Abstract

   Ear height is an important maize morphological trait that influences
   plant lodging resistance in the field, and is based on the number and
   length of internodes under the ear. To explore the effect of internodes
   on ear height, the internodes under the ear were analysed in four
   commercial hybrids (Jinsai6850, Zhengdan958, Xundan20, and Yuyu22) from
   different heterotic groups in China. The eighth internode, which is the
   third aboveground extended internode, exhibited high-parent or over
   high-parent heterosis and contributed considerably to ear height. Thus,
   the proteome of the eighth internode was examined. Sixty-six protein
   spots with >1.5-fold differences in accumulation (P < 0.05) among the
   four hybrids were identified by mass spectrometry and data analyses.
   Most of the differentially accumulated proteins exhibited additive
   accumulation patterns, but with epistatic effects on heterosis
   performance. Proteins involved in phenylpropanoid and benzoxazinoid
   metabolic pathways were observed to influence indole-3-acetic acid
   biosynthesis and polar auxin transport during internode development.
   Moreover, indole-3-acetic acid content was positively correlated with
   the eighth internode length, but negatively correlated with the extent
   of the heterosis of the eighth internode length.

Introduction

   Heterosis refers to a phenomenon in which the survival and performance
   of a hybrid are superior to those of the genetically distinct parents.
   As a multigenic complex trait, heterosis has been attributed to
   dominance, over-dominance, or epistasis based on quantitative genetic
   theory. Lippman and Zamir^[36]1 reported that the magnitude and rate of
   vegetative growth, flowering time, yield, and resistance to biotic and
   abiotic environmental stresses, which are regulated by many biochemical
   pathways, influence heterosis to some extent. Additionally, heterosis
   is trait-dependent, implying that the underlying mechanisms are active
   within the biological context of specific traits^[37]2,[38]3. Genome
   structural variations^[39]4,[40]5, epigenetic modifications^[41]6,
   diverse global expression trends^[42]7,[43]8, specific protein
   functions, differential accumulations, and post-transcriptional
   modifications^[44]7,[45]9–[46]13 have been associated with the
   heterosis of specific organs and developmental stages at the molecular
   level.

   Plant lodging in the field not only decreases the final yield, it also
   hinders the mechanical harvesting of maize (Zea mays)^[47]14. The ratio
   of ear height to plant height is an important indicator influencing
   plant stalk lodging resistance, especially during the grain-filling
   stage^[48]7. Thus, ear height has been widely studied as an important
   morphological trait, not only because it influences plant lodging
   resistance in the field, but also because of its association with high
   heterosis at the quantitative trait locus (QTL) mapping level. Several
   stable QTLs for plant height and ear height were detected on
   chromosomes 1 and 10 in two F[2:3] populations^[49]9. Moreover, five
   QTL clusters for plant height and ear height were identified in 11
   recombinant inbred line populations based on 787 single nucleotide
   polymorphisms (SNPs)^[50]15. Seven SNPs out of 14,401 were
   significantly related to ear height according to association mapping
   results with 159 maize inbred lines, and were co-localised to a QTL
   region for ear height^[51]16. Different studies revealed diverse
   mechanisms related to plant height and ear height heterosis. Some
   studies confirmed that additive and partial dominance were the major
   QTL effects on plant height and ear height^[52]17,[53]18. Meanwhile,
   Song et al.^[54]19 concluded that dominance, over-dominance, and
   epistasis contributed to the heterosis of plant height and ear height.

   Ear height is based on the basal five or six internodes (depending on
   the genetic background), which do not elongate and remain
   underground^[55]4 as well as the elongating internodes under the ears.
   The first to sixth internodes normally do not elongate. The internodes
   begin to elongate from the seventh internode, with the extent of the
   elongation influenced by various hormones and other factors. Therefore,
   the number and length of the extended aboveground internodes under the
   ear determine the final ear height. For any hybrid, superior ear height
   is reflected by longer internodes rather than a greater number of
   internodes in comparisons with the inbred lines^[56]5. The molecular
   mechanism regulating ear height heterosis should be characterised in
   detail for a specific internode rather than for the entire plant.
   Therefore, as an initial study, the sixth to eleventh extended
   internodes under the ear were phenotyped, and the eighth internode was
   used for a proteomic analysis. The objectives of this study were to (1)
   elucidate the contribution of each extended internode to ear height and
   the associated heterosis and (2) explore the molecular regulatory
   mechanism related to internode heterosis.

Methods

Internode sampling

   Four elite Chinese commercial maize hybrids and their parental lines
   [Jinsai6850 (P2 × A50), Zhengdan958 (Zheng58 × Chang7-2), Xundan20
   (Xun9058 × Xun928), and Yuyu22 (Zong3 × Yu87-1)] were used in this
   study. These hybrids have been classified into the following three main
   heterotic groups in China: Reid × TangSPT (Zhengdan958 and Xundan20),
   Lancaster × Lvdahonggu (Jinsai6850), and Zi330 × Tem-tropic I
   (Yuyu22)^[57]6. Seeds of the four hybrids and their corresponding
   parental lines were sowed on the farm of Henan Agricultural University
   (Zhengzhou, China; E113°42′, N34°48′) in the summer of 2015. The plots
   consisted of eight rows, each 4 m long, with in-row and inter-row
   spacings of 25 and 75 cm, respectively. Only the middle rows were
   sampled to avoid edge effects. The sixth internode is the first
   aboveground internode because the first five internodes are compressed
   in the crown under the soil line for these hybrids and inbred lines. To
   ensure accurate sampling times, the fifth and tenth leaves were marked
   in the field. Plant height (from soil level to the top of the main
   tassel branch), ear height (from soil level to the ear), and the
   lengths of the sixth to eleventh internodes (first to sixth extended
   aboveground internodes under the ear) were measured for each genotype
   at the adult plant stage. Measurements were completed with five
   replicates, with average values calculated for each genotype. Data were
   analysed with the PROC MIXED model of the SAS 8.0 statistical software
   package. The eighth internode of four hybrids and their parental lines
   were sampled when the internodes elongated by about 1 cm (i.e., rapidly
   growing internodes). Three replicates of internodes were collected (10
   plants per biological replicate) and immediately frozen in liquid
   nitrogen. Samples were stored at −80 °C for a subsequent protein
   extraction and indole-3-acetic acid (IAA) quantification. At least
   three replicates of each material were prepared to eliminate probable
   variations due to biological differences among individual plants. The
   length of each leaf-inserted internode was measured after flowering.

Total protein extraction and two-dimensional gel electrophoresis analysis

   The collected eighth internodes (1 cm; approximately 1 g) were ground
   to a powder in liquid nitrogen. Debris were removed, after which an
   equal amount of ground material for each of the 10 plants per
   biological replicate was mixed for each genotype. Samples were
   resuspended with 10 mL pre-cooled trichloroacetic acid (TCA) buffer
   (10% w/v TCA in acetone with 0.07% β-mercaptoethanol) and vortexed for
   2 h at 20 °C. After centrifuging samples at 15,000 × g for 30 min, the
   supernatant was discarded and the precipitate was rinsed four times
   with 10 mL chilled buffer (80% acetone with 0.07% β-mercaptoethanol)
   and a centrifugation at 15,000 × g for 10 min. The final precipitate
   was freeze-dried under a vacuum, after which the dried protein pellet
   was resuspended in a buffer [8 M urea, 2 M thiourea, 4% (w/v) CHAPS,
   and 40 mM dithiothreitol], with 20 µL buffer added for every 1 mg
   precipitate. Protein concentrations were determined based on a standard
   curve prepared using bovine serum albumin as a standard. For each
   biological replicate, 800 μg total protein was analysed by
   two-dimensional electrophoresis, with three technical replicates.
   Immobilised pH gradient strips (24 cm, Immobiline DryStrips; Bio Rad,
   Hercules, CA, USA) with a linear gradient of pH 4–7 were rehydrated
   with the protein solution for 16 h at 50 V. The isoelectric focusing
   was completed at 250 V for 30 min (slow), 250 V for 2 h (rapid), 500 V
   for 2 h (rapid), 1,000 V for 2 h (rapid), 9,000 V for 5 h (linear),
   10,000 V for 10 h (rapid), and a constant 500 V for the final 12 h at
   20 °C. The strips were then immediately equilibrated with 10 mL Buffer
   1 (0.375 M Tris-HCl, pH 8.8, 6 M urea, 20% glycerol, 4% SDS, and 2%
   DTT) for 10 min followed by 10 mL Buffer 2 (0.375 M Tris-HCl, pH 8.8,
   6 M urea, 20% glycerol, 4% SDS, and 2.5% iodoacetamide) for 10 min. The
   DryStrips were embedded at the top of 12% polyacrylamide gels, and the
   proteins were separated in the second dimension at a constant voltage
   of 50 V for 30 min. A constant 200 V was maintained until the
   electrophoresis was completed.

Protein analysis by mass spectrometry

   After the electrophoretic separation of proteins, the gels were stained
   with Coomassie brilliant blue G250 and photographed with the PowerLook
   2100 XL scanner (UMAX). Protein spots were detected and matched with
   the default parameters of the “spot detection wizard” function in the
   PDQuest 8.0 program. The “find spot centers” function was used with
   default auto-noise smoothing and background subtraction. A Gaussian
   model was applied to generate a master gel for each image file. All
   gels were matched to selected reference master gels and normalised in
   the automated mode followed by manual group correction. The following
   normalisation parameters were used: “total quantity in valid spots”,
   “total density in gel image”, “mean of log ratios”, and “local
   regression model”. Only protein spots that were consistent between
   replicates were retained. The average intensity of each protein spot,
   with a fixed effect for genotype, was determined based on an analysis
   of variance (ANOVA) model. The differences between the hybrid and
   parental values as well as between the hybrid and mid-parent values
   were determined. Protein spots with significant differences in the
   maximum intensities (P < 0.05) between the hybrids and their parental
   lines (i.e., at least 1.5-fold difference) were further analysed by
   mass spectrometry (MS).

   The lists of theoretical peptides for each peptide mass fingerprint
   combined with tandem MS data were used to search the Viridiplantae
   database (1,093,002 sequences) maintained by the National Center for
   Biotechnology Information (downloaded 12 December 2012). The database
   was searched for homologous sequences using the MASCOT 2.2 program
   ([58]www.matrixscience.com; Matrix Science). The search criteria were
   as follows: peptide mass tolerance of 100 ppm; maximum of a single
   missed tryptic cleavage; fragment mass tolerance of 0.4 Da;
   carbamidomethylation by cysteine residues as the fixed modification;
   and oxidation by methionine residues as the dynamic modification. Only
   proteins with a MASCOT score >60 with 95% confidence and at least two
   matched peptides were accepted. Unique and top-ranked peptides were
   selected for filtering homologous proteins. Gene Ontology (GO)
   functional annotations and the theoretical Mr/pI for the identified
   proteins were retrieved from [59]http://www.geneontology.org/ and
   [60]http://www.expasy.ch/tools/pi_tools.html, respectively.
   Additionally, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment analysis was conducted using Blast2GO.

Data analysis

   Protein spots with average intensities that did not significantly
   deviate from the mid-parent values (P > 0.05) were considered to
   correspond to additively accumulated proteins. Meanwhile, protein spots
   with average intensities that significantly deviated from the
   mid-parent values were considered to correspond to nonadditively
   accumulated proteins, which were categorised according to the method
   described by Hoecker et al.^[61]10. We used “ + ” and “−” to indicate
   that the protein spot intensity for the F[1] hybrid was similar to that
   of the high-parent and low-parent, respectively. Additionally, “ ++ ”
   and “− −” indicated that the protein spot intensity for the F[1] hybrid
   was significantly different from that of the high-parent and
   low-parent, respectively. Furthermore, “ + −” indicated the protein
   spot intensity identified for the F[1] hybrid was between that of the
   mid-parent and high-parent or between that of the mid-parent and
   low-parent.

Transcript profiling

   Total RNA was extracted from the eighth internode of each hybrid and
   corresponding inbred lines using the TRIzol reagent (Invitrogen, USA).
   Primers specific for several genes related to the phenylpropanoid and
   benzoxazinoid metabolic pathways were designed using Primer Premier 5.0
   for a quantitative real-time polymerase chain reaction analysis
   (qRT-PCR) (Table [62]S1). Primers were also designed for the actin
   gene, which was used as an internal standard
   (5′-CGATTGAGCATGGCATTGTCA-3′ and 5′-CCCACTAGCGTACAACGAA-3′). Briefly,
   the RNA was reverse transcribed to cDNA using M-MLV reverse
   transcriptase (Promega, USA). The qRT-PCR (25 µL reaction volume) was
   completed with the SYBR Premix Ex Taq™ Kit (Takara, Dalian, China) and
   the iQ5 real-time PCR system (BioRad, USA). Experiments were conducted
   in triplicate, and the average expression level value for each gene was
   calculated. Differences in the mean expression levels between hybrids
   were tested by a one-way ANOVA (significance level of 0.05).

Quantification of indole-3-acetic acid

   Three replicates were analysed for each sample under the same
   conditions. To quantify IAA, each 500-mg sample was homogenised with
   1 mL 80% methanol (in H[2]O) and centrifuged at 15,000 × g for 10 min.
   The subsequent steps of the IAA quantification procedure were completed
   as previously described^[63]11.

Results

Internode effects on ear height heterosis

   For the hybrids, ear height, which was determined based on the extended
   internodes under the ear, was the main factor influencing the heterosis
   of plant height (from soil level to the top of the main tassel branch),
   with contributions of 48% for Jinsai6850, Zhengdan958, and Yuyu22, and
   56% for Xundan20 (Fig. [64]1, Table [65]S2). Ear height is also
   correlated with plant lodging resistance. Thus, characterising the
   heterosis of extended internodes under the ear may be useful for
   decreasing the centre of gravity for plants, with potentially important
   implications for improving plant lodging resistance. The heterosis
   performance of ear height was mainly influenced by the sixth
   (2.67–5.68%) to eleventh extended internodes (12.04–14.12%). For the
   extended internodes, a significant difference (P < 0.05) in the
   contribution to ear height (i.e., proportion of the ear height
   contributed by the internode length) was detected between the sixth and
   seventh internodes, and between the seventh and eighth internodes
   (Table [66]S2). Additionally, there were no significant differences
   among the eighth to eleventh internodes. Therefore, the eighth
   internode contributed more to the ear height than the sixth and seventh
   internodes. During the mature plant stage, the sixth internode was too
   short (3.52–6.88 cm) to be considered for the final ear height
   (107.20–131.62 cm) and plant height (221.30–271.90 cm). However,
   according to the ANOVA results, the eighth internode was the only
   extended internode that contributed to ear height similarly to the
   internode above in all hybrids except for Xundan20 (P = 0.009), and
   exhibited high- (Jinsai6850) or over high-parent (Zhengdan958,
   Xundan20, and Yuyu22) heterosis (Fig. [67]2; Table [68]S2). An analysis
   of the hormone contents in neighbouring internodes indicated that the
   eighth internode underwent less extensive changes than the ninth to
   eleventh internodes. Moreover, a correlation analysis revealed a
   relationship between the length of the eighth internode and plant
   height or ear height for most tested materials, with the extent of the
   correlation differing among the diverse materials (Table [69]S3). Thus,
   the proteome of the eighth internode was analysed to clarify the
   genetic basis of heterosis.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Trait performance and mid-parent heterosis in four hybrids.

Figure 2.

   [72]Figure 2
   [73]Open in a new tab

   Comparison of internode length and plant height between the hybrids and
   parental inbred lines.

   The eighth internode, ear height, and plant height exhibited the
   following heterosis expression pattern for each hybrid: high-parent
   expression (+) in Jinsai6850 and over high-parent expression pattern
   (++) in Zhengdan958, Xundan20, and Yuyu22. The degree of the mid-parent
   heterosis and high- or over high-parent heterosis decreased from the
   sixth to eleventh internodes for Zhengdan958 and Jinsai6850. However,
   the highest mid-parent and high- or over high-parent heterosis for the
   ninth internode were observed in Xundan20 (57.36 and 52.56%) and Yuyu22
   (54.11 and 52.39%) (Fig. [74]2; Table [75]S2). In other words, the
   extent of the heterosis was inconsistent with the heterosis performance
   for the extended internodes, ear height, and plant height.

Differentially accumulated proteins in the eighth internode

   The proteome of the eighth internode was analysed to identify
   differentially accumulated proteins among the four hybrids and their
   parents. A total of 206 (196 and 182), 201 (205 and 198), 180 (229 and
   141), and 219 (235 and 232) differentially accumulated protein spots
   were detected in Jinsai6850 (A50 × P2), Zhengdan958
   (Zheng58 × Chang7–2), Xundan20 (Xun9058 × Xun928), and Yuyu22
   (Zong3 × Yu87–1), respectively (Fig. [76]3). Moreover, 15, 16, 22, and
   13 protein spots with >1.5-fold difference (P < 0.05) were excised from
   gels and identified by MS, respectively (Fig. [77]4, Table [78]S4). The
   majority (54/66) of these protein spots were additively accumulated in
   the four hybrids, especially in Xundan20 (21/22). The non-additively
   accumulated protein spots exhibited different patterns in the four
   hybrids, even for the hybrids in the same heterotic group [i.e.,
   Zhengdan958 (+, ++, and +−) and Xundan20 (−)] (Fig. [79]4,
   Table [80]S4). The 66 protein spots (peptides are listed in
   Table [81]S5) were represented by 46 GenBank accessions. Additionally,
   some GenBank accessions were identified in the same hybrid twice with
   different accumulation patterns, likely because of distinct
   post-translational modifications of the corresponding proteins. GenBank
   accessions gi|195613358 (GRMZM2G014844_T01; EC 3.2.1.21) and
   gi|226499080 (GRMZM2G033555_T01; EC1.1.1.219) were identified in all
   hybrids. However, gi|195613358 accumulated differently in the four
   hybrids (protein spots 2 and 3 in Jinsai6850, 16 in Zhengdan958, 42 in
   Xundan20, and 63 in Yuyu22), while the accumulation patterns for
   gi|226499080 were similar in the four hybrids (protein spot 10 in
   Jinsai6850, 27 in Zhengdan958, 50 in Xundan20, and 65 in Yuyu22)
   (Fig. [82]4, Table [83]S4).

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   Two-dimensional maps of differentially accumulated proteins in the
   eighth internode of the hybrids and parental inbred lines. Proteins
   that accumulated differentially in hybrids and parental inbred lines
   are numbered.

Figure 4.

   Figure 4
   [86]Open in a new tab

   Heat map and abundance patterns of differentially accumulated proteins
   in the eighth internode of the hybrids and parental lines. Note:
   Differences in accumulation are presented as a continuous range of
   colours. Red corresponds to the maximum accumulation (grey value:
   449574); green corresponds to the minimum accumulation (grey value: 0);
   and yellow corresponds to the median accumulation (grey value: 224787).

Functional analysis of the identified proteins

   The 66 protein spots were classified into the following six GO
   categories: metabolism, genetic information processing, environmental
   information processing, organismal systems, cellular processes, and
   unknown (Fig. [87]5, Table [88]S6). Metabolism was the largest
   category, with 74% (49/66) of the differentially accumulated protein
   spots, implying metabolic activities are critical for internode
   development and heterosis. The metabolism category was further divided
   into seven subcategories, including glutathione metabolism,
   phenylpropanoid metabolism, and benzoxazinoid metabolism, each
   comprising 10 protein spots. Additionally, two protein spots
   (gi|195613358 and gi|226499080) related to phenylpropanoid metabolism
   were differentially accumulated in all hybrids, while four protein
   spots (gi|162463414, gi|195655583, gi|195624066, and gi|162459230)
   involved in benzoxazinoid metabolism were differentially accumulated in
   certain hybrids. Carbohydrate metabolism (10 protein spots), energy
   metabolism, nucleotide metabolism, amino acid metabolism, and
   metabolism of cofactors and vitamins were the other metabolism
   subcategories.

Figure 5.

   [89]Figure 5
   [90]Open in a new tab

   Functional classification of all differentially accumulated proteins in
   the eighth internode of the hybrids and parental lines. Proteins were
   classified based on a maize protein database. Note: Proportions of
   different metabolic activities were calculated based on the number of
   identified proteins.

Relationships among indole-3-acetic acid content, gene expression level, and
internode length

   The IAA concentrations of the eighth internode were 147.87, 169.24,
   253.72, and 328.27 ng/g in Xundan20, Zhengdan958, Yuyu22, and
   Jinsai6850, respectively (Table [91]1). The Pearson correlation
   coefficients indicated a positive correlation between IAA concentration
   and the eighth internode length (0.976^**) and ear height (0.212^*). In
   contrast, a negative correlation was observed between IAA concentration
   and the extent of the heterosis of the eighth internode length
   (−0.654^*), ear height (−0.166^*), and plant height (−0.273^**)
   (Table [92]2). These results implied that high IAA levels may induce
   the elongation of internodes, while inhibiting the heterosis of
   internode length.

Table 1.

   Indole-3-acetic acid (IAA) content in hybrids and the relative
   expression levels of key genes influencing IAA biosynthesis and polar
   distribution.
   Hybrids The 8^th internode length (cm) Relative expression level IAA
   (ng/g) Ear height (cm) Plant height (cm) Heterosis degree (Mid-parent)
   BX2 BX5 FLS DFR Ear height (%) Plant height (%) The 8^th internode
   length (%)
   Xundan20 11.50 1.21 1.11 0.91 1.73 147.87 127.74 227.40 52.0 31.3 29.5
   Zhengdan958 12.06 1.00 1.00 1.00 1.00 169.24 107.20 221.30 39.9 26.0
   45.3
   Yuyu22 13.34 0.95 1.07 0.67 1.65 253.72 131.62 272.90 87.7 43.7 34.3
   Jinsai6850 13.82 0.52 0.81 2.62 0.97 328.27 121.09 251.0 24.8 17.9 20.3
   [93]Open in a new tab

Table 2.

   Pearson correlation coefficient analysis of indole-3-acetic acid (IAA)
   content, gene expression level, eighth internode length, and heterosis
   extent.
   IAA Heterosis degree The 8^th internode length Ear height Plant height
   The 8^th internode Ear height Plant height
   IAA −0.654^* −0.166^* −0.273^** 0.976^** 0.212^* 0.699
   BX2 −0.936^**
   BX5 −0.790^**
   FLS 0.746^** −0.717^** −0.763^* −0.827^**
   DFR −0.398^**
   [94]Open in a new tab

   Note: ^*Significant at the 0.05 level; ^**Significant at the 0.01
   level.

   The expression profiles of the key genes related to the phenylpropanoid
   and benzoxazinoid metabolic pathways were examined by qRT-PCR analysis
   (Figs [95]6 and [96]7). In the phenylpropanoid metabolic pathway,
   phenylalanine ammonia lyase (GRMZM2G170692_T01; EC 4.3.1.24) is the
   first rate-limiting enzyme. The corresponding gene was highly expressed
   in Yuyu22 (partial dominance, +−) and Zhengdan958 (above high-parent,
   ++), and expressed at relatively low levels in Jinsai6850 (low-parent,
   −) and Xundan20 (below low-parent, − −). The genes encoding cinnamate
   4-hydroxylase (GRMZM2G147245_T01; EC 1.14.13.11), cinnamoyl-CoA
   reductase (GRMZM2G033555_T01; EC 1.2.1.44), and cinnamyl-alcohol
   dehydrogenase (GRMZM5G844562; EC 1.1.1.195) exhibited similar
   expression trends in the four hybrids, but were associated with
   different heterosis patterns (Fig. [97]6). Dihydroflavonol is the
   competitive substrate of flavonol synthase (FLS; EC 1.14.11.23) and
   dihydroflavonol 4-reductase (DFR; EC 1.1.1.219). Our qRT-PCR analysis
   revealed that the FLS and DFR expression levels exhibited the opposite
   trends in the four hybrids, which was consistent with their network
   functions in the phenylpropanoid metabolic pathway. The IAA content of
   the eighth internode was positively correlated with the FLS expression
   level (0.746^**), but negatively correlated with the DFR expression
   level (−0.398^**) (Table [98]2). Moreover, the DFR expression level and
   the accumulation of the encoded protein were almost inversely related
   in the four hybrids (i.e., the highest transcript levels were observed
   in Xundan20, followed by Yuyu22, Zhengdan958, and Jinsai6850, while the
   highest protein levels were observed in Zhengdan958, followed by
   Jinsai6850, Yuyu22, and Xundan20). These results indicated there was a
   lack of consistency between gene expression and protein accumulation.
   The IAA biosynthetic pathway and benzoxazinoid metabolic pathway
   (Fig. [99]7) compete for free indole. The cytochrome P450 enzyme
   subfamily (BX2 to BX5) converts indole into benzoxazinoids. The genes
   encoding these enzymes were highly expressed in Xundan20, and the
   expression levels were negatively correlated with IAA content. This
   suggested that IAA biosynthesis was restricted by BX2 and BX5.

Figure 6.

   [100]Figure 6
   [101]Open in a new tab

   Summary of the phenylpropanoid metabolic pathway as well as the
   relative expression levels and patterns of key genes in the hybrids.
   Genes with the associated EC number are provided. Accessions encode the
   differentially accumulated proteins identified in this study. PAL,
   phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CCR,
   cinnamoyl-CoA reductase; CAD, cinnamyl-alcohol dehydrogenase; FLS,
   flavonol synthase; DFR, dihydroflavonol 4-reductase; CHS, chalcone
   synthase; CHI, chalcone isomerase; and F3H, flavanone 3-hydroxylase.

Figure 7.

   [102]Figure 7
   [103]Open in a new tab

   Summary of the benzoxazinoid metabolic pathway as well as the relative
   expression levels and patterns of key genes in the hybrids. Genes with
   the associated EC number are provided. Accessions encode the
   differentially accumulated proteins identified in this study. BX1,
   benzoxazinless1; BX2 to BX5, cytochrome P450 enzymes; BX8 and BX9,
   UDP-glucosyltransferases; BX6, dioxygenase; BX7, O-methyltransferase;
   and DIBOA, 2,4-dihydroxy-2H-1,4-benzoxazin-3(4 H)-one.

Discussion

Heterosis performance and differentially accumulated proteins

   Dominance, over-dominance, and pseudo over-dominance at a single locus
   and epistasis at two loci have contributed to the heterosis of
   different traits according to quantitative genetic
   analyses^[104]12,[105]13,[106]20,[107]21. In this study, dominant and
   over-dominant effects were observed to differentially affect specific
   internodes in certain hybrids (Table [108]S2). Ear height and plant
   height were influenced by a dominant effect in Jinsai6850, but an
   over-dominant effect in Zhengdan958, Xundan20, and Yuyu22. The
   over-dominant effect on maize plant height was also detected in several
   other studies^[109]22–[110]24. The mechanism underlying heterosis has
   been analysed at transcriptional and translational levels^[111]2.
   Additive, dominant, and over-dominant effects on gene expression or
   protein accumulation differentially contribute to the heterosis of a
   particular trait at specific developmental stages^[112]2. The
   overwhelming additive accumulation of differentially accumulated
   proteins in the four hybrids was associated with a non-additive effect
   on the heterosis performance of the eighth internode (Fig. [113]4,
   Tables [114]S2 and [115]4). Similar results were also reported for
   maize embryos at 6 days after fertilisation^[116]25, while another
   study concluded that the moderate transcriptional expression of the
   SINGLE FLOWER TRUSS locus could result in an over-dominant effect on
   yield^[117]26. These results proved that an additive interaction at the
   translational level may lead to a non-additive effect on phenotypic
   performance due to an epistatic interaction between different proteins.

Regulation of indole-3-acetic acid affects internode development and
heterosis

   Certain metabolic and physiological pathways are reportedly relevant
   for heterosis. Goff^[118]27 and Ni et al.^[119]28 emphasised the
   effects of the overall energy-use efficiency and increased energy input
   for the manifestation of multigenic heterosis. Gibberellins, which are
   important plant hormones, help regulate stem
   elongation^[120]29–[121]33. The metabolic and signalling pathways
   associated with these hormones are believed to influence plant height
   heterosis in wheat^[122]5. This has been supported by similar studies
   on the role of gibberellins in the heterosis of biomass accumulation in
   maize^[123]34–[124]36 and rice^[125]37. Auxin, which is another
   important plant hormone, regulates cell elongation, division,
   differentiation, and morphogenesis^[126]38. Thus, auxin may be involved
   in internode development and heterosis.

   Indole-3-acetic acid, which is the most potent native auxin^[127]39,
   induces most of the auxin effects in intact plants. An appropriate
   balance between IAA biosynthesis and efflux [polar auxin transport
   (PAT)] must be maintained in living plants because auxin is toxic at
   excessive concentrations. Additionally, ethylene accumulates in
   response to high auxin doses, resulting in inhibited elongation, leaf
   abscission, and even plant death^[128]40,[129]41. In this study, the
   benzoxazinoid and phenylpropanoid metabolic pathways were emphasised
   because of their effects on IAA biosynthesis and PAT.

   For most monocotyledonous plant species, IAA biosynthesis is regulated
   by indole, which is an intermediate product of the benzoxazinoid
   pathway^[130]42. We observed that the BX2 (−0.936^**) and BX5
   (−0.790^**) expression levels were negatively correlated with IAA
   contents in the eighth internode, but were positively correlated with
   the eighth internode length. These results implied that balanced IAA
   metabolism influenced by the benzoxazinoid pathway is important for
   internode length heterosis. Another secondary metabolite, flavonol,
   which resembles the synthetic auxin transport inhibitor
   naphthylphthalamic acid, regulates PAT^[131]43. Flavonol negatively
   regulates PAT by competing with auxin efflux carriers, such as PIN and
   ABCB (PGP proteins) in vivo^[132]44–[133]47. Polar auxin transport
   refers to the complex and coordinated active transport of auxin
   molecules from cell to cell throughout the plant. Cell growth (division
   and expansion) and the generation of auxin gradients throughout the
   plant require PAT^[134]48,[135]49. Moreover, PAT allows stems to turn
   toward light sources (phototropism) and roots to grow in response to
   gravity (gravitropism), while mediating other tropisms associated with
   differential cell growth on different sides of an organ^[136]49. In
   this study, the expression of the gene encoding FLS, which controls the
   first committed step of the flavonol branch of phenylpropanoid
   metabolism, was negatively correlated with the extent of the mid-parent
   heterosis of ear height and plant height in the four hybrids
   (Fig. [137]6, Table [138]2). These results are consistent with those of
   previous studies involving Arabidopsis thaliana, in which a mutant
   incapable of synthesising flavonoids exhibited enhanced auxin transport
   and developmental abnormalities, including an increased number of
   inflorescences, decreased plant height, increased secondary root
   development^[139]44, and delayed gravitropism^[140]47. Both studies
   suggested that increased basipetal auxin transport interfered with the
   formation of an auxin gradient. Additionally, the negative regulation
   by flavonols was required for plant development and heterosis.
   Conserved regulators, such as MYB, basic helix-loop-helix, and
   WD40-type transcription factors, are indirectly involved in the final
   step of flavonoid translocation through the vacuolar membrane^[141]50.
   They also regulate the activation of structural genes, such as BZ2 in
   maize^[142]51, AN9 in petunia^[143]52, and TT19 in A. thaliana^[144]53,
   which encode glutathione S-transferases^[145]54,[146]55 involved in
   cell detoxification. Seven of the identified differentially accumulated
   protein spots (10.6%) were involved in glutathione metabolism. These
   results imply that cell detoxification during IAA transport and
   distribution is critical.

   Polar auxin transport was inhibited in Jinsai6850, resulting in the
   over-accumulation of IAA in the eighth internode, a decrease in the
   extent of heterosis, and a greater abundance of GST proteins for
   detoxifying cells. In Yuyu22, only a few flavonols were needed to
   regulate PAT, which revealed the suitable IAA contents and extensive
   heterosis. In Zhengdan958 and Yuyu22, which belong to the same
   heterotic group, IAA content and flavonol regulation exhibited similar
   trends. Therefore, the heterosis extent and performance were consistent
   in the eighth internode.

Conclusions

   In different heterotic combinations, additive effects at a single locus
   and epistatic effects at two loci are primarily responsible for the
   non-additive effects on the heterosis performance of the eighth
   internode length and ear height. Regarding epistatic interactions, an
   integrated metabolic pathway helps regulate each intermediate. A
   regulatory network involving IAA biosynthesis and PAT, which can be
   affected by the phenylpropanoid and benzoxazinoid metabolic pathways,
   should be included in the molecular mechanism underlying the
   development of internode heterosis (Fig. [147]8).

Figure 8.

   [148]Figure 8
   [149]Open in a new tab

   Model of the development and heterosis of internodes. The development
   and heterosis of internodes are mainly regulated by an auxin gradient,
   which is the result of a balance between polar auxin transport (PAT)
   and auxin biosynthesis. In plant cells and organs, PAT is negatively
   regulated by flavonols, which are biosynthesised in the cytoplasm and
   stored in the vacuole. Transmembrane transport of flavonols to the
   vacuole is indirectly regulated by structural genes (e.g., BZ2)
   encoding glutathione S-transferases. Additionally, flavonol
   biosynthesis and BZ2 expression are regulated by a network of
   transcription factors. Once PAT is inhibited, auxin will accumulate in
   cells and organs to toxic levels. Moreover, excessive auxin
   concentrations induce ethylene production, which negatively regulates
   plant development. Thus, detoxification by benzoxazinoid metabolites
   and the competition for indole between the indole-3-acetic acid
   biosynthesis pathway and the benzoxazinoid metabolic pathway are
   required to protect cells and organs. Therefore, flavonols, which are
   synthesised in the phenylpropanoid metabolic pathway, and
   benzoxazinoids are important regulators of the development and
   heterosis of internodes via a complex network of key structural genes
   and transcription factors.

Electronic supplementary material

   [150]Table S1^ (11.4KB, xlsx)
   [151]Table S2^ (15.4KB, xlsx)
   [152]Table S3^ (14KB, xls)
   [153]Table S4^ (36.5KB, xls)
   [154]Table S5^ (231.5KB, xls)
   [155]Table S6^ (39KB, xls)

Acknowledgements