Abstract A long period of silk viability is critical for a good seed setting rate in maize (Zea mays L.), especially for inbred lines and hybrids with a long interval between anthesis and silking. To explore the molecular mechanism of silk viability and its heterosis, three inbred lines with different silk viability characteristics (Xun928, Lx9801, and Zong3) and their two hybrids (Xun928×Zong3 and Lx9801×Zong3) were analyzed at different developmental stages by a proteomic method. The differentially accumulated proteins were identified by mass spectrometry and classified into metabolism, protein biosynthesis and folding, signal transduction and hormone homeostasis, stress and defense responses, and cellular processes. Proteins involved in nutrient (methionine) and energy (ATP) supply, which support the pollen tube growth in the silk, were important for silk viability and its heterosis. The additive and dominant effects at a single locus, as well as complex epistatic interactions at two or more loci in metabolic pathways, were the primary contributors for mid-parent heterosis of silk viability. Additionally, the proteins involved in the metabolism of anthocyanins, which indirectly negatively regulate local hormone accumulation, were also important for the mid-parent heterosis of silk viability. These results also might imply the developmental dependence of heterosis, because many of the differentially accumulated proteins made distinct contributions to the heterosis of silk viability at specific developmental stages. Introduction The maize silk is functionally equivalent to the stigma and style of a typical pistil. It is a specialized elongated tissue that begins to senesce about 8–10 days after it emerges from the husks [[35]1]. In normal conditions, pollination is completed within 1–2 days after silk emergence. Thus, a period of 8–10 days of silk viability is normally sufficient for seed setting. However, in hybrid seed production where plants are emasculated, a female parent with a long period of silk viability is critical for a good seed set. Thus, elucidating the molecular mechanisms of silk viability is necessary for the production of elite inbred lines and hybrid selection, which in turn contribute to maize hybrid seed production and field production. Before losing viability, the maize silk can receive viable pollen, promotes pollen germination, guides pollen tube navigation, and aids pollen–ovule interactions [[36]2,[37]3]. Thus, silk viability has wide-ranging implications other than the specific function of supporting pollen germination through to fertilization. Under conducive conditions, compatible pollen grains hydrate and germinate pollen tubes. The tubes elongate via tip growth, penetrate the cell layers of the stigma, and enter the trichome, navigating within the transmitting tracts of the silk [[38]4]. Usually, only a single pollen tube grows through the micropyle and eventually reaches the ovule where fertilization occurs [[39]5]. All of these biological processes are critical for reproduction. Thus, several studies have analyzed the molecular mechanisms of pollen adhesion, pollen germination, pollen tube guidance, and pollen–ovule interactions [[40]2, [41]3]. In pistil-interacting pollen tubes, genes involved in signal transduction, transcription, and pollen tube growth are highly expressed [[42]6]. Cysteine-rich peptides (CRPs) for cell–cell communications were shown to be important for pollen–pistil interactions in several studies [[43]7–[44]10]. Genes involved in amino acid and lipid transport were shown to be important for, and unique to, reproductive processes in maize silks [[45]11]. The cytosolic free Ca^2+ concentration is another important factor in normal pollen tube growth and morphology [[46]12]. A Ca^2+ channel in pollen is formed by glutamate receptor-like proteins, which are regulated by D-serine in the pistil [[47]13]. Nitric oxide, which has negative chemotropic activity in lily pollen tubes and is involved in pollen tube guidance in Arabidopsis, might function in tip-growth downstream of Ca^2+ signaling [[48]14]. For normal pollen tube growth, lipid transfer protein 5 (related to lily stigma cysteine-rich adhesin) and cysteine-rich receptor-like kinases were shown to be important for stigma-mediated reproductive processes in Arabidopsis [[49]15] and maize [[50]11], respectively. Normal pH and K^+ homeostasis in the pollen tube are important for guidance of the pollen tube to the ovule [[51]16]. cAMP was shown to play a second messenger role in regulating pollen tube growth and reorientation [[52]17]. Receptor-like kinase proteins that maintain pollen tube integrity [[53]18] and pollen tube attractants secreted by synergid cells [[54]19] are also required to guide pollen tube growth to the embryo sacs to complete fertilization. A defensin-like cysteine-rich peptide protein encoded by ZmES4 was shown to cause pollen tube burst in mature maize synergid cells by opening K^+ channels [[55]20]. Although many studies have identified key genes and molecules in the mechanisms of pollen–pistil interactions, pollen tube guidance, and pollen–ovule interactions in maize, few have focused on the molecular basis of silk viability, especially the genes and proteins related to silk viability before pollination. In practice, silks of maize hybrids always have much longer period time to accept pollen, complete double fertilization, and obtain seeds than their parental lines. Such as, silk viability of the improved lines of the local germplasm TangSPT that extensive used in maize breeding in China only has 5–6 days, while, 7–8 days silk viability were kept for their hybrid combinations with other lines from different heterotic group ([56]S1 Table). However, the molecular mechanism of silk viability and its heterosis remain largely unstudied. The aims of this study were to: (1) identify key proteins related to silk viability at different silk developmental stages using a proteomics approach using three inbred lines, (2) illuminate the potential molecular mechanism of silk viability heterosis using two different hybrid combinations and its corresponding inbred lines, and (3) identify the common factors regulating both silk viability and its heterosis. Results Silk viability evaluated by seed setting rate To evaluate differences in silk viability, the seed setting rate in the ear mid-base region (5–15 rounds from the ear base) was analyzed in three inbred lines and two hybrids ([57]Table 1). At the five sampling stages, D[4], D[6], D[8], D[10], and D[12], the average seed setting rate of three biological replications for the inbred lines Xun928 and Lx9801 was 99.3%, 97.4%, 95.0%, 26.0%, 13.5%, and 96.3%, 89.3%, 78.8%, 16.3%, 1.9%, respectively. The inbred line Zong3 sustained a high seed setting rate of 100% for all sampling stages. The ANOVA results showed that the difference of seed setting rate was significant between the parental inbred lines and their corresponding hybrids (P < 0.05), except for D[4] between the inbred line Xun928 (P = 0.079), Zong3, and their hybrid combination Xun928×Zong3. The different sampling stages of Xun928, Lx9801, Xun928×Zong3, and Lx9801×Zong3 also showed significant differences (P < 0.01). Thus, least-significant difference (LSD) multiple comparisons were performed and showed that the seed setting rate between each sampling stage was significantly different both for Xun928 and Lx9801. However, non-significant difference was detected between D[4] and D[6] both for the two hybrids Xun928×Zong3 and Lx9801×Zong3. Table 1. Analysis of variance for mid-base setting rate of inbred lines and corresponding hybrid combinations. Sampling stage [58]^f Xun928 Lx9801 Zong3 Xun928×Zong3 Lx9801×Zong3 P-value [59]^h P-value [60]^i D[4] 99.3% [61]^a 96.3% [62]^a 100.0% 100.0% [63]^a 100.0% [64]^a 0.079 3.97E-4[65]^** D[6] 97.4% [66]^b 89.3% [67]^b 100.0% 100.0% [68]^a 99.6% [69]^a 0.028[70]^* 1.91E-06[71]^** D[8] 95.0% [72]^c 78.8% [73]^c 100.0% 95.6% [74]^b 80.3% [75]^b 7.32E-4[76]^** 9.00E-07[77]^** D[10] 26.0% [78]^d 16.3% [79]^d 100.0% 84.6% [80]^c 70.4% [81]^c 2.84E-09[82]^** 1.81E-10[83]^** D[12] 13.5% [84]^e 1.9% [85]^e 100.0% 80.2% [86]^d 66.9% [87]^d 3.09E-11[88]^** 3.6E-11[89]^** P-value [90]^g 4.10E-15[91]^** 7.10E-16[92]^** 1.25E-10[93]^** 1.52E-12[94]^** [95]Open in a new tab a, b, c, d, and e represent the results of LSD analysis at 0.05 significant level. ^f: D[4], D[6], D[8], D[10], and D[12] represents days after silk emerged above ligule of the husk outer leaf. Setting percentage was calculated by averaging three biological replications and each replication. Each replication consisted of 10 intact ears. ^g: P-value is significance level among different sampling stages. ^h: P-value is significance level between hybrids Xun928×Zong3 and its two parental lines at each sampling stage. ^i: P-value is significance level between hybrids Lx9801×Zong3 and its two parental lines at each sampling stage. *: Significant at 0.05 level. **: Significant at 0.01 level. The seed setting rate significantly decreased from D[8] to D[10] in both Xun928 and Lx9801 and the two hybrids. However, the decrease in the seed setting rate was slower in the hybrids than in the two inbred lines because of heterosis in the hybrids. Compared with those of the parental inbred lines, the seed setting rate of the hybrids fell between the mid-parent and high-parent values; i. e, seed setting rate showed partial dominant heterosis. Based on the phenotype of seed setting rate and heterostic degree, only the sampling stages with a significant difference at the 0.01 level were used for the proteomic analysis. Thus, the hybrids at D[8], D[10], and D[12] were used in the proteomic analysis of heterosis, and the inbred lines Xun928, Lx9801, and Zong3 at stages D[6], D[8], D[10], and D[12] were used in the proteomic analysis of silk viability ([96]Table 1). Differentially accumulated proteins For the 2-DE analysis, only protein spots that showed the same trend in the three biological replicates were retrieved ([97]S1 and [98]S2 Figs). After normalization and ANOVA, only 3, 7, and 16 differentially accumulated protein spots were obtained for the inbred lines Xun928, Lx9801, and Zong3, respectively. These protein spots, which showed maximum changes more than 1.5-fold (P < 0.05) during the four sampling stages, were manually excised and analyzed by MS ([99]Table 2 and [100]S2 Table). Among the 26 differentially accumulated protein spots, 17 and 7 protein spots showed the lowest and the highest levels at D[6], respectively ([101]S3 Table). Meanwhile, 8 and 4 out of the 14 protein spots corresponding to D[6] showed the highest and the lowest levels at D[12], respectively. Among them, protein spot 46 gradually accumulated during silk development, while protein spot 61 gradually diminished. Table 2. Differentially accumulated proteins identified by MS during silk development at four sampling stages in three inbred lines. Spot No.[102] ^a Fold change[103] ^b Line name Accession number[104] ^c Protein name[105] ^d Theoretical MW/PI Protein Score[106] ^e Protein Score C. I.%[107] ^f Pep. Count Gene name[108] ^g Gene position[109] ^h P-value Amino acid metabolism 8 4.1 Xun928 gi|414869037 putative methionine synthase family protein 84781.4/5.54 783 100 8 GRMZM2G149751 1:176865419–176870974 0 64 18.2 Zong3 gi|195636806 methylthioribose-1-phosphate isomerase [Zea mays] 38662.3/5.70 192 100 4 GRMZM2G139533 4:12797382–12792141 1.30E-148 Biosynthesis of other secondary metabolites 16 2.1 Xun928 gi|195635735 GDSL-motif lipase/hydrolase-like protein [Zea mays] 41926.6/6.81 338 100 3 GRMZM2G700208 10:33361096–33356952 1.10E-39 53 6.5 Zong3 gi|1706374 Dihydroflavonol-4-reductase; 39172.9/5.48 1,060 100 11 GRMZM2G026930 3:216387831–216386092 9.70E-112 63 5.7 Zong3 gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 592 100 11 GRMZM2G162755 6:120060819–120059072 3.80E-46 Carbohydrate metabolism 3 3.1 Xun928 gi|414872668 alcohol dehydrogenase1 [Zea mays] 41572.9/6.15 162 100 2 GRMZM2G442658 1:274053872–274050420 4.00E-154 65 2.5 Zong3 gi|413917002 putative glyoxalase family protein [Zea mays] 32438.4/5.82 322 100 5 GRMZM2G181192 10:60096710–60090400 8.20E-115 Stress and defense response 26 4.1 Lx9801 gi|12004294 T cytoplasm male sterility restorer factor 2 [Zea mays] 59750.9/6.69 369 100 2 GRMZM2G058675 9:34129896–34143610 2.90E-229 41 1.6 Zong3 gi|414873866 salt tolerance protein isoform 3 [Zea mays] 35309.2/4.92 221 100 4 GRMZM2G352415 1:297098531–297096881 1.50E-108 42 1.5 Zong3 gi|414873866 salt tolerance protein isoform 3 [Zea mays] 35309.2/4.92 377 100 6 GRMZM2G352415 1:297098531–297096881 1.50E-108 Cell motility 48 2.2 Zong3 gi|414879552 putative actin family protein [Zea mays] 41920.9/5.18 691 100 7 GRMZM2G053284 3:175945092–175942615 7.50E-192 Carbon fixation 40 2.9 Zong3 gi|414874045 RuBisCO large subunit-binding protein subunit 61418.8/5.20 557 100 7 GRMZM2G434173 1:300072472–300068298 9.10E-203 Glycan biosynthesis and metabolism 56 3.8 Zong3 gi|12585309 Phosphoglucomutase, cytoplasmic 1; 63285.9/5.46 475 100 10 GRMZM2G109383 5:10871874–10866206 2.90E-261 62 2.7 Zong3 gi|12585310 Phosphoglucomutase, cytoplasmic 2; 63229.9/5.47 541 100 10 GRMZM2G023289 1:267953598–267959889 2.70E-267 Nucleotide metabolism 46 3.1 Zong3 gi|414868742 adenine phosphoribosyl transferase 1 [Zea mays] 19507.4/5.14 321 100 5 GRMZM2G131907 1:164655824–164658422 8.60E-72 Signal transduction 61 3.0 Zong3 gi|28373358 Chain A, Crystal Structure Of The Maize Zm-P60.1 Beta-Glucosidase 58185.4/5.44 280 100 9 GRMZM2G016890 10:34240659–34245626 1.20E-127 Unknown 49 3.1 Zong3 gi|223975961 unknown [Zea mays] 34782.7/5.59 296 100 3 GRMZM2G175562 6:88908479–88909745 1.50E-136 19 2.8 Lx9801 gi|194689886 unknown [Zea mays] 17198.5/8.64 502 100 4 GRMZM2G152775 6:69732633–69729284 1.10E-41 37 3.7 Lx9801 gi|241920977 hypothetical protein SORBIDRAFT_01g020010 61322.1/5.67 373 100 9 GRMZM2G458208 5:31026372–31014140 8.10E-250 35 3.7 Lx9801 gi|257676173 unnamed protein product [Oryza sativa] 79272.0/7.19 430 100 3 GRMZM2G149751 1:176865419–176870974 2.1E-315 47 2.0 Zong3 gi|414884326 hypothetical protein ZEAMMB73_606346 [Zea mays] 31549.8/5.36 210 100 3 GRMZM2G051771 7:34010380–34006572 2.40E-122 66 3.2 Zong3 gi|219973780 unnamed protein product [Zea mays] 38084.1/6.00 760 100 11 GRMZM2G700188 7:108416493–108414610 2.30E-103 33 4.1 Lx9801 gi|224031021 unknown [Zea mays] 68688.9/5.36 1,230 100 18 GRMZM2G421857 4:235992822–235986865 2.60E-308 34 9.8 Lx9801 gi|194708072 unknown [Zea mays] 68547.2/5.46 236 100 8 GRMZM2G033208 9:22791995–22794986 1.10E-307 32 3.1 Lx9801 gi|293336560 hypothetical protein LOC100381658 [Zea mays] 60422.9/5.47 628 100 8 GRMZM2G003385 3:185895426–185890963 1.70E-256 43 2.1 Zong3 gi|257738098 unnamed protein product [Zea mays] 35967.1/5.35 973 100 10 GRMZM2G108153 2:25459872–25458525 3.40E-54 [110]Open in a new tab Notes ^a Spot No. corresponds to labels in 2-DE map. ^b Maximum fold changes between different developmental stages were calculated by ANOVA. ^c GenBank accession number of protein spot. ^d Protein name in NCBI database. ^e Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. ^f Confidence interval of the identified proteins. ^g Gene name retrieved from maize sequence ([111]http://ensembl.gramene.org/Zea_mays/Info/Index) by cDNA blast. ^h Physical position determined by blast function in MaizeGDB. For the heterosis analysis, 46, 47, and 37 protein spots with maximum changes of more than two-fold (P < 0.05) between the hybrid Xun928×Zong3 and its corresponding parents were retrieved at D[8], D[10], and D[12], respectively ([112]Table 3 and [113]S2 Table). The corresponding numbers of protein spots with more than two-fold changes between the hybrid Lx9801×Zong3 and its two parents were 24, 37, and 24, respectively. Out of the 215 differentially accumulated proteins, about 57% (122 protein spots) were additively accumulated and 43% (93 protein spots) were non-additively accumulated in the two hybrids. Among the non-additively accumulated proteins, five interaction patterns were observed ([114]Table 3); “−”, “− −”, “+”, “+ −”, and “+ +”, accounting for about 34% (32 protein spots), 3% (3 protein spots), 42% (39 protein spots), 12% (11 protein spots), and 9% (8 protein spots) of the non-additively accumulated proteins, respectively. The “− −” pattern was only detected in the hybrid Xun928×Zong3 at D[12], and the “+ +” pattern was only detected at D[10] and D[12] in the two hybrids. The “+ −” pattern was found in the hybrid Xun928×Zong3 at D[8] and D[12] and the hybrid Lx9801×Zong3 at D[10] and D[12]. The other two major non-additive accumulation patterns “+” and “−” were well distributed across the three sampling stages in each hybrid. Table 3. Differentially accumulated proteins identified by MS between hybrids and corresponding inbred lines during different sampling stages. Spot No.[115] ^a Fold change[116] ^b Heterotic pattern[117] ^c Hybrid name[118] ^d Accession number[119] ^e Protein name[120] ^f Theoretical MW/PI Protein Score[121] ^g Protein Score C. I.% [122]^h Pep. Count Gene name[123] ^i Gene position[124] ^j P-value Amino acid metabolism 270 2.2 A XZ-D[12] gi|195613424 N-acetyltransferase [Zea mays] 24346.3/5.95 208 100 7 9:152823282–152822299 1.40E-46 277 Y/N + LZ-D[12] gi|195619648 IN2-1 protein [Zea mays] 27425.1/4.97 382 100 15 GRMZM2G162486 9:139006399–139008602 1.90E-76 80 Y/N + XZ-D[8] gi|413923160 arginine decarboxylase isoform 1 [Zea mays] 71110.8/5.18 945 100 22 GRMZM2G374302 4:144866060–144868200 5.80E-137 121 Y/N A LZ-D[8] gi|413933557 putative methionine synthase family protein isoform 1 84907.5/5.83 435 100 15 GRMZM2G112149 5:15569743–15564425 0.00E+00 148 Y/N + XZ-D[10] gi|413950042 methionine adenosyltransferase [Zea mays] 46391.5/6.03 884 100 17 GRMZM2G117198 8:129153090–129156433 1.30E-189 194 2.9 A LZ-D[10] gi|413950043 methionine adenosyltransferase [Zea mays] 43389.9/5.57 577 100 15 GRMZM2G117198 8:129153090–129156433 1.30E-189 238 3.4 + + XZ-D[12] gi|413950043 methionine adenosyltransferase [Zea mays] 43389.9/5.57 407 100 14 GRMZM2G117198 8:129153090–129156433 1.30E-189 155 Y/N A XZ-D[10] gi|414869037 putative methionine synthase family protein 84781.4/5.54 472 100 19 GRMZM2G149751 1:176865419–176870974 0.00E+00 239 4.7 A XZ-D[12] gi|414869037 putative methionine synthase family protein 84781.4/5.54 466 100 19 GRMZM2G149751 1:176865419–176870974 0.00E+00 257 2.5 A XZ-D[12] gi|414886469 triosephosphate isomerase [Zea mays] 32685.8/6.14 555 100 15 GRMZM5G852968 7:143564086–143560301 6.60E-113 137 Y/N + LZ-D[8] gi|50086699 betaine aldehyde dehydrogenase [Zea mays] 55739.3/5.34 262 100 10 GRMZM2G146754 4:79470324–79474910 4.40E-203 284 Y/N A LZ-D[12] gi|50086699 betaine aldehyde dehydrogenase [Zea mays] 55739.3/5.34 347 100 13 GRMZM2G146754 4:79470324–79474910 4.40E-203 Biosynthesis of other secondary metabolites 289 Y/N - LZ-D[12] gi|195613588 tropinone reductase 2 [Zea mays] 28173.5/5.99 401 100 11 GRMZM2G152258 2:231084833–231086155 5.50E-61 163 16.5 A XZ-D[10] gi|195655583 O-methyltransferase ZRP4 [Zea mays] 40662.7/5.74 367 100 12 GRMZM2G385313 2:127725353–127723913 1.10E-46 171 Y/N A XZ-D[10] gi|195655583 O-methyltransferase ZRP4 [Zea mays] 40662.7/5.74 413 100 14 GRMZM2G385313 2:127725353–127723913 1.10E-46 236 4.2 + + XZ-D[12] gi|195655583 O-methyltransferase ZRP4 [Zea mays] 40662.7/5.74 271 100 10 GRMZM2G385313 2:127725353–127723913 1.10E-46 243 6.1 + XZ-D[12] gi|195655583 O-methyltransferase ZRP4 [Zea mays] 40662.7/5.74 358 100 15 GRMZM2G385313 2:127725353–127723913 1.10E-46 89 Y/N + - XZ-D[8] gi|413920184 O-methyltransferase ZRP4 [Zea mays] 39557.4/5.52 509 100 8 GRMZM2G349791 4:1300771–1299504 1.00E-43 118 8.4 - LZ-D[8] gi|413920184 O-methyltransferase ZRP4 [Zea mays] 39557.4/5.52 672 100 14 GRMZM2G349791 4:1300771–1299504 1.00E-43 150 Y/N A XZ-D[10] gi|413920184 O-methyltransferase ZRP4 [Zea mays] 39557.4/5.52 797 100 16 GRMZM2G349791 4:1300771–1299504 1.00E-43 272 Y/N + XZ-D[12] gi|413920184 O-methyltransferase ZRP4 [Zea mays] 39557.4/5.52 430 100 13 GRMZM2G349791 4:1300771–1299504 1.00E-43 291 3.9 A LZ-D[12] gi|413920184 O-methyltransferase ZRP4 [Zea mays] 39557.4/5.52 662 100 15 GRMZM2G349791 4:1300771–1299504 1.00E-43 79 Y/N A XZ-D[8] gi|104303692 UDP-glucose flavonoid-3-O-glucosyltransferase [Zea mays] 49229.9/5.33 593 100 16 GRMZM2G165390 9:11781405–11779704 1.20E-60 197 Y/N - LZ-D[10] gi|162463323 anthocyanidin 3-O-glucosyltransferase [Zea mays] 49251.8/5.39 105 100 9 GRMZM2G165390 9:11781402–11779647 1.80E-62 99 Y/N A XZ-D[8] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 934 100 21 GRMZM2G162755 6:120060819–120059072 3.80E-46 117 Y/N + LZ-D[8] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 140 100 9 GRMZM2G162755 6:120060819–120059072 3.80E-46 122 Y/N A LZ-D[8] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 411 100 17 GRMZM2G162755 6:120060819–120059072 3.80E-46 123 6.8 A LZ-D[8] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 560 100 20 GRMZM2G162755 6:120060819–120059072 3.80E-46 159 4.8 A XZ-D[10] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 419 100 19 GRMZM2G162755 6:120060819–120059072 3.80E-46 200 Y/N A LZ-D[10] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 457 100 19 GRMZM2G162755 6:120060819–120059072 3.80E-46 201 6.3 A LZ-D[10] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 668 100 20 GRMZM2G162755 6:120060819–120059072 3.80E-46 212 10.1 + + LZ-D[10] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 518 100 16 GRMZM2G162755 6:120060819–120059072 3.80E-46 241 Y/N A XZ-D[12] gi|413944345 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52096.3/5.83 519 100 17 GRMZM2G162755 6:120060819–120059072 3.80E-46 119 Y/N A LZ-D[8] gi|413944348 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52266.8/5.36 517 100 17 GRMZM2G383404 6:120201779–120203533 6.90E-47 152 Y/N A XZ-D[10] gi|413944348 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52266.8/5.36 653 100 16 GRMZM2G383404 6:120201779–120203533 6.90E-47 231 Y/N A LZ-D[10] gi|413944348 anthocyanidin 3-O-glucosyltransferase [Zea mays] 52266.8/5.36 393 100 15 GRMZM2G383404 6:120201779–120203533 6.90E-47 202 5.3 A LZ-D[10] gi|414590349 anthocyanidin 3-O-glucosyltransferase [Zea mays] 47063.1/5.84 626 100 17 GRMZM2G180283 2:202537696–202536156 9.90E-60 76 9.2 A XZ-D[8] gi|414881303 anthocyaninless1 [Zea mays] 39100.8/5.48 1,110 100 20 GRMZM2G026930 3:216387830–216386070 4.20E-111 138 12.9 A LZ-D[8] gi|414881303 anthocyaninless1 [Zea mays] 39100.8/5.48 957 100 15 GRMZM2G026930 3:216387830–216386070 4.20E-111 188 18.8 A XZ-D[10] gi|414881303 anthocyaninless1 [Zea mays] 39100.8/5.48 917 100 18 GRMZM2G026930 3:216387830–216386070 4.20E-111 224 15.1 + LZ-D[10] gi|414881303 anthocyaninless1 [Zea mays] 39100.8/5.48 966 100 20 GRMZM2G026930 3:216387830–216386070 4.20E-111 285 Y/N A LZ-D[12] gi|414881303 anthocyaninless1 [Zea mays] 39100.8/5.48 857 100 17 GRMZM2G026930 3:216387830–216386070 4.20E-111 96 3.6 - XZ-D[8] gi|195635735 GDSL-motif lipase/hydrolase-like protein [Zea mays] 41926.6/6.81 76 99.614 5 GRMZM2G700208 10:33361036–33356952 1.10E-39 255 Y/N - XZ-D[12] gi|413920639 chitinase 1 [Zea mays] 31165.5/4.97 116 100 5 GRMZM2G358153 4:12097106–12098325 0.00017 275 Y/N + LZ-D[12] gi|413920639 chitinase 1 [Zea mays] 31165.5/4.97 80 99.807 4 GRMZM2G358153 4:12097106–12098325 0.00017 127 Y/N + LZ-D[8] gi|414878829 glutathione S-transferase4 [Zea mays] 23879.7/5.96 378 100 8 GRMZM2G034083 3:128543799–128546078 9.70E-199 216 Y/N + - LZ-D[10] gi|414878829 glutathione S-transferase4 [Zea mays] 23879.7/5.96 369 100 8 GRMZM2G034083 3:128543799–128546078 9.70E-199 298 Y/N A LZ-D[12] gi|414878829 glutathione S-transferase4 [Zea mays] 23879.7/5.96 314 100 6 GRMZM2G034083 3:128543799–128546078 9.70E-199 217 Y/N + LZ-D[10] gi|162460516 Glutathione transferase III(b) [Zea mays] 23865.6/5.96 267 100 5 GRMZM2G146246 3:155017244–155015982 1.00E-52 Carbohydrate metabolism 223 Y/N A LZ-D[10] gi|20385155 NADPH-dependent reductase [Zea mays] 39172.9/5.48 739 100 19 GRMZM2G026930 3:216387831–216386092 9.70E-112 74 Y/N A XZ-D[8] gi|195643366 APx2—Cytosolic Ascorbate Peroxidase [Zea mays] 27298.8/5.28 890 100 13 GRMZM2G140667 2:219260991–219258343 1.10E-96 140 Y/N A LZ-D[8] gi|195643366 APx2—Cytosolic Ascorbate Peroxidase [Zea mays] 27298.8/5.28 629 100 13 GRMZM2G140667 2:219260991–219258343 1.10E-96 190 Y/N A XZ-D[10] gi|195643366 APx2—Cytosolic Ascorbate Peroxidase [Zea mays] 27298.8/5.28 762 100 13 GRMZM2G140667 2:219260991–219258343 1.10E-96 268 Y/N A XZ-D[12] gi|195643366 APx2—Cytosolic Ascorbate Peroxidase [Zea mays] 27298.8/5.28 684 100 15 GRMZM2G140667 2:219260991–219258343 1.10E-96 287 Y/N A LZ-D[12] gi|195643366 APx2—Cytosolic Ascorbate Peroxidase [Zea mays] 27298.8/5.28 766 100 13 GRMZM2G140667 2:219260991–219258343 1.10E-96 104 Y/N A XZ-D[8] gi|413942605 6-phosphogluconate dehydrogenase isoenzyme B 53102.9/5.93 638 100 24 GRMZM2G127798 6:57906012–57903707 3.00E-243 176 Y/N A XZ-D[10] gi|413942605 6-phosphogluconate dehydrogenase isoenzyme B 53102.9/5.93 460 100 18 GRMZM2G127798 6:57906012–57903707 3.00E-243 211 Y/N + + LZ-D[10] gi|413942605 6-phosphogluconate dehydrogenase isoenzyme B 53102.9/5.93 401 100 14 GRMZM2G127798 6:57906012–57903707 3.00E-243 247 Y/N A XZ-D[12] gi|413942605 6-phosphogluconate dehydrogenase isoenzyme B 53102.9/5.93 408 100 15 GRMZM2G127798 6:57906012–57903707 3.00E-243 177 Y/N A XZ-D[10] gi|413944222 ppi-phosphofructokinase [Zea mays] 61640.1/6.3 291 100 18 GRMZM2G059151 6:115378780–115372697 2.50E-239 232 Y/N + LZ-D[10] gi|413949561 sucrose-phosphatase1 [Zea mays] 47669.2/5.8 255 100 10 GRMZM2G055489 8:115837557–115832290 6.90E-134 173 Y/N A XZ-D[10] gi|414865246 putative alcohol dehydrogenase superfamily 41850.2/6.1 551 100 14 GRMZM2G154007 1:19329011–19326354 4.80E-110 95 Y/N A XZ-D[8] gi|414866238 APx1-Cytosolic Ascorbate Peroxidase [Zea mays] 27467.9/5.64 393 100 11 GRMZM2G137839 1:43682927–43685858 3.20E-113 149 4.0 + XZ-D[10] gi|414868220 putative enolase family protein isoform 1 [Zea mays] 48369.6/5.59 743 100 19 GRMZM2G048371 1:125087719–125081660 4.40E-210 105 Y/N A XZ-D[8] gi|414871407 GDP-mannose 3,5-epimerase 1 [Zea mays] 43337.3/5.99 405 100 17 GRMZM2G124434 1:240922252–240925270 3.80E-188 160 5.1 A XZ-D[10] gi|414872918 UDP-glucose 6-dehydrogenase [Zea mays] 56749.2/6.23 648 100 19 GRMZM2G328500 1:278196990–278199108 4.20E-237 165 5.2 A XZ-D[10] gi|414872919 UDP-glucose 6-dehydrogenase isoform 1 [Zea mays] 53495.4/5.71 407 100 17 GRMZM2G328500 1:278196990–278199108 4.20E-237 204 Y/N + LZ-D[10] gi|414878099 ATP-citrate synthase [Zea mays] 46948.1/5.58 380 100 16 GRMZM2G034083 3:128543602–128547481 9.70E-199 292 Y/N + LZ-D[12] gi|414878099 ATP-citrate synthase [Zea mays] 46948.1/5.58 354 100 16 GRMZM2G034083 3:128543602–128547481 9.70E-199 86 Y/N A XZ-D[8] gi|75172084 Sucrose-phosphatase 1; 47583/5.48 634 100 18 GRMZM2G055489 8:115838485–115832372 2.60E-141 120 Y/N A LZ-D[8] gi|75172084 Sucrose-phosphatase 1; 47583/5.48 436 100 17 GRMZM2G055489 8:115838485–115832372 2.60E-141 151 Y/N A XZ-D[10] gi|75172084 Sucrose-phosphatase 1; 47583/5.48 466 100 16 GRMZM2G055489 8:115838485–115832372 2.60E-141 Cell structure 220 3.8 A LZ-D[10] gi|8928413 Tubulin beta-4 chain 50784.1/4.86 771 100 21 GRMZM2G066191 5:14874004–14870370 1.90E-209 262 4.5 A XZ-D[12] gi|8928413 Tubulin beta-4 chain 50784.1/4.86 561 100 20 GRMZM2G066191 5:14874004–14870370 1.90E-209 283 Y/N + - LZ-D[12] gi|8928413 Tubulin beta-4 chain 50784.1/4.86 675 100 21 GRMZM2G066191 5:14874004–14870370 1.90E-209 Stress and defense response 164 Y/N + XZ-D[10] gi|162459030 legumin-like protein [Zea mays] 38046.6/5.79 611 100 12 GRMZM5G801289 8:132711366–132712942 3.30E-118 207 Y/N + LZ-D[10] gi|162459030 legumin-like protein [Zea mays] 38046.6/5.79 494 100 12 GRMZM5G801289 8:132711366–132712942 3.30E-118 246 5.0 A XZ-D[12] gi|162459030 legumin-like protein [Zea mays] 38046.6/5.79 653 100 11 GRMZM5G801289 8:132711366–132712942 3.30E-118 98 Y/N A XZ-D[8] gi|195629806 legumin-like protein [Zea mays] 38076.6/5.79 872 100 12 GRMZM2G005552 8:132711342–132712943 4.20E-118 244 Y/N A XZ-D[12] gi|195629806 legumin-like protein [Zea mays] 38076.6/5.79 750 100 13 GRMZM2G005552 8:132711342–132712943 4.20E-118 111 4.8 + - XZ-D[8] gi|413934538 peroxidase 39 isoform 2 [Zea mays] 35814.3/7.59 654 100 14 GRMZM2G085967 5:47608191–47611493 8.40E-97 115 Y/N A XZ-D[8] gi|413934538 peroxidase 39 isoform 2 [Zea mays] 35814.3/7.59 663 100 15 GRMZM2G085967 5:47608191–47611493 8.40E-97 213 Y/N A LZ-D[10] gi|413934538 peroxidase 39 isoform 2 [Zea mays] 35814.3/7.59 378 100 13 GRMZM2G085967 5:47608191–47611493 8.40E-97 214 Y/N A LZ-D[10] gi|413934538 peroxidase 39 isoform 2 [Zea mays] 35814.3/7.59 459 100 17 GRMZM2G085967 5:47608191–47611493 8.40E-97 248 3.6 - XZ-D[12] gi|413934538 peroxidase 39 isoform 2 [Zea mays] 35814.3/7.59 516 100 13 GRMZM2G085967 5:47608191–47611493 8.40E-97 183 Y/N A XZ-D[10] gi|75994013 pathogenesis-related protein 5 [Zea mays subsp. mays] 18061.3/4.87 216 100 2 GRMZM2G402631 1:178036019–178035437 4.50E-38 110 Y/N A XZ-D[8] gi|414870957 secretory protein [Zea mays] 24524.1/4.82 242 100 6 GRMZM2G153208 1:228705641–228704654 1.70E-66 147 Y/N - XZ-D[10] gi|414870957 secretory protein [Zea mays] 24524.1/4.82 333 100 7 GRMZM2G153208 1:228705641–228704654 1.70E-66 Energy metabolism 87 Y/N A XZ-D[8] gi|28172915 cytosolic 3-phosphoglycerate kinase [Zea mays] 31662.8/5.01 850 100 16 GRMZM2G382914 5:84861898–84858488 1.70E-128 235 Y/N A XZ-D[12] gi|28172915 cytosolic 3-phosphoglycerate kinase [Zea mays] 31662.8/5.01 494 100 18 GRMZM2G382914 5:84861898–84858488 1.70E-128 88 Y/N A XZ-D[8] gi|413935733 phosphoglycerate kinase isoform 3 [Zea mays] 42469.8/5.65 988 100 23 GRMZM2G382914 5:84858478–84861909 8.60E-175 237 Y/N + XZ-D[12] gi|413935733 phosphoglycerate kinase isoform 3 [Zea mays] 42469.8/5.65 839 100 26 GRMZM2G382914 5:84858478–84861909 8.60E-175 199 5.8 - LZ-D[10] gi|11467189 ATP synthase CF1 alpha subunit [Zea mays] 55729.4/5.87 692 100 28 GRMZM2G385622 2:200755769–200756023 2.90E-245 145 2.8 - XZ-D[10] gi|162464321 malate dehydrogenase, cytoplasmic [Zea mays] 35909.3/5.77 574 100 16 GRMZM2G415359 1:231403594–231398465 1.40E-156 182 2.2 - XZ-D[10] gi|195612184 ATP synthase delta chain [Zea mays] 21269.3/5.72 143 100 6 GRMZM2G171628 2:202325467–202328755 1.60E-70 71 Y/N A XZ-D[8] gi|195640660 formate dehydrogenase 1 [Zea mays] 41678.2/6.32 455 100 13 GRMZM2G049811 9:49450491–49446497 1.10E-156 124 Y/N - LZ-D[8] gi|414589713 putative oxidoreductase, aldo/keto reductase 36998/6.07 457 100 11 GRMZM2G087507 2:188279947–188276596 1.70E-84 Carbon fixation 67 3.0 A XZ-D[8] gi|11467200 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit 53294.6/6.33 842 100 27 GRMZM2G469277;GRMZM2G062854 6:160979914–160980168 3.10E-241 106 Y/N + XZ-D[8] gi|11467200 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit 53294.6/6.33 575 100 18 GRMZM2G469277;GRMZM2G062854 6:160979914–160980168 3.10E-241 178 7.4 A XZ-D[10] gi|11467200 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit 53294.6/6.33 577 100 20 GRMZM2G469277;GRMZM2G062854 6:160979914–160980168 3.10E-241 Protein biosynthesis and folding 91 2.7 A XZ-D[8] gi|413922095 retrotransposon protein Ty1-copia subclass [Zea mays] 28923.7/6.31 253 100 8 GRMZM2G073079 4:70093844–70091439 6.10E-39 94 4.3 A XZ-D[8] gi|413919640 glycine-rich protein 2b [Zea mays] 20576.1/5.92 469 100 10 GRMZM5G895313 10:143487212–143487775 2.40E-35 161 Y/N A XZ-D[10] gi|413932420 putative translation elongation/initiation factor family 46309.1/6.85 244 100 14 GRMZM2G313678 5:1033629–1037919 7.50E-176 72 Y/N A XZ-D[8] gi|162464130 eukaryotic translation initiation factor 5A [Zea mays] 17713.8/5.61 536 100 10 GRMZM2G144030 7:163045095–163042293 1.30E-70 146 Y/N A XZ-D[10] gi|162464130 eukaryotic translation initiation factor 5A [Zea mays] 17713.8/5.61 205 100 10 GRMZM2G144030 7:163045095–163042293 1.30E-70 112 3.4 A XZ-D[8] gi|414887577 elongation factor 1-delta 1 [Zea mays] 24951.4/4.39 392 100 10 GRMZM2G031545 7:165426108–165423624 1.40E-64 251 Y/N A XZ-D[12] gi|414887577 elongation factor 1-delta 1 [Zea mays] 24951.4/4.39 148 100 7 GRMZM2G031545 7:165426108–165423624 1.40E-64 70 Y/N A XZ-D[8] gi|414589578 chaperonin isoform 1 [Zea mays] 25558.7/8.67 465 100 11 GRMZM2G399284 2:184583814–184578857 1.20E-67 258 Y/N A XZ-D[12] gi|414589578 chaperonin isoform 1 [Zea mays] 25558.7/8.67 220 100 8 GRMZM2G399284 2:184583814–184578857 1.20E-67 260 Y/N A XZ-D[12] gi|414589578 chaperonin isoform 1 [Zea mays] 25558.7/8.67 213 100 13 GRMZM2G399284 2:184583814–184578857 1.20E-67 279 Y/N A LZ-D[12] gi|414589578 chaperonin isoform 1 [Zea mays] 25558.7/8.67 291 100 13 GRMZM2G399284 2:184583814–184578857 1.20E-67 252 3.4 A XZ-D[12] gi|145666464 protein disulfide isomerase [Zea mays] 56921/5.01 783 100 31 GRMZM2G393320 4:14881672–14877150 5.30E-214 229 Y/N A LZ-D[10] gi|413933376 proteasome subunit beta type [Zea mays] 23240/5.6 95 99.994 10 GRMZM2G111566 5:12576599–12579751 5.20E-81 288 Y/N - LZ-D[12] gi|413933376 proteasome subunit beta type [Zea mays] 23240/5.6 224 100 10 GRMZM2G111566 5:12576599–12579751 5.20E-81 Glycan biosynthesis and metabolism 267 Y/N - - XZ-D[12] gi|195629642 lichenase-2 precursor [Zea mays] 35062.1/5.68 72 98.78 6 GRMZM2G137535 6:142506341–142502273 5.70E-75 290 Y/N + + LZ-D[12] gi|195629642 lichenase-2 precursor [Zea mays] 35062.1/5.68 234 100 5 GRMZM2G137535 6:142506341–142502273 5.70E-75 90 3.8 A XZ-D[8] gi|34588146 Alpha-1,4-glucan-protein synthase 41690.8/5.75 715 100 21 GRMZM2G073725 1:248925143–248922623 1.20E-186 Lipid metabolism 198 Y/N - LZ-D[10] gi|12620877 lipoxygenase [Zea mays] 96587.2/5.71 419 100 28 GRMZM2G109056 1:264266453–264290362 0.00E+00 281 Y/N + - LZ-D[12] gi|413933924 epoxide hydrolase 2 isoform 1 [Zea mays] 35469.8/5.09 118 100 7 GRMZM2G032910 5:24638574–24636922 5.20E-49 Nucleotide metabolism 225 Y/N + LZ-D[10] gi|414589043 pyrimidine-specific ribonucleoside hydrolase rihB 34329.6/5.36 254 100 5 GRMZM2G104999 2:166013604–166017041 1.60E-116 266 Y/N + - XZ-D[12] gi|414589043 pyrimidine-specific ribonucleoside hydrolase rihB 34329.6/5.36 161 100 4 GRMZM2G104999 2:166013604–166017041 1.60E-116 286 Y/N + LZ-D[12] gi|414589043 pyrimidine-specific ribonucleoside hydrolase rihB 34329.6/5.36 260 100 7 GRMZM2G104999 2:166013604–166017041 1.60E-116 Plant hormone biosynthesis and signal transduction 107 2.9 A XZ-D[8] gi|195645942 cytokinin-O-glucosyltransferase 2 [Zea mays] 53489.2/5.86 286 100 11 GRMZM2G083130 3:135786100–135784404 1.10E-94 274 Y/N + - LZ-D[12] gi|413924376 acc oxidase [Zea mays] 35699.8/4.97 217 100 12 GRMZM2G126732 4:177660129–177657861 1.60E-109 168 4.0 A XZ-D[10] gi|414867333 allene oxide cyclase 4 [Zea mays] 25932.4/9.05 140 100 8 GRMZM2G077316 1:76741280–76740459 3.50E-59 299 Y/N - LZ-D[12] gi|195652523 ABA-responsive protein [Zea mays] 29250.4/6.14 263 100 8 GRMZM2G106622 10:14307592–14310405 5.10E-74 193 2.4 - XZ-D[10] gi|21730839 Chain C, Crystal Structure Of Auxin-Binding Protein 1 In Complex With 1-Naphthalene Acetic Acid 18552.2/5.24 157 100 7 GRMZM2G116204 3:133916933–133921384 6.00E-87 263 Y/N - XZ-D[12] gi|413925162 putative O-Glycosyl hydrolase superfamily protein [Zea mays] 83731.8/5.78 294 100 15 7:144964081–144966447 2.60E-221 69 Y/N A XZ-D[8] gi|413939100 pyrophosphate-energized proton pump1, partial [Zea mays] 16441.6/9.6 90 99.981 3 GRMZM2G090718 5:210647780–210643103 3.60E-41 254 Y/N A XZ-D[12] gi|413944231 gibberellin receptor GID1L2 [Zea mays] 34820.6/5 463 100 12 GRMZM2G156310 6:115727511–115728876 2.80E-57 Unknown 187 2.8 + XZ-D[10] gi|414589057 hypothetical protein ZEAMMB73_276484 [Zea 46817.3/5.45 645 100 20 GRMZM2G084881 2:166253571–166248891 1.80E-170 265 11.2 A XZ-D[12] gi|414589057 hypothetical protein ZEAMMB73_276484 [Zea 46817.3/5.45 543 100 20 GRMZM2G084881 2:166253571–166248891 1.80E-170 109 Y/N A XZ-D[8] gi|414870644 hypothetical protein ZEAMMB73_435161 [Zea mays] 14648.4/5 158 100 5 GRMZM2G078022 1:220950014–220945296 4.80E-31 126 Y/N A LZ-D[8] gi|414584728 hypothetical protein ZEAMMB73_474117 [Zea mays] 28283.2/5.83 269 100 9 GRMZM2G165535 2:964185–961339 1.70E-105 128 5.4 A LZ-D[8] gi|414876731 hypothetical protein ZEAMMB73_561858 [Zea mays] 33565.8/5.96 521 100 13 GRMZM2G120304 3: 31902191–31898927 8.30E-51 169 4.6 + XZ-D[10] gi|414876731 hypothetical protein ZEAMMB73_561858 [Zea mays] 33565.8/5.96 297 100 9 GRMZM2G120304 3: 31902191–31898927 8.30E-51 113 Y/N A XZ-D[8] gi|414587271 hypothetical protein ZEAMMB73_690514 [Zea mays] 27760.2/5.07 542 100 16 GRMZM5G806182 2:54474692–54473385 114 Y/N A XZ-D[8] gi|414587271 hypothetical protein ZEAMMB73_690514 [Zea mays] 27760.2/5.07 425 100 11 GRMZM5G806182 2:54474692–54473385 135 Y/N A LZ-D[8] gi|414587271 hypothetical protein ZEAMMB73_690514 [Zea mays] 27760.2/5.07 699 100 15 GRMZM5G806182 2:54474692–54473385 179 Y/N + XZ-D[10] gi|414587271 hypothetical protein ZEAMMB73_690514 [Zea mays] 27760.2/5.07 132 100 8 GRMZM5G806182 2:54474692–54473385 180 Y/N A XZ-D[10] gi|414587271 hypothetical protein ZEAMMB73_690514 [Zea mays] 27760.2/5.07 496 100 14 GRMZM5G806182 2:54474692–54473385 245 Y/N A XZ-D[12] gi|414588073 hypothetical protein ZEAMMB73_739939 [Zea mays] 38290.9/8.95 202 100 6 GRMZM2G330377 2:73644958–73649833 1.50E-19 108 4.0 A XZ-D[8] gi|414587991 hypothetical protein ZEAMMB73_919014 [Zea mays] 42290.4/6.53 524 100 14 GRMZM2G050131 2:102982712–102984376 0.014 81 Y/N A XZ-D[8] gi|414879972 hypothetical protein ZEAMMB73_969630 [Zea mays] 60422.9/5.47 990 100 21 GRMZM2G003385 3:185895426–185890963 1.70E-256 233 Y/N A XZ-D[12] gi|195642378 hypothetical protein [Zea mays] 42563.1/5.51 209 100 12 GRMZM2G072909 9:65142126–65155480 9.30E-130 189 Y/N - XZ-D[10] gi|195640116 hypothetical protein [Zea mays] 27609.9/5.12 292 100 9 GRMZM2G430600 7:174328795–174326866 9.90E-55 116 Y/N A LZ-D[8] gi|195620226 hypothetical protein [Zea mays] 17568/5.51 514 100 10 GRMZM2G041258 6:106123618–106122830 6.40E-28 228 Y/N A LZ-D[10] gi|195640026 hypothetical protein [Zea mays] 17528.9/5.35 150 100 9 GRMZM2G041258 6:106123618–106122833 3.90E-28 210 5.4 + + LZ-D[10] gi|413950795 hypothetical protein ZEAMMB73_038317 [Zea mays] 46509.6/6.11 478 100 21 GRMZM2G432128 8:147229052–147224985 1.30E-196 219 Y/N A LZ-D[10] gi|413944150 hypothetical protein ZEAMMB73_357615 [Zea mays] 35990.9/5.03 970 100 14 GRMZM2G701082 6:113741028–113738663 4.50E-15 221 Y/N A LZ-D[10] gi|413944150 hypothetical protein ZEAMMB73_357615 [Zea mays] 35990.9/5.03 546 100 16 GRMZM2G701082 6:113741028–113738663 4.50E-15 282 Y/N A LZ-D[12] gi|413944150 hypothetical protein ZEAMMB73_357615 [Zea mays] 35990.9/5.03 830 100 13 GRMZM2G701082 6:113741028–113738663 4.50E-15 157 7.3 A XZ-D[10] gi|413921122 hypothetical protein ZEAMMB73_482448 [Zea mays] 87111.7/5.78 430 100 23 4:28145897–28146077 0 174 Y/N A XZ-D[10] gi|413939433 hypothetical protein ZEAMMB73_631326 [Zea mays] 53487.6/6 284 100 15 GRMZM5G806449 5:215337548–215346511 6.10E-197 259 Y/N A XZ-D[12] gi|413948610 hypothetical protein ZEAMMB73_645738 [Zea mays] 18144.2/5.3 156 100 4 GRMZM2G045664 8:74457615–74456935 2.30E-46 278 Y/N + LZ-D[12] gi|413948610 hypothetical protein ZEAMMB73_645738 [Zea mays] 18144.2/5.3 206 100 4 GRMZM2G045664 8:74457615–74456935 2.30E-46 166 3.8 + + XZ-D[10] gi|413952229 hypothetical protein ZEAMMB73_660929 [Zea mays] 12646.2/7.9 179 100 4 GRMZM2G159643 8:171408802–171405316 1.10E-48 186 Y/N A XZ-D[10] gi|413935382 hypothetical protein ZEAMMB73_705624 [Zea mays] 55418.5/6.53 465 100 16 GRMZM2G370852 5:72953434–72949542 5.70E-217 218 3.5 - LZ-D[10] gi|413949747 hypothetical protein ZEAMMB73_945417 [Zea mays] 49720.5/6.99 602 100 21 GRMZM2G083016 8:120301530–120303746 5.30E-191 73 Y/N +- XZ-D[8] gi|413955418 hypothetical protein ZEAMMB73_953540 [Zea mays] 21571.6/5.26 345 100 8 GRMZM2G314769 9:122457581–122458680 2.10E-22 75 3.5 + - XZ-D[8] gi|413955418 hypothetical protein ZEAMMB73_953540 [Zea mays] 21571.6/5.26 165 100 4 GRMZM2G314769 9:122457581–122458680 2.10E-22 141 Y/N A LZ-D[8] gi|413955418 hypothetical protein ZEAMMB73_953540 [Zea mays] 21571.6/5.26 185 100 7 GRMZM2G314769 9:122457581–122458680 2.10E-22 191 Y/N A XZ-D[10] gi|413955418 hypothetical protein ZEAMMB73_953540 [Zea mays] 21571.6/5.26 173 100 4 GRMZM2G314769 9:122457581–122458680 2.10E-22 226 Y/N + LZ-D[10] gi|413955418 hypothetical protein ZEAMMB73_953540 [Zea mays] 21571.6/5.26 92 99.99 4 GRMZM2G314769 9:122457581–122458680 2.10E-22 280 Y/N A LZ-D[12] gi|413955418 hypothetical protein ZEAMMB73_953540 [Zea mays] 21571.6/5.26 95 99.994 5 GRMZM2G314769 9:122457581–122458680 2.10E-22 139 Y/N - LZ-D[8] gi|226496343 uncharacterized protein LOC100273624 [Zea mays] 28169.5/6 187 100 7 GRMZM2G152258 2:231084849–231086151 5.00E-60 132 Y/N A LZ-D[8] gi|194700784 unknown [Zea mays] 65656.1/6.31 103 100 16 GRMZM2G162688 5:189872033–189878985 3.10E-201 103 Y/N - XZ-D[8] gi|194701170 unknown [Zea mays] 38872.7/7.52 441 100 6 GRMZM2G053206 2:232657306–232656057 2.00E-25 130 Y/N - LZ-D[8] gi|194701170 unknown [Zea mays] 38872.7/7.52 228 100 5 GRMZM2G053206 2:232657306–232656057 2.00E-25 172 Y/N - XZ-D[10] gi|194701170 unknown [Zea mays] 38872.7/7.52 503 100 7 GRMZM2G053206 2:232657306–232656057 2.00E-25 234 Y/N - XZ-D[12] gi|223949117 unknown [Zea mays] 41017.8/5.28 136 100 11 GRMZM2G047564 9:88481641–88468222 3.50E-123 129 Y/N A LZ-D[8] gi|223950161 unknown [Zea mays] 41693.3/6.32 306 100 11 GRMZM2G049811 9:49450407–49446403 2.10E-157 134 Y/N A LZ-D[8] gi|223950161 unknown [Zea mays] 41693.3/6.32 273 100 14 GRMZM2G049811 9:49450407–49446403 2.10E-157 209 Y/N + LZ-D[10] gi|223950161 unknown [Zea mays] 41693.3/6.32 441 100 18 GRMZM2G049811 9:49450407–49446403 2.10E-157 261 3.5 - - XZ-D[12] gi|223975961 unknown [Zea mays] 34782.7/5.59 657 100 14 GRMZM2G175562 6:88908479–88909745 1.50E-136 185 Y/N A XZ-D[10] gi|219886829 unknown [Zea mays] 40504.2/4.99 225 100 14 GRMZM2G105019 3:217552207–217545545 1.20E-154 153 2.4 + XZ-D[10] gi|223975139 unknown [Zea mays] 57479.1/5.46 376 100 15 GRMZM2G161868 3:202435255–202437504 7.40E-256 85 Y/N A XZ-D[8] gi|194699562 unknown [Zea mays] 50961.1/5.17 709 100 21 GRMZM2G372068 2:226478163–226476497 1.80E-101 264 Y/N + - XZ-D[12] gi|194699562 unknown [Zea mays] 50961.1/5.17 355 100 14 GRMZM2G372068 2:226478163–226476497 1.80E-101 203 Y/N - LZ-D[10] gi|194692156 unknown [Zea mays] 39808.2/5.82 195 100 7 GRMZM2G055489 8:115838429–115832290 3.60E-112 293 Y/N - LZ-D[12] gi|194692156 unknown [Zea mays] 39808.2/5.82 420 100 17 GRMZM2G055489 8:115838429–115832290 3.60E-112 192 Y/N - XZ-D[10] gi|194702634 unknown [Zea mays] 24981.6/5.46 218 100 8 GRMZM2G096153 1:7863369–7862135 2.00E-49 215 Y/N + + LZ-D[10] gi|223947673 unknown [Zea mays] 34283.6/7.75 158 100 10 GRMZM2G014397 10:10731158–10736508 6.70E-120 68 Y/N A XZ-D[8] gi|224029461 unknown [Zea mays] 28955.8/5.08 386 100 13 GRMZM2G148769 2:221504148–221501546 3.20E-88 136 Y/N A LZ-D[8] gi|224029461 unknown [Zea mays] 28955.8/5.08 195 100 9 GRMZM2G148769 2:221504148–221501546 3.20E-88 181 Y/N A XZ-D[10] gi|224029461 unknown [Zea mays] 28955.8/5.08 299 100 13 GRMZM2G148769 2:221504148–221501546 3.20E-88 256 Y/N A XZ-D[12] gi|224029461 unknown [Zea mays] 28955.8/5.08 201 100 12 GRMZM2G148769 2:221504148–221501546 3.20E-88 276 Y/N + LZ-D[12] gi|224029461 unknown [Zea mays] 28955.8/5.08 238 100 9 GRMZM2G148769 2:221504148–221501546 3.20E-88 167 2.6 + XZ-D[10] gi|194697638 unknown [Zea mays] 64089/4.8 270 100 16 GRMZM2G048324 1:70432507–70436832 8.00E-156 97 Y/N + XZ-D[8] gi|218319054 unnamed protein product [Zea mays] 27952.1/5.47 509 100 11 GRMZM2G038075 5:3146159–3147709 1.50E-74 125 Y/N + LZ-D[8] gi|218319054 unnamed protein product [Zea mays] 27952.1/5.47 346 100 10 GRMZM2G038075 5:3146159–3147709 1.50E-74 205 5.8 A LZ-D[10] gi|218319054 unnamed protein product [Zea mays] 27952.1/5.47 161 100 10 GRMZM2G038075 5:3146159–3147709 1.50E-74 250 Y/N - XZ-D[12] gi|257737930 unnamed protein product [Zea mays] 37624.5/4.98 178 100 8 1:77358025–77358598 1.60E-93 206 Y/N - LZ-D[10] gi|295415566 unnamed protein product [Zea mays] 34182/5.91 359 100 14 GRMZM2G005887 1:177054888–177059486 2.80E-128 230 Y/N + LZ-D[10] gi|295421207 unnamed protein product [Zea mays] 34240/5.68 420 100 17 GRMZM2G005887 1:177054888–177059486 1.90E-129 92 Y/N + XZ-D[8] gi|296515149 unnamed protein product [Zea mays] 29489.9/5.76 779 100 16 GRMZM2G038075 5:3146159–3147709 3.10E-74 271 Y/N + - XZ-D[12] gi|296515149 unnamed protein product [Zea mays] 29489.9/5.76 262 100 13 GRMZM2G038075 5:3146159–3147709 3.10E-74 175 Y/N A XZ-D[10] gi|298547953 unnamed protein product [Zea mays] 53307.3/5.92 516 100 19 GRMZM2G145715 3:153967866–153964586 2.90E-238 142 4.3 + XZ-D[10] gi|257696214 unnamed protein product [Zea mays] 39454.2/7.12 402 100 12 GRMZM2G137839 1:43682852–43685890 1.50E-113 143 4.9 + XZ-D[10] gi|257696214 unnamed protein product [Zea mays] 39454.2/7.12 479 100 15 GRMZM2G137839 1:43682852–43685890 1.50E-113 170 Y/N + XZ-D[10] gi|257640662 unnamed protein product [Zea mays] 39196.1/6.09 684 100 19 GRMZM2G328094 8:6457224–6458504 1.90E-113 208 Y/N + LZ-D[10] gi|257640662 unnamed protein product [Zea mays] 39196.1/6.09 388 100 17 GRMZM2G328094 8:6457224–6458504 1.90E-113 249 Y/N A XZ-D[12] gi|257676371 unnamed protein product [Zea mays] 23715.2/4.65 175 100 3 GRMZM2G175076 3:231979651–231978427 2.80E-41 82 Y/N A XZ-D[8] gi|257333572 unnamed protein product [Zea mays] 64533.6/6.13 498 100 22 GRMZM2G016890 10:34240678–34245632 1.10E-128 83 9.5 A XZ-D[8] gi|257333572 unnamed protein product [Zea mays] 64533.6/6.13 658 100 23 GRMZM2G016890 10:34240678–34245632 1.10E-128 84 Y/N + XZ-D[8] gi|257333572 unnamed protein product [Zea mays] 64533.6/6.13 557 100 23 GRMZM2G016890 10:34240678–34245632 1.10E-128 154 Y/N A XZ-D[10] gi|257333572 unnamed protein product [Zea mays] 64533.6/6.13 260 100 18 GRMZM2G016890 10:34240678–34245632 1.10E-128 195 Y/N - LZ-D[10] gi|257333572 unnamed protein product [Zea mays] 64533.6/6.13 468 100 26 GRMZM2G016890 10:34240678–34245632 1.10E-128 294 Y/N - LZ-D[12] gi|257333572 unnamed protein product [Zea mays] 64533.6/6.13 372 100 19 GRMZM2G016890 10:34240678–34245632 1.10E-128 196 Y/N - LZ-D10 gi|257714156 unnamed protein product [Zea mays] 64551.6/6.23 360 100 22 GRMZM2G016890 10:34240678–34245632 4.60E-128 295 Y/N - LZ-D[12] gi|257714156 unnamed protein product [Zea mays] 64551.6/6.23 334 100 17 GRMZM2G016890 10:34240678–34245632 4.60E-128 77 Y/N A XZ-D[8] gi|296511817 unnamed protein product [Zea mays] 46789.3/5.45 722 100 19 GRMZM2G084881 2:166253571–166248891 6.90E-171 78 Y/N A XZ-D[8] gi|219972638 unnamed protein product [Zea mays] 46039.3/5.21 562 100 13 GRMZM2G098239 2:10633629–10631020 2.40E-60 253 3.9 - - XZ-D[12] gi|257725861 unnamed protein product [Zea mays] 60702.2/5 386 100 22 6:142273504–142277935 7.60E-215 93 Y/N A XZ-D[8] gi|219746454 unnamed protein product [Zea mays] 25750.4/5.52 282 100 10 GRMZM2G434541 3:206915357–206915753 2.70E-52 [125]Open in a new tab Notes ^a Spot No. corresponds to labels in 2-DE map. ^b Maximum fold changes among three groups (female line, male line, and hybrid combination) were calculated by ANOVA.Y/N means that difference between them was present or absent. ^c Heterotic patterns were examined following Hoecker et al. (2008). “A” indicates protein spots that had no significant difference in average spot intensities with midparent value at 0.05 level. Average spot intensities of proteins that deviated significantly from the mid-parent value of the parental lines at the 0.05 cut-off level were defined as non-additive proteins. Based on this premise, “+” and “-” represented protein spot intensities identified in F[1] hybrids that were similar to the high parent and low parent values, respectively. “+ +” and “- -” represented the protein spot intensities identified in F[1]hybrids that were significantly different from the high parent and the low parent values, respectively. “+ -” represented the protein spot intensities identified in F[1] hybrids that fell in between the mid-parent and the high parent or the mid-parent and the low parent values. ^d Hybrids Xun928×Zong3 and Lx9801×Zong3 are abbreviated as XZ and LZ, respectively. D[8], D[10], and D[12] represent sampling stages. ^e GenBank accession number of protein spot. ^f Protein name in NCBI database. ^g Proteins scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. ^h Confidence interval of the identified protein. ^i Gene name retrieved from maize sequence ([126]http://ensembl.gramene.org/Zea_mays/Info/Index) by cDNA blast. ^j Physical position determined by blast function in MaizeGDB. Differentially accumulated proteins identified as important for silk viability and its heterosis Three proteins differentially accumulated during silk development were identified, including gi|413944345 (protein spot 63 in Zong3), gi|414869037 (protein spot 8 in Xun928), and gi|195635735 (protein spot 16 in Xun928). These three proteins also differentially regulated the heterosis of silk viability in the two hybrids (Tables [127]2 and [128]3). gi|413944345, which differentially regulated silk development in the common paternal line Zong3, showed differential accumulation in the two hybrids Xun928×Zong3 and Lx9801×Zong3 at almost all sampling stages. gi|414869037 and gi|195635735, which were specific for silk development in the inbred line Xun928, contributed to the heterosis of silk viability only in the hybrid Xun928×Zong3 (protein spots 155 and 239 at D[10] and D[12]; protein spot 96 at D[8]), and not in Lx9801×Zong3. The functional category analysis showed that these three proteins were involved in anthocyanin biosynthesis, methionine metabolism, and suberin biosynthesis. Additionally, the proteins gi|195643366 (APx2, cytosolic ascorbate peroxidase; protein spots 74, 190, 268), gi|413942605 (6-phosphogluconate dehydrogenase isoenzyme B, protein spots 104, 176, 247), and gi|413920184 (O-methyltransferase ZRP4, protein spots 89,150, 272) regulated the heterosis of silk viability at all sampling stages only in the hybrid Xun928×Zong3. The proteins gi|414878829 (glutathione S-transferase 4, protein spots 127, 216, 298) and gi|414881303 (anthocyaninless1, protein spots 138, 224, 285), affected the heterosis of silk viability at all sampling stages only in the hybrid Lx9801×Zong3 ([129]Table 3). Three out of these five proteins were involved in secondary metabolism in the phenylpropanoid pathway. The proteins gi|195629642 (lichenase-2 precursor, protein spots 267, 290) and gi|413920639 (chitinase 1, protein spots 255, 275) contributed to silk viability heterosis only at the late silk developmental stage (D[12]) of the two hybrids. Functional category and KEGG pathway enrichment classifications of differentially accumulated proteins The differentially accumulated proteins associated with silk viability and its heterosis was in similar functional categories. Unknown proteins comprised a large proportion of the differentially accumulated proteins, accounting for 38% and 40% of the proteins related to silk viability and its heterosis, respectively. The proteins involved in metabolism group accounted for the largest proportion of the differentially accumulated proteins, accounting for 43% of proteins related to silk viability and 42% of proteins related to the heterosis ([130]Fig 1). Proteins involved in protein biosynthesis and folding, including transcription, translation, folding, sorting and degradation, were the second most abundant group and were specific to the heterosis of silk viability. Other important categories, based on protein abundance, were stress and defense response, plant hormone biosynthesis and signal transduction, and cellular processes. In the largest category, metabolism, there were six and eight subcategories of proteins involved in silk viability and its heterosis, respectively. Among them, methionine metabolism and flavonoid metabolism were important for both silk viability and its heterosis (Tables [131]2 and [132]3, [133]Fig 1), and lipid metabolism and energy metabolism were specific to the heterosis of silk viability. Fig 1. Functional categories of differentially accumulated proteins in the inbred lines and its corresponding hybrid combinations. [134]Fig 1 [135]Open in a new tab The protein–protein interaction networks involved in silk viability and its heterosis were analyzed by searching the String database ([136]Fig 2). Three proteins were implicated in silk viability and its heterosis: gi|413944345 (KOG1192), gi|414869037 (KOG2263) and gi|195635735 (NOG293481). These proteins had only one or two interacting proteins and were distributed at a remote node in the network. Some reductases or dehydrogenases ([137]S4 Table) were located at the interaction nodes and played an important role in the protein–protein interaction networks both for silk viability and its heterosis; for example, KOG1502-KOG2450-KOG0022 in the interaction network for silk viability and KOG1502-KOG1577-KOG2450-KOG0022-KOG0725 in the interaction network for the heterosis of silk viability. The two important branches for the heterosis of silk viability were ATP energy production (KOG1758-KOG1353-KOG1350-KOG1626-) and protein metabolism (KOG0177-KOG0179-KOG0863-KOG1688-) ([138]Fig 2B). Additionally, two glutathione S-transferases (gi|195619648 KOG0406, gi|162460516 KOG0867) might play a crucial role in providing energy and proteins for the entire protein–protein interaction network for the heterosis of silk viability. Meanwhile, Kinases (KOG1367, KOG2440), enolase (KOG2670), and isomerase (KOG1643) increased the complexity of the protein–protein interaction network for the heterosis of silk viability compared with the network for silk viability, which was complicated by cell cytoskeleton proteins ([139]Fig 2A). Fig 2. Protein–protein interaction networks obtained in silico using String database, with COG functions. [140]Fig 2 [141]Open in a new tab Discussion Comparison of protein categories related to phenotypes of the inbred lines and their corresponding hybrids The results in this study revealed that several functional categories of proteins corresponded to the seed setting rate phenotype of the inbred lines and its corresponding hybrids. For the inbred lines, proteins involved in flavonoid metabolism, methionine metabolism and cytokinin signaling, made the highest contributions to contributed the highest silk viability in the inbred line Zong3 ([142]Table 2). Compared to the inbred line Lx9801, the stronger silk viability of the inbred line Xun928 was attributed to proteins involved in methionine metabolism, and these proteins also contributed to the heterosis of silk viability in the hybrids, but the proportions of their contributions differed. Proteins involved in flavonoid metabolism were important for heterosis of silk viability at all sampling stages in the two hybrids ([143]Table 3). However, proteins involved in methionine metabolism contributed differently to the heterosis of silk viability at different developmental stages of silks in the two hybrids: at D[10] and D[12] for Xun928×Zong3, and at D[8] and D[10] for Lx9801×Zong3. These results implied that proteins contributing to silk viability were not always as important for the heterosis of silk viability in the hybrids. Compared with the hybrid Lx9801×Zong3 at the three sampling stages, the hybrid Xun928×Zong3 accumulated more proteins involved in protein biosynthesis and folding, stress and defense responses, signal transduction and cell detoxification in response to genetic and environmental changes. Thus, the hybrid Xun928×Zong3 showed stronger heterosis than the hybrid Lx9801×Zong3. Potential regulation networks revealed by differentially accumulated proteins related to silk viability and its heterosis Methionine metabolism and salvage cycle Nutrient supply is a basic requirement for successful fertilization. The nutrients in pollen, however, can support only about 2 cm of tube growth in the maize silk [[144]21]. Thus, the maize silk must provide enough nutrients to support pollen tube growth over a longer distance. Consistent with this, many differentially accumulated proteins were related to cysteine and methionine metabolism (Tables [145]2 and [146]3). Proteins involved in methionine supply were important for silk viability. Methionine functions not only as a building block for protein synthesis, but also as a signaling molecule in communicating intracellular metabolic events to receptors on the cell surface. Therefore, methionine could supply appropriate signals to support pollen tube growth and guidance in the maize silk. In plants, methionine synthase (MeSe EC 2.1.1.12; protein spots 8, 121, 155, 239) catalyzes the terminal step of the methionine synthesis pathway by transferring a methyl group to homocysteine (Hcy), producing methionine. However, this de novo synthesis is energetically expensive and highly tissue-specific. To save energy consumption, about 80% of the methionine is recycled [[147]22]. S-Adenosylmethionine (AdoMet)-dependent transferase (protein spots 148, 194, 238) plays a critical role in methionine recycling by transferring the methyl group from AdoMet to S-adenosylhomocysteine (AdoHcy). This is not the only methionine recycling pathway in plants ([148]S3 Fig). Methylthioribose-1-phosphate isomerase (protein spot 64), which catalyzes the phosphorylated methylthioribose (MTR) to methylthioribulose-1-P, is the first and ubiquitous enzyme for methionine recycling. The accumulation of adenine (Ade), a by-product of MTR formation, inhibits methionine recycling. On the other hand, Ade is also a substrate for phosphoribosyl Transferase 1 (APT1) (protein spot 46), which regulates cytokinin levels by converting active cytokinin forms to inactive ones. Loss of APT1 activity leads to excess accumulation of cytokinins, inducing a myriad of cytokinin-regulated responses, such as delayed leaf senescence, anthocyanin accumulation, and downstream gene expression [[149]23]. AdoMet, as the major product of methionine metabolism, is an important cofactor that modulates various biological activities [[150]24,[151]25]. As the major methyl-group donor, AdoMet can regulate transmethylation reactions at the levels of DNA metabolism, RNA metabolism, and protein post-translational modifications [[152]26]. AdoMet is also involved in metabolic and developmental regulation, since it is a substrate for thesynthesis of nicotianamine, ethylene (1-aminocyclopropane-1-carboxylate synthase), and polyamines [[153]22]. AdoMet metabolism is complicated by its interaction with plant growth hormones such as cytokinins and auxins [[154]27]. Thus, AdoMet is involved in regulating plant developmental by fine-tuning gene transcription, cell proliferation, and the production of secondary metabolites [[155]28,[156]29]. In this study, positive regulators of nutrients production were identified in the inbred lines Zong3 (protein spots 46, 64) and Xun928 (protein spot 8), but not in the inbred line Lx9801. In the hybrid combinations, relatively more positive regulators (protein spots 148, 155, 238, 239) were identified at the late developmental stages (D[10] and D[12]) in Xun928×Zong3. However, only two positive regulators (protein spots 121, 194) were identified in hybrid Lx9801×Zong3 and differentially accumulated at D[8] and D[10]. These results were consistent with the stronger silk viability of the inbred lines Zong3 and Xun928 than that of Lx9801, as well as the high seed setting rates (84.6% for D[10] and 80.2% for D[12]) and mid-parent heterotic degrees (34.4% for D[10] and 41.4% for D[12]; data not shown) during the late sampling stages in the hybrid Xun928×Zong3. For comparison, Lx9801×Zong3 showed seed setting rates of 70.4% and 66.9% at D[10] and D[12]; and mid-parent heterotic degrees of 21.1% and 31.3% at D[10] and D[12] (Tables [157]1–[158]3). Photosystem and energy metabolism Photosynthesis provides fuel for plant growth by converting light energy into chemical energy. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), catalyzes the first major step of the Calvin cycle (carbon fixation) to produce energy-rich carbohydrates. This reaction uses ATP as an energy source and NADPH as reducing power, and is often the rate-limiting step in photosynthesis [[159]30]. In all eukaryotes, Rubisco is an oligomer consisting of eight large subunits bound to eight small subunits. The large subunits (protein spots 67, 106, 178) contain the enzymatically active substrate binding sites and are synthesized in the chloroplast. The small subunits are synthesized in precursor form by cytoplasmic ribosomes. Assisted by the RuBisCO large subunit-binding protein (protein spot 40), mature small subunits assemble with large subunits to form the oligomeric holoenzyme in the stroma. Several studies have shown that increased expression levels of RuBisCO subunits could increase photosynthetic efficiency by increasing catalytic activity and/or by decreasing the oxygenation rate [[160]31]. ATP synthase (EC 3.6.3.14), a key enzyme in energy metabolism, is widely involved in oxidative and photosynthetic phosphorylation and plays an important role in many processes in plants. It consists of two rotary motors: the membrane-integrated CF[o] and the hydrophilic CF[1]. CF[o] mainly participates in proton transport through thylakoids, whereas CF[1] contains the nucleotide binding, catalytic, and regulatory sites of the ATP complex [[161]32]. CF[1] contains five subunits: α (protein spot 199), β, γ, δ (protein spot 182), and ε [[162]32,[163]33]. The gene encoding the CF[1] α subunit, atpA, was shown to be related to cold resistance, and the transcript level of atpA was positively correlated with ATP synthase activity [[164]34]. Mutation of the atpA gene in a cytoplasmic male sterile line caused an energy supply shortage during flower development, resulting in abnormal microspore development compared with its maintainer [[165]35]. In this study, proteins involved in energy metabolism differentially accumulated in the inbred line Zong3 (protein spot 40) and the hybrid Xun928×Zong3 at D[8] and D[10] (protein spots 67, 106, 178, 182). A sufficient energy supply may be important to support stronger silk viability of Zong3, compared with those of Xun928 and Lx9801, and the stronger heterosis of silk viability in Xun928×Zong3 than in Lx9801×Zong3. Protein metabolism and cell senescence Proteins have a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Many elaborate regulation mechanisms are involved in converting DNA sequences into functional proteins. Translation, the assembly of proteins by ribosomes, is an essential part of the protein biosynthetic pathway and requires initiation and elongation complexes [[166]21]. Eukaryotic translation initiation factor 5A (eIF-5A) (protein spots 72, 146) not only regulates protein synthesis but also acts as an important determinant of cell proliferation and senescence. In dividing and dying cells, different isoforms of eIF-5A execute its biological switching function in response to physiological and environmental cues [[167]36–[168]38]. The elongation factor-1 (EF1) complex (protein spots 112, 161, 251) is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. EF1A is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site of the ribosome. EF1 delta (protein spots 112, 251), functions as a guanine nucleotide exchange factor in regenerating active EF1A-GTP from inactive EF1A-GDP. During and after protein synthesis, polypeptide chains often fold into their native secondary and tertiary structures, whether they are used in the cell or secreted. To achieve their final correct states, cellular and secreted proteins require the help of several other folding proteins or chaperones. Protein disulfide isomerase (protein spot 252) catalyzes protein-folding, allowing proteins to reach their final correctly folded state without enzymatic disulfide shuffling [[169]39]. Unneeded or damaged proteins are transferred to proteasomes, an active complex composed of α subunits and β subunits (protein spots 229, 288), to be degraded into amino acids that are used to synthesize new proteins. At all sampling stages, many differentially accumulated proteins involved in protein biosynthesis and correct folding were identified in the hybrid Xun928×Zong3. However, more proteins involved in proteasomes differentially accumulated during the late sampling stages (D[10] and D[12]) in the hybrid Lx9801×Zong3. These results implied that the hybrid Lx9801×Zong3 might consume more resources during normal metabolism, which weakened its silk viability, especially at the late silk developmental stages. During normal plant development, the insoluble polyesters suberin and cutin form extracellular lipophilic barriers to prevent membrane leakiness [[170]40]. However, membranes become leaky when the cell begins to senescence. This process is usually accompanied by the accumulation of proteins involved in lipid metabolism. In this study, O-methyltransferase (protein spots 89, 150, 163, 171, 236, 243, 272), the first rate-limiting enzyme in suberin synthesis, and GDSL-motif lipase/hydrolase (protein spot 96), an enzyme involved in the hydrolysis and transfer of activated monomers in cutin synthesis [[171]41], differentially accumulated in the hybrid Xun928×Zong3. Proteins involved in lipid metabolism only differentially accumulated in the hybrid Lx9801×Zong3. These results implied that membrane leakiness might occur earlier in Lx9801×Zong3 than in Xun928×Zong3 at the late silk developmental stages. Thus, silk viability was lost earlier in Lx9801×Zong3 than in Xun928×Zong3. This pattern of protein accumulation might also explain the faster decrease in the seed setting rate and the weaker heterotic degree in the hybrid Lx9801×Zong3 at the late silk developmental stages. Phenylpropanoid metabolism and plant hormones regulation The plant hormone auxin regulates cell elongation, division, differentiation, and morphogenesis. Many proteins involved in elaborate temporal and spatial regulation of auxin metabolism, transport, and signaling have been identified. Auxin-binding protein 1 (ABP1: protein spot 193) mediates cell elongation and, directly or indirectly, cell division. In previous studies, ectopic and inducible expression of ABP1 conferred auxin-dependent cell expansion in tobacco cells that normally lack auxin responsiveness [[172]42] and antisense suppression of ABP1 eliminated auxin-induced cell elongation and reduces cell division. A homozygous null mutation of ABP1 was embryo-lethal in Arabidopsis [[173]43]. Auxin-induced swelling of proteoplasts and intact guard cells can also be attributed to ABP1 [[174]44,[175]45]. Pyrophosphate-energized vacuolar membrane proton pump 1 (protein spot 69) facilitates auxin transport and regulates auxin-mediated developmental processes by modulating apoplastic pH [[176]46]. Flavonoids in the phenylpropanoid pathway are another regulator of active auxin and have species-specific roles in nodulation, fertility, defense, and ultraviolet protection. Flavonols have been shown to negatively regulate the polar auxin transport (PAT) by competing for free auxin with auxin efflux carriers such as PIN and ABCB (PGP proteins) in vivo [[177]47]. Dihydroflavonol 4-reductase (DFR4, EC1.1.1.219: protein spot 53) and UDP-glucoside: flavonoid glucosyltransferase (EC 2.4.1.115: protein spots 63, 76, 79, 99, 117, 119, 122, 123, 138, 152, 159, 188, 197, 200, 201, 202, 212, 224, 231, 241, 285) are the first and last enzymes in the anthocyanin biosynthetic pathway, respectively. Their differential accumulation may be related to competition for the dihydroflavonol substrate with the flavonol branch, and thus, could indirectly affect PAT. The relatively higher contents of anthocyanin biosynthetic enzymes corresponded to higher levels of glutathione S-transferase-like proteins (protein spots 127, 216, 217, 298), which transport anthocyanins from the ER to the vacuole [[178]48] in the hybrid Lx9801×Zong3 at the three sampling stages. Cytokinin is another important hormone that regulates cell proliferation and differentiation. The two active forms of cytokinins are the isopentenyl adenine (iP)-type and the zeatin-type [[179]49]. The metabolic regulation of cytokinin includes biosynthesis, interconversion, inactivation and degradation [[180]50]. β-glucosidase (EC 3.2.1.21, protein spot 61) encoded by Zm-p60.1 catalyzes the release of active cytokinins from their inactive storage and transport forms (cytokinin-O-glucosides) [[181]51]. Over-expression of Zm-p60.1 disrupted zeatin homeostasis in intact transgenic plants, rendering them hypersensitive to exogenous zeatin [[182]51]. Inactive cytokinin can also be generated by cytokinin-O-glucosyltransferase (protein spot 107) [[183]52,[184]53]. Studies of maize transformants harboring zeatin O-glucosyltransferase have shown that zeatin O-glucosylation affects root formation, leaf development, chlorophyll content, senescence, and male flower differentiation through developmental modifications [[185]53]. Proteins involved in the regulation of hormone levels (ABP1, pyrophosphate-energized vacuolar membrane proton pump 1; anthocyanin biosynthesis pathway; and cytokinin-O-glucosyltransferase) were identified as being important for both silk viability and its heterosis. These proteins differentially accumulated in the hybrid Xun928×Zong3, whereas only those involved in anthocyanin biosynthesis differentially accumulated in the hybrid Lx9801×Zong3. The flexibility of the systems regulating hormone levels may explain the increase in silk viability in the hybrid Xun928×Zong3, resulting in the high seed setting rate and strong heterosis. In summary, proteins gi|413944345, gi|414869037, and gi|195635735 were attractive and might be related with silk viability as well as its heterosis. Significant correlation (r = 0.827^* for gi|413944345; r = -0.365^* for gi|414869037; r = 0.556^* for gi|195635735) was detected between these protein spots accumulation level and seed setting rate. Thus, we could propose the following hypotheses regarding proteins related to silk viability and its heterosis: methionine salvage, protein synthesis, and ATP supply function as positive regulators of silk viability, and therefore, contribute to strong silk viability and its heterosis in Zong3 and Xun928×Zong3, respectively. Active fatty acid metabolism, a signal for cell wall degradation, and anthocyanins, which negatively regulate local hormone accumulation, were related to weaker silk viability in Lx9801 and Lx9801×Zong3. The metabolism of cutin and suberin, which were derived from phenylpropanoid precursors, might confer stronger silk viability (Zong3 and Xun928) and stronger heterosis of silk viability in hybrids by slowing the silk aging process, especially during the late stages of silk development. Conclusions In this study, the heterosis of silk viability could be mainly attributed to additive accumulation of differentially regulated proteins, although proteins that accumulated in a non-additive manner made a similar contribution. Simple additive and dominant effects at a single locus, as well as complex epistatic interactions of metabolic pathway genes at two or more loci, resulted in partially dominant silk viability heterosis in the hybrids. For silk viability, most important differentially accumulated proteins were those involved in methionine metabolism for nutrient supply, phenylpropanoid metabolism for hormone homeostasis, protein biosynthesis and metabolism for genetic information processing, and carbon fixation for energy generation. Materials and Methods Plant materials Three typical inbred lines, Zong3, Xun928, and Lx9801, with different silk viability were used in this study. Among more than one hundred inbred lines, the silk viability of the inbred line Zong3 was extremely high. The inbred lines Xun928 and Lx9801 had relatively weak silk viability. To assay the heterosis of silk viability in different genetic backgrounds, two hybrids, Xun928×Zong3 and Lx9801×Zong3 were created in this study. The two hybrids and the three inbred lines were planted on the farm of Henan Agricultural University (Zhengzhou, China; E 113°42′, N 34°48′) in summer of 2013, when the daily average temperature was 14.3°C. The annual average rainfall is 640.9 mm in this region. Each plot consisted of ten 5-m-long rows, with 20 cm of in-row spacing and 67 cm of inter-row spacing. Only the middle rows were sampled to avoid edge effects. Before the silks emerged from the husk, ear shoots were totally covered with bags to avoid pollen contamination. To evaluate the silking time accurately, the silking time of each ear of the materials was recorded in the field. Day 1 (D[1]) was marked as the day that the silks emerged above the ligule of the outer leaf of the husk. Silks were removed from the mid-base region of each ear at D[4], D[6], D[8], D[10], and D[12] and immediately frozen in liquid nitrogen in the field. Each sample was collected with three biological replications and 10 ears were mixed for each replication. At the same time, the ear for each sample was saturation-pollinated by hand on the sampling day to measure the seed setting rate. Pollination was completed between 9 and 10 a.m. (below 37°C) to ensure consistent pollination efficiency. The ears were harvested at physiological maturity, and only the seeds at the mid-base (5–15 rounds from the base) were used to calculate the seed setting rate of the cob according to silk development characteristics [[186]54]. The seed setting rate was calculated by dividing the total number of spikelets by the number of fully grown seeds. Protein extraction and MS identification Each genotype was assayed with three biological replications corresponding to each sampling stage. Frozen silks (1 cm, approx. 1.0 g) of each biological replication (mixture of silks from ten different plants) were fully ground in liquid nitrogen and then extracted in 10 mL pre-cooled trichloroacetate (TCA) buffer (10% w/v TCA in acetone with 0.07% β-mercaptoethanol) with vortexing for 2 h at 20°C. After centrifugation at 15,000 × g for 30 min, the supernatant was discarded and the precipitate was rinsed with 10 mL chilled buffer (80% acetone with 0.07% β-mercaptoethanol) four times by centrifuging for 10 min at 15,000 × g. The final cleaned precipitate was freeze-dried under a vacuum. The dried protein pellet per 1 mg was resuspended in 20 μL buffer (8 M urea, 2 M thiourea, 4% (w/v) CHAPS and 40 mM dithiothreitol (all from Solarbio)). The protein was quantified using a Bio-Rad protein assay with bovine serum albumin as a standard and used for two-dimensional gel electrophoresis (2-DE). Three technical replications were assayed for each biological replication. For each technical replication, equal amounts of total protein extract (800 μg) were used for isoelectric focusing (IEF). Immobilized dry strips (24 cm, Imobiline drystrips, Bio Rad, Hercules, CA, USA) with a linear gradient of pH 4–7 were rehydrated for 16 h at 50 V. The IEF conditions for separating proteins were as followes: slow 250 V for 30 min, rapid 250 V for 2 h, rapid 500 V for 2 h, rapid 1,000 V for 2 h, linear 9,000 V for 5 h, rapid 10,000 V for 10 h, and a constant 500 V for the final 12 h at 20°C. Strips were immediately equilibrated in 10 mL of two types of SDS equilibration buffer for 15 min each. Buffer 1 contained 0.375 M Tris-HCl pH 8.8, 6 M urea, 20% glycerol, 4% SDS, and 2% DTT and buffer 2 contained 0.375 M Tris-HCl pH 8.8, 6 M urea, 20% glycerol, 4% SDS, and 2.5% iodoacetamide. IPG gel strips with the proteins were embedded into the top of a polyacrylamide gel (12%) after equilibration and separated at a constant voltage of 50 V for 30 min. Then, a constant voltage of 200 V was maintained until the electrophoresis was finished. Digital images of the gels stained with Coomassie brilliant blue G250 were obtained with a scanner (UMAX Power Look 2100 XL). Spot detection and matching was performed with the default parameters using the “spot detection wizard” function in PDQuest 8.0 software. The “find spot centers” function was used with default auto-noise smoothing and background subtraction. A Gaussian model was selected to generate a master gel for each image file. All the gels were matched to the reference master gels selected and normalized in automated mode followed by manual group correction. The normalization parameters were “total quantity in valid spots”, “total density in gel image”, “mean of log ratios”, and “local regression model”. After normalization, ANOVA was used to calculate the significance of differences in the relative abundance of protein in individual spot features among the developmental stages of a certain inbred line, as well as among hybrids and their corresponding inbred lines at each developmental stage. For protein spots further assayed by MS, the maximum intensity variation criterion was set to ≥ 1.5-fold and ≥ 2-fold (P < 0.05) among different sampling stages for each inbred line, and between hybrid and parental inbred lines at each sampling stage, respectively. The selected proteins were excised manually from gels, subjected to in-gel digestion with trypsin, and then destained using 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile for 15 min at room temperature. The discolored spots were vacuum-dried and incubated with modified porcine trypsin at 37°C overnight. After centrifugation, the supernatant was collected and vacuum-dried, and then the precipitate was re-dissolved in 60% acrylonitrile/0.1% trifluoroacetic acid (TFA) (100 μL) for 15 min to obtain the peptides. Then, a 0.3 mL peptide sample and 0.3 mL matrix consisting of 10 mg/mL α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% TFA was analyzed on a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The parameters of the MS were set with 4000 Series Explorer software (Applied Biosystems). The lists of theoretical peptide MS from each peptide-map-fingerprinting (PMF) combined with MS/MS were used to search the NCBI (National Center for Biotechnology Information) database without repetition for homologous sequences using MASCOT 2.2 software ([187]www.matrixscience.com). The search criteria were as follows: 1) peptide mass tolerance of 100 ppm; 2) maximum of a single missed tryptic cleavage; 3) fragment mass tolerance of 0.4 Da; and 4) carbamidomethylation by cysteine residues as fixed modifications and oxidation by methionine residues as dynamic modifications. Only proteins with a MASCOT score > 60 with 95% confidence and at least two matched peptides were accepted. Gene Ontology (GO) annotations and the theoretical Mr/pI for the identified proteins were retrieved from [188]http://www.geneontology.org/ and [189]http://www.expasy.ch/tools/pi_tools.html, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was carried out using the blast function in BLAST2GO. The protein–protein interaction network was analyzed by the publicly available program STRING ([190]http://string-db.org/). Clusters of Orthologous Groups (COG) of proteins functions were used to construct the networks. Only an interaction networks with a high confidence (0.700 for silk viability or 0.900 for heterosis of silk viability) and no more than five interactors were retained. The eukaryotic orthologous groups (KOGs) were considered prime selections for a single protein spot. Data analysis Protein spots that had no significant difference in average spot intensity from the mid-parent value at the 0.05 level were considered additively accumulated (A). The accumulation pattern of each non-additive protein was classified as described by Hoecker et al. [[191]55]. Average spot intensities of proteins that deviated significantly from the mid-parent value of the parental lines at P < 0.05 level were defined as non-additive proteins. “+” and “−” were used to indicate that the protein spot intensity identified in the F[1] hybrid was similar to the high parent and low parent values, respectively. “+ +” and “− −” indicated that the protein spot intensity identified in the F[1] hybrid was significantly different from the high parent and low parent values, respectively. “+ −” indicated the protein spot intensity identified in the F[1] hybrid fell in between the mid-parent and high parent or mid-parent and low parent values. ANOVA, LSD, and correlation analysis were performed with the corresponding function in Excel 2007. Supporting Information S1 Fig. Representative 2-DE map of proteins related to silk viability at different developmental stages. (DOCX) [192]Click here for additional data file.^ (692.1KB, docx) S2 Fig. Representative 2-DE map of proteins related to the heterosis of silk viability in the hybrid combinations of Xun928×Zong3 and Lx9801×Zong3 at different developmental stages. (DOCX) [193]Click here for additional data file.^ (1.2MB, docx) S3 Fig. Recycling of methionine cited from Ravanel et al. [[194]28]. Enzymes: 1, cystathionine g-synthase; 2, cystathionine b-lyase; 3, methionine synthase; 4, AdoMet synthetase; 5, AdoMet-dependent methylase; 6, AdoHcy hydrolase; 7, 1-aminocyclopropane-1-carboxylicacid synthase; 8, AdoMet decarboxylase; 9, threonine synthase. Note that AVG inhibits both cystathionine g-synthase and 1-aminocyclopropane-1-carboxylic acid synthase. (TIF) [195]Click here for additional data file.^ (153.9KB, tif) S1 Table. Silk viability information of different inbred lines and their corresponding hybrids. (XLSX) [196]Click here for additional data file.^ (11.1KB, xlsx) S2 Table. Peptide information for protein spots with significant changes based on MS analysis. (XLSX) [197]Click here for additional data file.^ (350.8KB, xlsx) S3 Table. Levels of differentially accumulated proteins in the inbred lines. (XLSX) [198]Click here for additional data file.^ (13KB, xlsx) S4 Table. Differentially accumulated protein spots in protein–protein interaction networks simulated by the String software, with COG functions. (XLSX) [199]Click here for additional data file.^ (12.1KB, xlsx) Abbreviations CRPs cysteine-rich peptides Hcy homocysteine AdoMet S-adenosylmethionine MTA methylthioadenosine APT1 Adenine Phosphoribosyl Transferase 1 RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase ABP Auxin-binding protein 1 AdoHcy S-adenosylhomocysteine Ade adenine MS mass spectrometry IEF isoelectri focusing MALDI-TOF matrix-assisted laser desorption ionization-time of flight PMF peptide-map-fingerprinting GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes COG Clusters of Orthologous Groups KOGs eukaryotic orthologous groups LSD least-significant difference Data Availability All relevant data are within the paper and its Supporting Information files. Funding Statement This work was supported by grants from the National Natural Science Foundation of China (31271732), the National High Technology Research and Development Program of China (2014CB138203), the State Key Laboratory of Wheat and Maize Crop Science (SKL2014ZH-09) and the Shanghai Science and Technology Committee (11DZ2272100). References