当前位置:首页 >> 生物学 >>

Comparative proteome analyses of phosphorus responses in


The Plant Journal (2008) 55, 927–939

doi: 10.1111/j.1365-313X.2008.03561.x

Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus ef?ciency
Kunpeng Li, Changzheng Xu, Zhaoxia Li, Kewei Zhang, Aifang Yang and Juren Zhang* School of Life Science, Shandong University, 27 Shanda South Road, Jinan, Shandong 250100, China
Received 3 February 2008; revised 28 March 2008; accepted 7 May 2008; published online 10 July 2008. * For correspondence (fax +86 531 8856 4350; e-mail jrzhang@sdu.edu.cn).

Summary Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P ef?ciency in maize, comparative proteome analyses were performed on the roots of the low-Ptolerant mutant 99038 and wild-type Qi-319 grown under P-suf?cient (+P) or P-de?cient ()P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or )P conditions. We identi?ed 73 (+P) and 95 ()P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was signi?cantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-ef?cient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation. Keywords: comparative proteome, global protein expression, maize, root, low-P-tolerant mutant, phosphorus.

Introduction Phosphorus (P) is an essential macronutrient for plant growth and development, and plant growth is often limited by the lack of phosphorus nutrients due to the heterogeneous distribution and low mobility of P (Franco-Zorrilla et al., 2004; Raghothama, 1999). To cope with inadequate phosphate (Pi) supplies, plants have evolved many adaptive strategies, which include modi?cations in carbon metabolism bypassing steps that require P, increased production and secretion of organic acids, and the re-design of root ? ndez et al., 2007; Raghothama, 1999; morphology (Herna Wasaki et al., 2003, 2006). Among these useful traits, main? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd

tenance of root growth and re-programming of root architecture are critical for Pi uptake because Pi is only acquired ef?ciently by root systems with large surface areas (Lai et al., 2007). Zhu and Lynch (2004) demonstrated that variation in lateral rooting may be an important component of genetic variation for P ef?ciency in maize. Genetic studies on the characteristics of maize lateral roots, seminal roots and root hairs indicated that these complex root traits are closely associated with several genes or QTLs and/or loci (Zhu et al., 2005a,b, 2006). Recently, LPR1 (LOW PHOSPHATE ROOT1) was identi?ed as a major quantitative trait locus with a
927

928 Kunpeng Li et al. considerable effect on primary root growth arrest in response to Pi starvation. The LPR1 protein and its paralogue LPR2 enable Arabidopsis primary root cap cells to sense low-Pi environments, which triggers primary root growth arrest (Svistoonoff et al., 2007). There is growing evidence that the magnitude of Pistarvation responses is regulated both locally, dependent on external Pi supply, and systemically, dependent on the Pi status of the whole plant (Franco-Zorrilla et al., 2004; Jain et al., 2007). Plant Pi-starvation responses are a complex event. Several transcription factors that are involved in the response to Pi starvation have been characterized, including the MYB transcription factor PHR1, the bHLH transcription factor OsPTF1 and the WRKY75 transcription factor (Devaiah et al., 2007; Franco-Zorrilla et al., 2004; Rubio et al., 2001; Yi et al., 2005). Recently, some studies have suggested that sugars are not only metabolites but also signal molecules mediating multiple physiological responses (Rolland and Sheen, 2005), and that cross-talk between systemic Pi-starvation responses and sugar sensing occurs in Arabidopsis (Franco-Zorrilla et al., 2005; Jain et al., 2007; Karthikeyan et al., 2007; Liu et al., 2005; Mu ¨ ller et al., 2007). More interestingly, the studies in Arabidopsis suggested that ‘determinate‘ root development is correlated with root meristem exhaustion (Sanchez-Calderon et al., 2005), and the cell-division activity seems to determine the magnitude of expression of phosphate starvation-induced (PSI) genes (Lai et al., 2007). As a consequence, those factors that promote cell division, including sucrose, enhance the magnitude of the Pi-starvation response in Arabidopsis, and those inhibiting cell proliferation reduce the responses to Pi starvation (Desnos, 2007; Lai et al., 2007). Roots traits for nutrient ef?ciency may be associated with altered growth behaviour in adaptation to nutrient stress. The systemic regulation mechanism of the Pi-starvation response may be involved in adaptation to P de?ciency at the whole-plant level. Therefore, systemic studies are required to understand P-nutrient characteristics of roots. Wu et al. (2003) used microarrays to analyse gene expression pro?les in Arabidopsis, and found that expression of approximately 29% of the genes on the microarray was latered twofold or more during Pi starvation. Misson et al. (2005) used the Arabidopsis whole-genome Affymetrix GeneChip to analyse the variations in transcript abundance of 22 810 genes. They revealed a coordinated induction or suppression of 612 and 254 PSI genes, respectively. Moreover, several transcripteomic analyses of the response to Pi starvation have also been performed in other species, ? ndez including white lupin, rice, and common bean (Herna et al., 2007; Uhde-Stone et al., 2003; Wasaki et al., 2003, 2006). All these results indicated that the plant response to Pi starvation is a coordinated regulatory process. However, the metabolic changes that occur under Pi starvation cannot all be explained by transcripteomic data. It is expected that proteomic analysis will also provide useful information for understanding the response to Pi starvation in plants. Recently, we performed systematic proteome analysis of maize roots in response to Pi starvation, which revealed that the response to Pi starvation is a complicated process involving carbon metabolism, regulation of the cell cycle, signal transduction, etc (Li et al., 2007a). However, a comparative proteome analysis of the response to Pi starvation in maize genotypes with different tolerance to low-P conditions has not been performed. Although some aspects of the strategies plant use to cope with low-P environments are understood, the majority are not; a broad view of protein expression is necessary to elucidate all of the mechanisms fully. Previously, we obtained the low-P-tolerant mutant 99038 from Qi-319 by somaclonal variation. They have a common genetic background but contrasting root systems, especially under Pi de?ciency (Li et al., 2007b). In this study, comparative proteome analyses were performed for the roots of mutant 99038 and wild-type Qi-319 treated with 1000 lM (+P) or 5 lM ()P) KH2PO4 for 17 days. The objectives of this research were to (i) determine the changes in protein expression or metabolic pathways that cause the differences in root morphology and tolerance to low-P environments between the two genotypes, (ii) improve our understanding of the genetic control mechanisms for P ef?ciency in maize, and (iii) provide valuable information for further study regarding the functions of genes involved in the response to Pi starvation. Results Differential analyses of root protein expression pro?les Previously, we found that Coomassie brilliant blue (CBB) and silver staining methods produced different results for some proteins due to the different staining mechanisms (Li et al., 2007a). To better analyse the differences in the root proteome between Qi-319 and 99038, we used a combination of both silver and CBB staining (Figures 1 and 2). The number of spots detected and the number that showed differential accumulation in the two genotypes are summarized in Table S1. Under +P conditions, approximately 1215 protein spots were detected on 2-DE gels, and, of these, 135 (11.1%) were differentially expressed between Qi-319 and 99038. Under )P conditions, we detected approximately 1607 proteins, and, of these, 196 (12.1%) were differentially expressed between the two genotypes. Of the 135 spots that were differentially expressed under +P conditions, 72 indicated higher accumulation in 99038 and 63 indicated higher accumulation in Qi-319. Of the 196 spots that were differentially expressed under )P conditions, 118 indicated higher accumulation in 99038 and 78 indicated higher accumulation in Qi-319. In total, of the differentially accumulated proteins

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

Root proteome for phosphorus ef?ciency in maize 929
Figure 1. Comparison of 2-DE gel maps of root proteins of 99038 and Qi-319 treated with 1000 lM KH2PO4 (+P) for 17 days. Seedlings (4 days old) were cultured for 20 days in +P nutrient solution, and then grown for an additional 17 days under +P conditions. The root proteins were extracted, and 150 lg protein (for silver staining) (a) or 1 mg protein (for CBB staining) (b, c) were separated in the ?rst dimension using pH 5-8 or pH 3-6 IPG gels (17 cm), and then in the second dimension using a 12% polyacrylamide gel. Image analysis was performed using PDQuest software, version 7.2.0 (Bio-Rad). The spots marked with numbers (H1– H73) indicate proteins that were differentially accumulated in the roots of 99038 and Qi-319 under +P conditions identi?ed by MALDI-TOF MS.

(a)
97.0

IEF 5
H1

pI

8
99038 97.0

IEF 5
H15 H16 H17

pI

8

Qi-319
H18

66.0
H2 H3 H4 H8 H10

66.0
H21 H23 H20 H19 H25 H24 H22

45.0 Mr (kDa)

H9

H7 H11

Mr (kDa)

H6

H5

45.0

H27 H29

H28

H26

30.0

H12 H13 H14

30.0
H30

20.1

20.1

H31 H32

14.4

14.4

(b)
97.0

IEF 5
H34

pI

8

IEF 5
97.0
H53 H54

pI

8

99038
H33

Qi-319
H52 H55 H56 H58 H57

66.0

H36

66.0
H35 H38 H39 H41 H43 H40

45.0 Mr (kDa)

H37

45.0

H60 H62 H63

H59

H42 H46 H45 H44 H47

Mr (kDa)

H61 H64

30.0

H48 H49 H50 H51

30.0

20.1

20.1

14.4

14.4

(c)
97.0

IEF 3 pI 6

IEF 3
97.0

pI

6

99038

Qi-319
H68 H69

66.0

66.0

H70

H65
45.0 45.0

H71

Mr (kDa)

H66

30.0

H67
20.1 20.1

Mr (kDa)

H72

30.0

H73

14.4

14.4

under +P and )P conditions, 95 and 141 showed responses to Pi starvation in the roots of Qi-319 and/or 99038 (Table 1). Protein identi?cation and classi?cation To better understand the biological processes that result in the different root characteristics and tolerance to low-P stress between 99038 and Qi-319, some proteins that were

differentially accumulated in response to Pi starvation were analysed by MALDI-TOF MS and identi?ed by MASCOT. An example of a peptide mass ?ngerprinting spectrum is shown in Figure S1. The identi?ed proteins were classi?ed based on the Munich Information Center for Protein Sequence Arabidopsis thaliana database (MATDB; http://mips.gsf.de/ proj/thal/db/) (Schoof et al., 2002). In total, 73 proteins that differentially accumulated under +P conditions were

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

930 Kunpeng Li et al.

(a)
97.0

IEF 5
L2 L3 L4 L5 L1

IEF pI 8 5 97.0
L8 L6 L9 L10 L13 L11 L12 L30 L26

pI

8

99038
L27 L28 L24 L25 L29

Qi-319

66.0

L7

66.0

45.0 Mr (kDa)

45.0 Mr (kDa)
L36

L31

L32

L14 L16 L18 L17

L15 L20

L35

L34 L33

30.0
L21

L19 L22

30.0

20.1 14.4

L23

20.1 14.4

Figure 2. Comparison of 2-DE gel maps of root proteins of 99038 and Qi-319 treated with 5 lM KH2PO4 ()P) for 17 days. Seedlings (4 days old) were cultured for 20 days in +P nutrient solution, and then grown for an additional 17 days in )P conditions. The root proteins were extracted, and 150 lg protein (for silver staining) (a) or 1 mg protein (for CBB staining) (b, c) were separated in the ?rst dimension using pH 5-8 or pH 3-6 IPG gels (17 cm) and then in the second dimension using a 12% polyacrylamide gel. Image analysis was performed using PDQuest software, version 7.2.0 (Bio-Rad). The spots marked with numbers (L1L95) indicate proteins that were differentially accumulated in roots of 99038 and Qi-319 under )P conditions identi?ed by MALDI-TOF MS.

(b)
97.0 66.0

IEF 5 pI
L37 L38 L39 L40 L41 L43 L42 L45 L49 L50 L53 L51 L56 L57 L52 L58 L61 L60 L59 L62

IEF 8 5 97.0 66.0
L44 L46 L47 L48
L81 L66 L68 L75 L77 L73 L80

pI
L67 L71 L70 L72 L78 L79 L69

8

99038

Qi-319

L74 L76

45.0 Mr (kDa)

45.0 Mr (kDa)
L82 L84 L85 L86 L83

L54 L55

30.0

L65 L64

30.0
L90

L89

L88

L87

L63

20.1 14.4

20.1 14.4 IEF 3

L91

(c)
97.0

pI

6

99038
97.0

IEF 3

pI

6

Qi-319

66.0

66.0

45.0 Mr (kDa)
L92

45.0 Mr (kDa)

L93

L94

30.0

30.0
L95

20.1

20.1

14.4

14.4

identi?ed, and 95 that differentially accumulated under )P conditions were identi?ed. These proteins represented a large range of functional categories, and were involved in multiple pathways including carbon and energy metabolism, signal transduction, regulation of the cell cycle, and phytohormone metabolism (Tables S2 and S3). Of the 73 (+P) and 95 ()P) differentially accumulated proteins, 26 proteins with similar function were present under both P conditions, and 17 had consistently changing

trends that were involved in multiple pathways (Table 2). Thirteen proteins were involved in metabolism, all of which were involved in carbon metabolism, with the exception of S-adenosylmethionine synthetase, and nine of these (75%) had the same changing pattern under both P conditions in both genotypes (Table 2). Moreover, the vacuolar ATPase subunit and GTP-binding nuclear protein RAN-B1, which are involved in the maintenance of ion homeostasis, the regulation of cell proliferation and signal transduction, also

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

Root proteome for phosphorus ef?ciency in maize 931
Table 1 Number of differentially accumulated proteins between the two genotypes and in response to Pi starvation on 2-DE gels (Figures 1 and 2) Qi-319 + P versus 99038 + P No. proteins over-accumulated in 99038 No. proteins over-accumulated in Qi-319 Total no. differentially accumulated proteins No. differentially accumulated proteins in response to Pi starvationa
a

Qi-319 )P versus 99038 )P 118 78 196 141

72 63 135 95

Differentially accumulated proteins in response to Pi starvation means the proteins that accumulate differentially between both genotypes and are also differentially accumulated in Qi-319 + P versus Qi-319 )P and/or 99038 +P versus 99038 )P.

showed the same changing pattern under both P conditions in both genotypes. These proteins that were differentially accumulated in the two genotypes showed the same changing pattern under different P conditions, which may mostly result from genotype differences. Further analyses of identi?ed proteins indicated that 14 (+P) and 27 ()P) proteins were over-accumulated in response to Pi starvation in the two genotypes and showed higher over-accumulation in the low-P-tolerant mutant 99038 compared with Qi-319 (Table S4 and S5). These proteins showed an obvious functional dominance of carbon metabolism (16 proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, UDP-glucose pyrophosphorylase and pyruvate phosphate dikinase), and regulation of cell proliferation (four proteins, including GTP-binding nuclear protein RAN-B1, the importin a2 subunit and mini-chromosome maintenance protein). Based on these results, we conclude that there are considerable genotypic differences in carbon metabolism and regulation of cell proliferation between 99038 and Qi-319. Differentially accumulated proteins indicate differences in carbon metabolism between the low-P-tolerant mutant and the wild-type The abundances of several proteins that participate in carbon metabolism showed obvious differences between genotypes under +P or )P conditions. These proteins were involved in tricarboxylic acid cycle, pentose phosphate pathway, glycolysis, etc. Our results showed that, under )P conditions, the amount of malate dehydrogenase (spot L58), the pyruvate dehydrogenase E1a subunit (spot L13) and citrate synthase (spot L11) in the roots of 99038 plants increased signi?cantly compared with Qi-319, while the amount of NAD+-dependent isocitrate dehydrogenase subunit 1 (spot L31) and aconitate hydratase (spot L66)

decreased signi?cantly. The accumulation of malate dehydrogenase and pyruvate dehydrogenase complex proteins may accelerate the production of substrates for citrate synthesis. An increase in the amount of citrate synthase may accelerate the synthesis of citrate, while the decrease in isocitrate dehydrogenase and aconitate hydratase may reduce the utilization of citrate. The proteome data suggest that 99038 plants could produce more citrate than Qi-319 under Pi-starvation conditions. To verify these alterations, the activities of malate dehydrogenase, citrate synthase and aconitase were analysed. The activities of citrate synthase and malate dehydrogenase in the roots of 99038 were signi?cantly higher than those in Qi-319 (Table 3), which is consistent with the 2-DE results (Table S3). However, the activity of aconitase showed no signi?cant difference between the genotypes under both +P and )P conditions, which might result from differences in their activity state. To test this hypothesis, we also measured the amount of citrate in the root tissues and exudates. The amounts of citrate in the root tissues and exudates of 99038 were signi?cantly higher than in Qi-319 under both P conditions (P < 0.05), con?rming that 99038 plants could accumulate and secrete more citrate than Qi-319, especially under Pi-starvation conditions (Table 3). The amounts of cytosolic 6-phosphogluconate dehydrogenase (spots H3, L46 and L47) in roots of 99038 plants were also signi?cantly higher than those of Qi-319 under both P conditions. As an important regulatory enzyme in pentose phosphate pathway, its increase suggests that the pentose phosphate pathway might be enhanced in the roots of 99038 compared with Qi-319 under the different P conditions. This might be useful in order to satisfy the requirements of cells for intermediate products of sugar metabolism and reductants, and facilitate root development in 99038. Several proteins related to glycolysis, such as the pyruvate phosphate dikinase subunit (spot L37), UDP-glucose pyrophosphorylase (spot L43), sucrose synthase (spots H16, L67, L70 and L71), and pyruvate kinase-like proteins (spots L6 and L73), were also differentially accumulated between genotypes under +P and/or )P conditions. Pyruvate phosphate dikinase and UDP-glucose pyrophosphorylase can utilize pyrophosphate (PPi) and produce ATP or UTP. The increase in the amount of pyruvate phosphate dikinase and UDPglucose pyrophosphorylase in the roots of 99038 under )P conditions suggests that 99038 plants could use in vivo P more ef?ciently than Qi-319. Whether under +P or )P conditions, the amounts of sucrose synthase in the roots of 99038 plants were lower than in Qi-319. Given that sucrose synthase plays an important role in sucrose metabolism, the amounts of sucrose in the roots of both genotypes may vary. To further test the hypothesis, we performed sugar composition analysis by TLC. The proportion of sucrose in the total soluble sugars of 99038 roots was signi?cantly higher than in Qi-319 roots (Figure 3).

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

932 Kunpeng Li et al.
Table 2 Differentially accumulated proteins with similar function present in the roots of 99038 and Qi-319 under both +P and )P conditions 99038 +P/Qi-319 +P Spot numbera Metabolism Fructokinase-2 (ZmFRK2) Pyruvate phosphate dikinase Aconitate hydratase Isocitrate dehydrogenase subunit Methionine synthase Alcohol dehydrogenase 1 Alcohol dehydrogenase 2 Cytosolic 6-phosphogluconate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase, cytosolic 3 Phosphoglycerate mutase Endosperm C-24 sterol methyltransferase S-adenosylmethionine synthetase Sucrose synthase 2 Patternb 99038 )P/+P 1:2.04 2.00:1 1:1.24, 1:3.84 9.32:1 6.22:1 2.05:1 1:1.04 1:1.07 Qi-319 )P/+P 1.39:1 1.04:1 2.20:1, 1:8.33 1:1.33 3.56:1 1:1.35 2.05:1 3.73:1 99038 )P/Qi-319 )P Spot number Pattern 99038 )P/+P 1.02:1, 1:4.87 13.33:1 1.98:1 1:1.49 2.33:1 1.06:1 1:1.55 4.20:1, 3.70:1 1:4.64 5.30:1 1:2.56 1:2.02 1.00:1, 1.67:1, )P only 5.51:1 1:11.33 1:1.27 Qi-319 )P/+P 2.36:1, 2.25:1 4.61:1 4.14:1 2.13:1 6.78:1 2.15:1 1:2.74 2.83:1, 1.76:1 1:1.85 2.13:1 1:1.75 1:8.55 5.29:1, 13.12:1, )P only 1.21:1 1:3.60 3.79:1 Comparison of patternc

H43 H68 H1, H15 H63 H53 H25 H38 H3

Increase Decrease Increase, Decrease Decrease Decrease Decrease Increase Increase

L36, L83 L37 L66 L31 L68 L91 L54 L46, L47 L88 L39 L35 L49 L67, L70, L71 L50 L86 L26

Decrease Increase Decrease Decrease Decrease Decrease Increase Increase

Opposition Opposition Opposition, agreement Agreement Agreement Agreement Agreement Agreement

H61 H35 H26 H65 H16

Decrease Increase Decrease Increase Decrease

2.00:1 1:2.62 1:1.21 1:3.81 1.05:1

1.98:1 1.12:1 1:2.30 1.13:1 1:6.37

Decrease Increase Decrease Increase Decrease

Agreement Agreement Agreement Agreement Agreement

Protein synthesis Translational initiation factor H71 eIF-4A Elongation factor Tu H28 Protein fate Luminal-binding protein H69, (HSP70 homologue) H70 Defence/interaction with environment Anionic peroxidase H10 Glutathione-S-transferase H29, GST 21 H30 Cytosolic ascorbate H48 peroxidase Cellular organization Tubulin a3 chain H22 Transcription/cell cycle/signal transduction Glycogen synthase kinase-3 H7 homologue MsK-3 Protein kinase family protein H24 GTP-binding nuclear protein H13 RAN-B1 Transport Vacuolar ATPase subunit H36 Unclassi?ed/unknown protein Predicted P0016F11.25 gene H27 product Hypothetical protein H62
a

Decrease Decrease Decrease

12.56:1 1:2.76 7.97:1, 4.50:1 1:2.75 3.10:1, 5.07:1 1.21:1

2.53:1 1:7.56 4.05:1, 1.49:1 1.82:1 1:2.09, 2.82:1 2.19:1

Increase Decrease Decrease

Opposition Agreement Agreement

Increase Decrease Increase

L34 L20, L90 L63

Decrease Increase, Decrease Increase

1:2.75 2.84:1, 2.31:1 2.21:1

1.82:1 1:1.76, 5.76:1 1.25:1

Opposition Opposition, agreement Agreement

Decrease Increase Decrease Increase

1.07:1 1:1.69 2.49:1 1.17:1

1:2.04 2.28:1 1:1.19 3.94:1

L92 L33 L51 L19

Increase Decrease Increase Increase

)P only +P only 4.21:1 1:1.05

NDd 1.04:1 1.79:1 1:2.62

Opposition Opposition Opposition Agreement

Increase Decrease Decrease

1:1.31 5.51:1 8.32:1

2.61:1 1:1.40 1.77:1

L17 L85 L32

Increase Decrease Decrease

3.12:1 1:2.54 )P only

1.63:1 1:1.16 )P only

Agreement Agreement Agreement

Spot numbers correspond to the numbers in Tables S2 and S3. ‘Increase’ indicates signi?cance at P < 0.05 and an increase in amount at least twofold on the 99038 gel under +P or )P conditions; ‘decrease’ indicates signi?cance at P < 0.05 and a decrease in amount at least twofold on the 99038 gel under +P or )P conditions, compared to Qi-319. c Comparison of pattern: ‘agreement’ means that the pattern changed in the same direction (increase or decrease) between 99038 and Qi-319 under both +P and )P conditions; ‘opposition’ means that the pattern changed in different directions. d ND, not detected.
b

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

Root proteome for phosphorus ef?ciency in maize 933
Table 3 Activity of citrate synthase, malate dehydrogenase and aconitase, and amounts of citrate in the root tissue and exudates Qi-319 +P Aconitase activity (U) Malate dehydrogenase activity (U) Citrate synthase activity (U) The amounts of citrate in root tissue (lm g)1 root FW) The amounts of citrate in root exudates (lm plant)1 h)1) 8.57 ? 0.29b 2.06 ? 0.06c 1.74 ? 0.16c 4.35 ? 0.96d 0.080 ? 0.006c 99038 +P 9.75 ? 1.49ab 2.71 ? 0.35b 3.26 ? 0.47b 9.20 ? 0.36c 0.105 ? 0.011b Qi-319 )P 9.96 ? 0.62a 2.72 ? 0.08b 2.61 ? 0.19b 10.75 ? 0.71b 0.110 ? 0.080b 99038 )P 9.45 ? 0.23a 4.31 ? 0.69a 4.89 ? 0.87a 14.20 ? 0.43a 0.166 ? 0.018a

Values shown represent the means of nine seedlings ? SD. Different superscript letters are used to indicate means that differ signi?cantly (P < 0.05) within a row. The activities of aconitase, malate dehydrogenase and citrate synthase were assayed spectrophotometrically at A240, A340 and A412, respectively. 1 U = 100 DA min)1 mg protein)1.

(a)
Glucose Fructose Sucrose Lactose Raffinose

(b)
% sucrose contents in total soluble sugars 45 40 35 30 25 20 15 10 5 0
Qi-319-P 99038-P Qi-319-P 99038-P

a b b

a

1

2

3

4

5

6

Figure 3. Analyses of the proportions of sucrose in the roots of Qi-319 and 99038 plants under different P conditions. (a) TLC analyses of sucrose extracted from the roots of Qi-319 and 99038 plants. Ten micrograms of total soluble sugars were spotted onto the TLC foil. Lane 1, standard mixture (glucose, fructose, sucrose, lactose, raf?nose); lane 2, sucrose; lanes 3–6, sugar solution extracted from the roots of Qi-319 +P, 99038 +P, Qi-319 )P and 99038 )P plants, respectively. (b) Proportion of sucrose in total soluble sugars extracted from roots of Qi-319 and 99038 plants, measured at 390 nm by chromatographic scanning using a CS-930 dual-wavelength thin chromatographic scanner (Shimadzu). Results are means and SD from three experiments. Different letters indicate means that differ signi?cantly (P < 0.05).

Differentially accumulated proteins reveal differences in the regulation of cell proliferation between the genotypes The amounts of several proteins that participate in the regulation of cell proliferation showed obvious differences between the genotypes under +P or )P conditions. Under )P conditions, the amount of phosphoprotein phosphatase 2A isoform 4 (PP2A, spot L15) increased signi?cantly in 99038 roots compared to Qi-319. Genetic analysis in Arabidopsis has shown that PP2A plays important roles in hormonemediated growth regulation, the control of cell shape and plant morphology, regulation of the cell cycle and elongation of root cortex cells (Rashotte et al., 2001; Zhou et al., 2004). Arabidopsis rcn1 mutant seedlings (rcn1 encodes PP2A protein) showed an obvious reduction in PP2A activity, and the elongation of differential cells in root was arrested (Kwak et al., 2002). PP2A activity is required for regulation of auxin transport, and reduction in PP2A activity could alter lateral root growth (Rashotte et al., 2001). Therefore, PP2A may participate in the development of roots by regulating

the cell cycle, phytohormone transport or signal transduction. Under Pi starvation, the higher amounts of PP2A in the roots of 99038 compared to Qi-319 may be advantageous for maintenance of root cell division and growth. As a member of the AAA-ATPase family of molecular chaperones, CDC48 protein (cell division cycle protein 48) participates in the degradation of targeted proteins by promoting ubiquitination (Braun et al., 2002). Current studies have shown that it plays important roles in several ubiquitin-dependent processes, such as membrane fusion, transcription factor activation, spindle disassembly etc (Cao et al., 2003). CDC48 protein is also an important regulator of the cell cycle, and is required for the cell-cycle commitment point via degradation of the G1-CDK inhibitor (Jiang et al., 2004). The signi?cant increase in CDC48 (spot H33) in the roots of 99038 compared to Qi-319 is likely to accelerate root cell proliferation, and this is consistent with 99038 having better developed roots than Qi-319 (Li et al., 2007b). Under both +P and )P conditions, the abundance of GTPbinding nuclear protein RAN-B1 (Ran GTPase, spots H13 and L19) was greater in the roots of 99038 than in Qi-319. RAN-B1 is a member of the Ran GTPase superfamily, whose members are essential regulators of many aspects of cell division, such as nuclear reconstitution, DNA replication, spindle assembly, regulating mRNA transcription and splicing, as well as regulating the transport of proteins and RNA across the nuclear envelope (Jiang et al., 2004; Kalab et al., 1999; Moore and Blobel, 1993). Furthermore, under )P conditions, the levels of the importin a2 subunit (NLS receptor, spot L41) and mini-chromosome maintenance protein (MCM6, spot L1) in the roots of 99038 were increased compared to Qi-319. Importin proteins participate in nuclear membrane and spindle assembly as well as nuclear import. Ran GTPases and importins mediate the nuclear import of some proteins that regulate DNA replication, transcription and precursor processing, including MCM6, which regulates the cell cycle and gene expression (Jiang et al., 2004). Subcellular localization analysis of MCM6 showed that its transport between cytoplasm and nucleus was cell cycle-dependent. Some MCM proteins have phosphorylation sites for cell cycledependent protein kinase, which are important regulation domains. Therefore, it is likely that MCM6, Ran GTPase and

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

934 Kunpeng Li et al. the importins are involved in maize root development mediated by P nutrition by regulating the cell cycle, and their increase in 99038 roots promoted cell division, resulting in better developed roots in 99038 compared to Qi-319. Based on these results, we conclude that the difference in the regulation of cell proliferation between the genotypes may be an important reason for the better developed roots and greater tolerance of low-P stress in 99038 compared to Qi-319. To verify the putative alterations, we observed and compared the cell morphology and numbers of elongation and meristem zones in the roots of 99038 and Qi-319 under Pi starvation. The roots of 99038 displayed no obvious differences in cell morphology or size of elongation zones, but the number of cells in the meristem zones was significantly increased in 99038 compared to Qi-319 (Figure 4). The meristem cells were smaller and more tightly arrayed in 99038 than in Qi-319. The results demonstrate that, under Pi starvation, 99038 plants accelerate cell proliferation in the meristem compared to Qi-319 plants. Other differentially accumulated proteins The abundances of some root proteins, including vacuolar ATPase subunits, phytohormone metabolism proteins, 26S proteasome components, molecular chaperones, resistance proteins, secondary metabolism and cellular organizationassociated proteins, etc. displayed signi?cant differences between the genotypes under the different P conditions. Given their roles in maintaining ion homeostasis, regulating plant growth, mediating signal transduction, protein folding and assembly, regulating the cellular redox state etc, the differential accumulation in the genotypes indicated that there may be considerable differences in the metabolism and development of roots between 99038 and Qi-319. Analysis of selected transcripts of differentially accumulated proteins To assess transcripts of differentially accumulated proteins, real-time RT-PCR analyses were performed on 10 of the 73 proteins that showed differential accumulation under +P conditions and on 29 of the 95 proteins that showed differential accumulation under )P conditions. The magnitude of the changes in the levels of these transcripts in both genotypes and a comparison with their protein expression patterns are shown in Table S6. There was a low level of correlation between variations in mRNA and protein levels, and the mRNA variations were very weak compared to the protein variations. Six genes of the 39 genes that were analysed by real-time RT-PCR showed no signi?cant changes in transcript abundance. For the others, 21 genes displayed change trends at the transcript level that were consistent with the change trends in protein expression and

(a)

(b)

Cell number 480 ?m–1 root length

(e)

8 7 6 5 4 3 2 1 0

Figure 4. Comparison of the root cell number of mutant 99038 and wild-type Qi-319 plants treated with 5 lM KH2PO4 ()P) for 17 days. (a, b) Root tip fragments of 99038 (a) and Qi-319 (b) under )P conditions were stained with propidium iodide. Scale bar = 80 lm. (c, d) Root cells of the meristem zones of 99038 (c) and Qi-319 (d) stained with propidium iodide. Scale bar = 40 lm. (e, f) Root cell numbers in the elongation zone (e) and the meristem zone (f) for 99038 and Qi-319 under )P conditions. Results are means ? SD from three experiments (n = 15). The asterisk indicates a signi?cant difference at P < 0.05.

99038-P Qi-319-P Elongation zone (f)
Cell number 200 ?m–1 root length 12 10 8 6 4 2 0

*

(c)

(d)

99038-P Qi-319-P Meristem zone
? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

Root proteome for phosphorus ef?ciency in maize 935 12 genes had change trends in transcription that were the opposite to the change trends in protein expression. Discussion Comparative proteomic analyses of maize roots derived from a low-P-tolerant mutant and wild-type under different P conditions showed that differences in citrate secretion, sugar metabolism and root-cell proliferation were the main reasons for the better developed roots and higher Pi-uptake ability of the low-P-tolerant mutant compared to the wildtype, and indicated that the P-ef?cient root system has greater capability for mobilization of external Pi and greater cell-division activity in the root meristem under Pi starvation. Increased accumulation and secretion of citrate facilitated Pi solubilization and enhanced Pi uptake by the low-P-tolerant maize mutant Under Pi stress, plants allocate more carbon to the roots and regulate the expression of carbon metabolism genes to improve low-P adaptation, and these are very effective strategies (Mu ¨ ller et al., 2007; Vance et al., 2003; Wasaki et al., 2003; Wu et al., 2003). In general, plants produce and release more organic acids to ameliorate the rhizosphere environment and improve Pi uptake under Pi-starvation conditions (Raghothama, 1999). Physiological studies have suggested that citrate synthase plays a key role in organic acid secretion from roots (Hof?and et al., 1992; Takita et al., 1999), and, of the acids secreted, citrate is one of the most effective at solubilizing Pi (Hinsinger, 2001). Neumann et al. (1999) showed that, under Pi starvation, white lupin improves Pi uptake through the release of large amounts of organic acids, especially citrate, into the rhizospheric soil. Studies on transgenic tobacco and Arabidopsis showed that overexpression of citrate synthase increases the Pi-uptake ability of plants and improves growth under Pi starvation as a result of ? pez-Bucio enhanced citrate secretion (Koyama et al., 2000; Lo et al., 2000). Our studies indicate that, under Pi starvation, 99038 plants could produce and secrete more citrate than Qi319 plants, which facilitates Pi solubilization in the soil and leads to more ef?cient Pi uptake of 99038 plants. The results supported our earlier data from studies of Pi-uptake kinetics indicating that 99038 plants had higher Pi-acquisition ability than Qi-319 at various Pi levels (Li et al., 2007b). Based on these results, the difference in the accumulation and secretion of citrate between the genotypes may be an important factor for the higher Pi-uptake ability and low-P tolerance of 99038 plants compared to Qi-319. Therefore, a greater ability to mobilize environmental Pi resources through increasing exudation is one of the most important characteristics of P-ef?cient root systems in maize. Our data also showed that, under Pi starvation, several enzymes of the tricarboxylic acid cycle, which are involved in supplying the substrates for citrate production, and in synthesis and utilization of citrate, showed signi?cant differences with respect to the amount of protein and the activity between the low-P-tolerant mutant and the wildtype. The results suggest that the increased accumulation and secretion of citrate might be regulated coordinately, involving both upregulation of synthesis and reduced utilization or degradation of citrate. However, the molecular mechanism for the increased accumulation and secretion of citrate regulated by Pi starvation is still unclear, and more studies utilizing the low-P-tolerant mutant will be helpful in elucidating the underlying mechanism. Sugars, especially sucrose, are involved in root growth and development associated with P ef?ciency A recent study suggested that the response to Pi starvation in plants, including changes in root morphology and the expression of PSI genes require sugars, and the sugar signal is essential for completion of the PSI signalling pathway (Karthikeyan et al., 2007). Among sugars, sucrose has been shown to have pronounced effects on expression of PSI genes, and the expression of PSI genes was enhanced with increasing concentrations of sucrose under Pi starvation (Jain et al., 2007; Karthikeyan et al., 2007; Lai et al., 2007). Jain et al. (2007) also found that sucrose plays a pivotal role in the development of lateral roots and root hairs induced by Pi starvation, suggesting correlations between sucrose and root growth in response to Pi starvation. In this study, we found that the proportion of sucrose in the total soluble sugars of 99038 roots was signi?cantly higher than in Qi-319 roots (Figure 3), which was advantageous for lateral root initiation and growth in 99038 plants. A previous study had shown that, under Pi starvation, the number and mean length of lateral roots in the low-P-tolerant mutant 99038 were increased compared to Qi-319 (Li et al., 2007b), which is in agreement with proposed role of sucrose in mediating root growth in response to Pi starvation. However, the roles of sucrose in regulating the re-programming of roots in response to Pi starvation are only beginning to be unravelled, and further studies are needed. Cell division plays a pivotal role in root growth and development associated with Pi ef?ciency Recent studies in Arabidopsis suggest that, under long-term low-P stress, the inhibited root growth is closely correlated with root meristem exhaustion, indicating that root meristem maintenance is disturbed by Pi starvation (Desnos, 2007; Sanchez-Calderon et al., 2005). In this study, we found that some proteins that promote cell proliferation were overaccumulated in the roots of the low-P-tolerant mutant, and cell division in the root tips was signi?cantly accelerated under Pi starvation compared to Qi-319. These results

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

936 Kunpeng Li et al. indicate that exhaustion of the root meristem, induced by long-term Pi starvation, may be alleviated in the low-P-tolerant mutant 99038, which is advantageous for the maintenance of root growth during long periods of Pi de?ciency. These results are consistent with our previous report that root growth was less restrained in 99038 than in Qi-319 after 17 days of Pi starvation, in particular that the number and length of the lateral roots were signi?cantly increased compared with Qi-319 (Li et al., 2007b). These results show that root meristem maintenance is an important factor resulting in better developed roots and advantageous root morphology for Pi uptake in the low-P-tolerant mutant 99038 compared with Qi-319 under Pi starvation. Among the traits associated with P ef?ciency, maintenance of root growth and re-programming of the root architecture are critical for Pi uptake due to the low mobility of soil Pi. Therefore, regulation of cell proliferation by promoting root growth and development plays a vital role in the adaptability of plants to low-Pi environments. Recently, a study by Lai et al. (2007) suggested that cell division activity plays a central role in determining the magnitude of PSI gene expression, and that conditions that promote cell proliferation, including increased sucrose, enhance the Pi-starvation responses of plants due to increased P demand. Our data show that the proportion of sucrose in the total sugars in the roots of 99038 plants was signi?cantly greater than in Qi-319 plants, and there was an obvious acceleration in root cell proliferation in 99038 plants compared to Qi-319 plants. This increased the expression of PSI genes in the low-P-tolerant mutant under Pi starvation, improving its adaptability to low-P environments. Major genes for P ef?ciency may exist in maize Previously, we found that the low-P-tolerant mutant 99038 and Qi-319 plants showed no signi?cant differences in their shoots, but 99038 plants had more extensive root systems and greater low-P tolerance than Qi-319 (Li et al., 2007b). It is well known that generally there are only a few gene mutations in cell mutants selected under stress conditions in plant tissue culture. The low-P-tolerant mutant 99038 was obtained from Qi-319 by somaclonal variation, and may be considered a near-isogenic line of Qi-319. However, differential expression analyses of root protein showed that, under +P or )P conditions, over 10% of the root proteins on 2-DE gels were altered twofold or more between the two genotypes, suggesting that they may have different regulatory mechanisms for P nutrition. In conclusion, it is possible that there are a few major genes that play important roles in the complicated Pi-starvation response of maize plants, and that one or a few of these pivotal genes was mutated in 99038, causing differential accumulation of downstream proteins, and eventually resulting in greater low-P tolerance and better developed roots compared to Qi-319. Comparisons of the proteome and gene expression data Although the responses of transcript and protein could be linked, their development is temporally separated. mRNA expression levels do not directly represent the abundance of proteins produced (Shinano et al., 2005; Vyetrogon et al., 2007). Our results showed no clear correlations between changes in the abundance of protein and transcript, and this is consistent with previous results (Scott, 2005; Sperling, 2001; Vyetrogon et al., 2007). Moreover, our results also indicated that the mRNA variations were weak compared with the corresponding variations in protein. This is probably because post-translational modi?cations have occurred, or because of differences in mRNA and protein stability and turnover (Liu et al., 2006; Vyetrogon et al., 2007). These results indicate the importance of performing proteomic analyses, which provide critical information regarding simultaneous changes in protein accumulation at a given time in a given tissue, or in response to a given environment, leading to better understanding of these biological processes.

Experimental procedures Plant growth conditions and treatments
Seeds of the maize inbred line Qi-319 and mutant 99038 were surface-sterilized and germinated in the dark at 28°C. To eliminate the in?uence of different amounts of seed phosphorus in the genotypes on the plant’s response to Pi starvation, seedlings (4 days old) were transferred to +P (1000 lM KH2PO4) nutrient solution for 20 days. Then half of the seedlings continued to grow in +P conditions and the other were cultured in 5 lM KH2PO4 nutrient solution for 17 days ()P conditions). Under )P conditions, the 1000 lM KH2PO4 in the +P nutrient solution was substituted by 1000 lM KCl. The basal compositions of the nutrient solution and the culture conditions of seedlings were as described previously (Li et al., 2007b). Seedlings were positioned in a completely randomized design in a greenhouse, and three batches of seedlings were cultured separately to provide biological replicates.

Protein isolation
Root tip fragments (3 cm) from Qi-319 and 99038 under +P or )P conditions were sampled. Protein extraction by tricholoroacetic acid/acetone precipitation was performed as described previously (Li et al., 2007a). The pellet was dried and dissolved in protein solubilization buffer (7 M urea, 2 M thio-urea, 4% CHAPS, 0.5% v/v carrier ampholyte pH 3-10, 40 mM DTT, 1 mM PMSF). Protein concentrations were determined using the Bradford method (Bradford, 1976). The protein samples were kept at )80°C until used.

2-DE and image analysis
2-DE was performed at pH 3-6 and pH 5-8 (17 cm) using immobilized PH gradient (IPG) strips (Bio-Rad, http://www.bio-rad.com/) as previously described (Li et al., 2007a). Gels were stained with silver and CBB, and the experiment was repeated three times with

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

Root proteome for phosphorus ef?ciency in maize 937
independent samples. The images of the stained gels were acquired using a GS-800 calibrated densitometer (Bio-Rad) and analysed using PDQuest software, version 7.2.0 (Bio-Rad). After background subtraction and spot detection, spots were matched and normalized using the method of total density in the gel image. The statistical signi?cance of quantitative data was determined using Student’s t test (n = 3, P < 0.05) at a 95% con?dence level, and proteins with a twofold or more change at the con?dence level were considered differentially accumulated. were soaked in 0.5 mM CaCl2 solution at pH 6.0 for 16 h before collection of root exudates. Then plant roots were washed for 5 min in deionized water, 15 mg l)1 thymol solution and 0.5 mM CaCl2 solution in tum. The roots of the seedlings were then submerged in 200 ml 0.5 mM CaCl2 solution for 4 h. The solution was collected, and thymol was added to prevent bacterial decomposition. After ?ltration through a 0.45 lm pore-size membrane, the samples were dried using a ThermoSavant ModulyoD-230 dryer (Thermo Scienti?c, http://www.thermo.com/), and ?nally re-dissolved in 300 ll of 0.1 M HCl for further analysis. The amounts of citrate in the root tissue and exudates were determined by reverse-phase HPLC in ion-suppression mode. Samples (20 ll) were injected onto a 4.6 · 250 mm Atlantis? dC18 5 lm column (Waters, http://www.waters.com), and 18 mM KH2PO4 solution adjusted to pH 2.7 with H3PO4 was used for isocratic elution, with a ?ow rate 0.8 ml min)1 at 25°C and UV detection at 210 nm. The identi?cation of citrate was performed by comparing retention times and absorption spectra with known standards.

Protein identi?cation by MALDI-TOF MS
Protein spots were carefully excised from the gels. Protein digestion and extraction were performed according to the method described by Li et al. (2007a). The protein digests were mixed with one volume of 10 mg ml)1 CHCA (a-cyano-4-hydroxycinnamic acid) in 50% v/v acetonitrile and 0.1% TFA for spotting onto metal plates, and dried for MALDI-TOF MS analysis (AXIMACFRplus, Shimadzu, http:// www.shimadzu.com) analysis. Spectra were obtained under 19 kV accelerating voltage in re?ection mode, with an m/z range of 700–3500 Da. The peptide mass ?ngerprinting spectra obtained from MALDITOF MS were calibrated, and mono-isotopic peaks identi?ed by Kompact, version 2.4.0 (Kratos Analytical, http://www.kratos.com). After peak ?ltering using Peak Erazor (version 2.01, http:// www.gpmaw.com/), mono-isotopic peak lists were compared against National Center for Biotechnology Information (NCBI)nr and MSDB (MS protein sequence database) using MASCOT (http:// www.matrixscience.com). Searches were performed using Cys carbamidomethylation as the ?xed modi?cation, and oxidation of Met and pyroGlu formation of N-terminal Gln as variable modi?cations, allowing 50 ppm mass tolerance and one trypsin miscleavage. The identi?ed proteins ranked in the top two hits for at least six matched peptides, with total coverage of over 15%, a molecular weight search (MOWSE) score ?67 by the MASCOT program, and agreement of ?20% between the Mr determined from the gel and the predicted Mr.

Quanti?cation of total soluble sugars and sucrose
The total soluble sugars in the maize root tip fragments were extracted in boiling water for 1 h, and measured using anthrone reagent with glucose as the standard (Yemm and Willis, 1954). The sucrose contents were analysed by TLC. Total soluble sugars (10 lg) were spotted onto a silica gel GF254 TLC foil (Merck, http:// www.merck.com) and eluted using 1-propanol/acetic acid/water (10/10/1 v/v/v). Sugars were detected by spraying colour-developing agent (2 g diphenylamine, 2 ml aniline and 10 ml 85% phosphoric acid in 100 ml acetone) onto the plate and heating it at 95°C for 15 min. TLC images were analysed at 390 nm using a Cs-930 dual-wavelength chromatoscanner (Shimadzu). All the experiments were repeated three times with independent samples.

Imaging of root cell size Measurement of the activities of citrate synthase, malate dehydrogenase and aconitase
Root tip fragments (300 mg) were homogenized in 1.2 ml of cold extraction buffer, which contained 50 mM HEPES/KOH (pH 7.5), 5 mM MgCl2, 2 mM EDTA, 10% v/v glycerol, 0.2% Triton X-100, 14 mM DTT and 1 mM PMSF, using a mortar and pestle. The homogenate was centrifuged at 20 000 g for 15 min at 4°C, and the resulting supernatant was used in enzyme assays. Citrate synthase, malate dehydrogenase and aconitase were assayed spectrophotometrically at A412, A340 and A240 according to the methods described by Srere (1967), Gross (1977) and Kennedy et al. (1983), respectively. Protein was quanti?ed using the Bradford method (Bradford, 1976). To examine cell arrangement and size, root tip fragments were stained with 100 lg ml)1 propidium iodide solution (pH 7.2) containing 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, and observed under a laser scanning confocal microscope (Leica, http://www.leica.com) with an argon laser.

Real-time RT-PCR and gene expression data analysis
We used the Premier program, version 5.0 (Premier Biosoft International, http://www.premierbiosoft.com), to design the primers for ampli?cation of the cDNA sequences encoding the differentially accumulated proteins. For differentially accumulated proteins that were not identi?ed via a maize sequence, the closest maize paralogue was identi?ed via TBLASTN database searches of the NCBInr database. Total RNA was isolated from the same tissue used for proteomic analysis using the water-saturation phenol/guanidine isothiocyanate/chloroform method, and treated with RNase-free DNase (Takara, http://www.takara.com.cn) according to the manufacturer’s protocol. cDNA synthesis was performed using the RT reagent kit (Takara) according to the manufacturer’s protocol. Realtime RT-PCR was performed on a Chromo 4 real-time detection system (MJ Research, http://www.mjr.com) using a SYBR? RT-PCR kit (Takara), in a 10 ll reaction volume containing 5 ll of SYBR? Green I PCR mix, 0.2 ll of each forward and reverse primer, 1 ll of

Measurement of the amount of citrate in root tissue and exudates
Root tip fragments were homogenized in 1.2 ml of 0.1 M HCl per 300 mg fresh weight, and the homogenate was incubated at 75°C for 15 min. After centrifugation at 12 000 g for 15 min, the supernatant was used for measurement of citrate levels in the tissue. The root exudates were collected as described previously (Shen et al., 2001) with minor modi?cations. The roots of three plants

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

938 Kunpeng Li et al.
diluted cDNA template, and appropriate amounts of sterile doubledistilled H2O. Ampli?cation conditions were 2 min at 95°C, 40 cycles of 15 sec at 95°C, 30 sec at 55–60°C and 30 sec at 72°C. The magnitude of changes in the amounts of RNA transcripts were calculated by the 2?DDCt method (Livak and Schmittgen, 2001) with actin1 as an internal control. The PCR primer sets are shown in Table S6. All experiments were repeated at least three times with independent samples. Franco-Zorrilla, J.M., Gonzalez, E., Bustos, R., Linhares, F., Leyva, A. and Paz-Ares, J. (2004) The transcriptional control of plant responses to phosphate limitation. J. Exp. Bot. 55, 285–293. ?n, A.C., Leyva, A. and Paz-Ares, J. (2005) Franco-Zorrilla, J.M., Mart? Interaction between phosphate starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiol. 138, 847–857. Gross, G.G. (1977) Cell wall-bound malate dehydrogenase from horseradish. Phytochemistry, 16, 319–321. ? ndez, G., Ram? ? s-Lo ? pez, O. et al. (2007) Phos?rez, M., Valde Herna phorus stress in common bean: root transcript and metabolic responses. Plant Physiol. 144, 752–767. Hinsinger, P. (2001) Bio-availability of soil inorganic P in the rhizosphere as affected by root induced chemical changes: a review. Plant Soil, 237, 173–195. Hof?and, E., van den Boogaard, R. and Findenegg, G.R. (1992) Biosynthesis and root exudation of citric and malic acids in phosphate-starved rape plants. New Phytol. 122, 675–680. Jain, A., Poling, M.D., Karthikeyan, A.S., Blakeslee, J.J., Peer, W.A., Titapiwatanakun, B., Murphy, A.S. and Raghothama, K.G. (2007) Differential effects of sucrose and auxin on localized phosphate de?ciency-induced modulation of different traits of root system architecture in Arabidopsis. Plant Physiol. 144, 232–247. Jiang, Q., Lu, Z. and Zhang, C. (2004) Role of Ran GTPase in cell cycle regulation. Chinese Sci. Bull. 49, 535–541. Kalab, P., Pu, R.T. and Dasso, M. (1999) The Ran GTPase regulates mitotic spindle assembly. Curr. Biol. 9, 481–484. Karthikeyan, A.S., Varadarajan, D.K., Jain, A., Held, M.A., Carpita, N.C. and Raghothama, K.G. (2007) Phosphate starvation responses are mediated by sugar signaling in Arabidopsis. Planta, 225, 907–918. Kennedy, M.C., Emptage, M.H., Dreyer, J.L. and Beinert, H. (1983) The role of iron in the activation–inactivation of aconitase. J. Biol. Chem. 258, 11098–11105. Koyama, H., Kawamura, A., Kihara, T., Hara, T., Takita, E. and Shibata, D. (2000) Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil. Plant Cell Physiol. 41, 1030–1037. Kwak, J.M., Moon, J.H., Murata, Y., Kuchitsu, K., Leonhardt, N., DeLong, A. and Schroeder, J.I. (2002) Disruption of a guard cellexpressed protein phosphatase 2A regulatory subunit, RCN1, confers abscisic acid insensitivity in Arabidopsis. Plant Cell, 14, 2849–2861. Lai, F., Thacker, J., Li, Y. and Doerner, P. (2007) Cell division activity determines the magnitude of phosphate starvation responses in Arabidopsis. Plant J. 50, 545–556. Li, K., Xu, C., Zhang, K., Yang, A. and Zhang, J. (2007a) Proteomic analysis of roots growth and metabolic changes under phosphorus de?cit in maize (Zea mays L.) plants. Proteomics, 7, 1501–1512. Li, K., Xu, Z., Zhang, K., Yang, A. and Zhang, J. (2007b) Ef?cient production and characterization for maize inbred lines with lowphosphorus tolerance. Plant Sci. 172, 255–264. Liu, J., Samac, D.A., Bucciarelli, B., Allan, D.L. and Vance, C.P. (2005) Signaling of phosphorus de?ciency-induced gene expression in white lupin requires sugar and phloem transport. Plant J. 41, 257– 268. Liu, Y., Lamkemeyer, T., Jakob, A., Mi, G., Zhang, F., Nordheim, A. and Hochholdinger, F. (2006) Comparative proteome analyses of maize (Zea mays L.) primary roots prior to lateral root initiation reveal differential protein expression in the lateral root initiation mutant rum1. Proteomics, 6, 4300–4308. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2?DDCt method. Methods, 25, 402–408.

Acknowledgements
We thank Dr Lushan Wang (Shandong University, China) for assistance with protein analysis, Zhen Chen (Shandong University, China) for sugar analysis, and Dr Roberta Greenwood (Shandong University, China) for help in editing this manuscript. This work was supported by the Natural Science Foundation of China (grant number 30070487) and the National Key Technology R&D Program of China (grant number 2006AA10A107).

Supporting Information
Additional supporting information may be found in the online version of this article. Figure S1. Peptide mass ?ngerprinting of cytosolic 6-phosphogluconate dehydrogenase (spot L46, Figure 2b). Table S1. Total number of spots and number of differentially accumulated spots between genotypes on the 2-DE gels shown in Figures 1 and 2. Table S2. Identi?cation of differentially accumulated proteins in the roots of 99038 and Qi-319 plants treated with 1000 lM KH2PO4 (+P) for 17 days (shown in Figure 1). Table S3. Identi?cation of differentially accumulated proteins in the roots of 99038 and Qi-319 plants treated with 5 lM KH2PO4 ()P) for 17 days (shown in Figure 2). Table S4. Spot volumes of identi?ed proteins shown in Table S2 or Figure 1. Table S5. Spot volumes of denti?ed proteins shown in Table S3 or Figure 2. Table S6. Primers used for real-time RT-PCR, and detailed results. Please note: Blackwell publishing are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

References
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Braun, S., Matuschewski, K., Rape, M., Thoms, S. and Jentsch, S. (2002) Role of the ubiquitin-selective CDC48UFD1/NPL4 chaperone (segregase) in ERAD of OLE1 and other substrates. EMBO J. 21, 615–621. Cao, K., Nakajima, R., Meyer, H.H. and Zheng, Y. (2003) The AAAATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis. Cell, 115, 355–367. Desnos, T. (2007) Root branching responses to phosphate and nitrate. Curr. Opin. Plant Biol. 11, 1–6. Devaiah, B.N., Karthikeyan, A.S. and Raghothama, K.G. (2007) WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 143, 1789–1801.

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939

Root proteome for phosphorus ef?ciency in maize 939
? pez-Bucio, J., de La Vega, O.M., Guevara-Garc? ?a, A. and HerreraLo Estrella, L. (2000) Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nat. Biotechnol. 18, 450– 453. Misson, J., Raghothama, K.G., Jain, A. et al. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc. Natl Acad. Sci. USA, 102, 11934–11939. Moore, M.S. and Blobel, G. (1993) The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature, 365, 661– 663. Mu ¨ ller, R., Morant, M., Jarmer, H., Nilsson, L. and Nielsen, T.H. (2007) Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol. 143, 156–171. Neumann, G., Massonneau, A., Martinoia, E. and Romheld, V. (1999) Physiological adaptations to phosphorus de?ciency during proteoid root development in white lupin. Planta, 208, 373–382. Raghothama, K.G. (1999) Phosphate acquisition. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 665–693. Rashotte, A.M., DeLong, A. and Muday, G.K. (2001) Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth. Plant Cell, 13, 1683–1697. Rolland, F. and Sheen, J. (2005) Sugar sensing and signalling networks in plants. Biochem. Soc. Trans. 33, 269–271. ?n, A.C., Iglesias, J., Leyva, A. Rubio, V., Linhares, F., Solano, R., Mart? and Paz-Ares, J. (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular. Genes Dev. 15, 2122–2133. Sanchez-Calderon, L., Lopez-Bucio, J., Chacon-Lopez, A., CruzRamirez, A., Nieto-Jacobo, F., Dubrovsky, J.G. and Herrera-Estrella, L. (2005) Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana. Plant Cell Physiol. 46, 174–184. Schoof, H., Zaccaria, P., Gundlach, H., Lemcke, K., Rudd, S., Kolesov, G., Arnold, R., Mewes, H.W. and Mayer, K.F. (2002) MIPS Arabidopsis thaliana database (MAtDB): an integrated biological knowledge resource based on the ?rst complete plant genome. Nucleic Acids Res. 30, 91–93. Scott, C.P. (2005) Update on proteomics in Arabidopsis. Where do we go from here? Plant Physiol. 138, 591–599. Shen, H., Wang, X., Shi, W., Cao, Z. and Yan, X. (2001) Isolation and identi?cation of speci?c root exudates in elephantgrass in response to mobilization of iron- and aluminum-phosphates. J. Plant Nutr. 24, 1117–1130. Shinano, T., Nanamori, M., Dohi, M., Wasaki, J. and Osaki, M. (2005) Evaluation of phosphorus starvation inducible genes relating to ef?cient phosphorus utilization in rice. Plant Soil, 269, 81–87. Sperling, K. (2001) From proteomics to genomics. Electrophoresis, 22, 2835–2837. Srere, P.A. (1967) Citrate synthase. Meth. Enzymol. 13, 3–11. Svistoonoff, S., Creff, A., Reymond, M., Sigoillot-Claude, C., Ricaud, L., Blanchet, A., Nussaume, L. and Desnos, T. (2007) Root tip contact with low phosphate media reprograms plant root architecture. Nat. Genet. 39, 792–796. Takita, E., Koyama, H. and Hara, T. (1999) Organic acid metabolism in aluminum-phosphate utilizing cells of carrot (Daucus carota L.). Plant Cell Physiol. 40, 489–495. ?n ? ez, M., Li, A., Vance, C.P. Uhde-Stone, C., Zinn, K.E., Ramirez-Ya and Allan, D.L. (2003) Nylon ?lter arrays reveal differential gene expression in proteoid roots of white lupin in response to phosphorus de?ciency. Plant Physiol. 131, 1064–1079. Vance, C.P., Uhde-Stone, C. and Allan, D.L. (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol. 157, 423–447. Vyetrogon, K., Tebbji, F., Olson, D.J., Ross, A.R. and Matton, D.P. (2007) A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the selfincompatible species Solanum chacoense Bitt. Proteomics, 7, 232–247. Wasaki, J., Yonetani, R., Kuroda, S. et al. (2003) Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots. Plant Cell Environ. 26, 1515–1523. Wasaki, J., Shinano, T., Onishi, K. et al. (2006) Transcriptomic analysis indicates putative metabolic changes caused by manipulation of phosphorus availability in rice leaves. J. Exp. Bot. 57, 2049–2059. Wu, P., Ma, L., Hou, X., Wang, M., Wu, Y., Liu, F. and Deng, X.W. (2003) Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves. Plant Physiol. 132, 1260–1271. Yemm, E.W. and Willis, A.J. (1954) The estimation of carbohydrates in plant extracts by the anthrone. Biochem. J. 57, 508–514. Yi, K., Wu, Z., Zhou, J., Du, L., Guo, L., Wu, Y. and Wu, P. (2005) OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice. Plant Physiol. 138, 2087–2096. Zhou, H.W., Nussbaumer, C., Chao, Y. and DeLong, A. (2004) Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A. Plant Cell, 16, 709–722. Zhu, J. and Lynch, J.P. (2004) The contribution of lateral rooting to phosphorus acquisition ef?ciency in maize (Zea mays L.) seedlings. Funct. Plant Biol. 31, 949–958. Zhu, J., Kaeppler, S.M. and Lynch, J.P. (2005a) Mapping of QTLs for lateral root branching and length in maize (Zea mays L.) under differential phosphorus supply. Theor. Appl. Genet. 111, 688–695. Zhu, J., Kaeppler, S.M. and Lynch, J.P. (2005b) Mapping of QTL controlling root hair length in maize (Zea mays L.) under phosphorus de?ciency. Plant Soil, 270, 299–310. Zhu, J., Mickelson, S.M., Kaeppler, S.M. and Lynch, J.P. (2006) Detection of quantitative trait loci for seminal root traits in maize (Zea mays L.) seedlings grown under differential phosphorus levels. Theor. Appl. Genet. 113, 1–10.

? 2008 The Authors Journal compilation ? 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 927–939


赞助商链接
相关文章:
更多相关标签: