interacts with beta-glucosidases (Kittur et al. 2007), which may release IAA from its glycosidic form, thus influencing IAA availability and possibly inducing a hormonal effect (Pizzeghello et al. 2001). Phenylalanine ammonia-lyase (PAL) expression was also affected by HS. As it links the primary metabolism of aromatic amino acids with the secondary metabolism of the phenylpropanoid pathway, its downregulation can be interpreted as forcing carbon skeletons toward energy metabolism.

To the best of our knowledge, this is the first proteomic analysis of changes induced in maize root plasma membranes after exposure to HS. A total of 42 differentially expressed proteins were identified. We focused on the proteins that relate to energy and metabolism, and cellular transport, transport facilitation, and transport routes. The majority of the proteins identified were downregulated. HS appeared to affect sucrose metabolism, malate dehydrogenase, ATPases, and cytoskeletal proteins. In some cases, our data better illustrate effects already reported in more simple models (i.e., Nicotiana tabacum callus), whereas in other cases, they enable a more detailed description of previous findings, or help to explain earlier results reported on the level of enzyme activity.

HS concentration and time of incubation are major parameters in unraveling plant responses to HS. Given the theoretical and practical limitations of proteomics, future investigations should consider these issues. Moreover, to investigate completely the HS/maize system, changes on the m-RNA level in HS-treated plants should also be considered in future research.

The proteins analyzed in this study are only a small part of the maize proteome; many other HS target proteins remain to be identified. However, the HS-responsive proteins in the root PM of Z. mays reported in this work can serve as a platform for future work investigating plant reactions to HS.

Notes

Acknowledgments

The authors thank Dr. Ornella Francioso of the Dipartimento di Scienze e Tecnologie Agroambientali, Università degli Studi di Bologna, for the DRIFT characterization of humic substances. The authors are also grateful to Prof. Angelo Fontana (CRIBI) for fruitful discussions.

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Paolo Carletti
    • 1
  • Antonio Masi
    • 1
  • Barbara Spolaore
    • 2
  • Patrizia Polverino De Laureto
    • 2
  • Mariangela De Zorzi
    • 1
  • Loris Turetta
    • 1
  • Massimo Ferretti
    • 1
  • Serenella Nardi
    • 1
  1. 1.Department of Agricultural BiotechnologyUniversity of PaduaPadovaItaly
  2. 2.Centro di Ricerca Interdipartimentale per le Biotecnologie Innovative (CRIBI)University of PaduaPadovaItaly

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