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. 2015 Apr 8;115(6):879–894. doi: 10.1093/aob/mcv018

Transcript profiles in cortical cells of maize primary root during ethylene-induced lysigenous aerenchyma formation under aerobic conditions

Hirokazu Takahashi 1, Takaki Yamauchi 1, Imene Rajhi 2, Naoko K Nishizawa 3,4, Mikio Nakazono 1,*
PMCID: PMC4407059  PMID: 25858325

Abstract

Background and Aims Internal aeration is important for plants to survive during periods of waterlogging, and the ability to form aerenchyma contributes by creating a continuous gas space between the shoots and the roots. Roots of maize (Zea mays) react to prolonged waterlogging by forming aerenchyma in root cortical cells by programmed cell death (PCD) in response to ethylene. The aim of this study was to understand the molecular mechanisms of ethylene-induced aerenchyma formation by identifying genes that are either up- or downregulated by ethylene treatment in maize root cortical cells.

Methods Three-day-old maize seedlings were treated with ethylene for several hours under aerobic conditions. Cortical cells were isolated from the primary roots using laser microdissection (LM), and transcript profiles with and without ethylene treatment were compared by microarray. In addition, the effect on ethylene-induced aerenchyma formation of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, was examined in order to assess the involvement of reactive oxygen species (ROS).

Key Results A total of 223 genes were identified whose transcript levels were significantly increased or decreased by ethylene treatment in root cortical cells under aerobic conditions. Subsequent tissue-specific quantitative reverse-transcription PCR analyses revealed that ethylene increased the transcript levels of genes related to ethylene signalling in all of the root tissues examined (stelar cells, cortical cells and outer cell layers), whereas it increased the transcript levels of genes related to cell wall modification and proteolysis specifically in the cortical cells. DPI treatment inhibited the ethylene-induced aerenchyma formation and suppressed expression of some cell wall modification-related genes.

Conclusions Several genes related to cell wall modification and proteolysis are specifically up- or downregulated in cortical cells during lysigenous aerenchyma formation under aerobic conditions with ethylene treatment. The results suggest that ethylene is perceived in stelar cells, cortical cells and outer cell layers in the maize primary root, and that the cortical cell-specific PCD is controlled downstream of ethylene perception through subsequent gene expression, which is partly regulated by ROS, in the cortical cells.

Keywords: Lysigenous aerenchyma, maize, Zea mays, ethylene, waterlogging, laser microdissection, microarray, reactive oxygen species, ROS, programmed cell death, PCD, transcript profile, primary root, anaerobic stress

INTRODUCTION

Excess water as a result of waterlogging or submergence can be fatal for non-wetland plants. Oxygen diffusion in water is 10 000 times slower than in air (Armstrong, 1979). Thus, waterlogging or submergence causes oxygen deficiency and damages the submerged part of a plant (Colmer and Voesenek, 2009). Plants have a variety of adaptive mechanisms to avoid oxygen deficiency under waterlogged or submerged conditions, one of which is aerenchyma formation (Colmer and Voesenek, 2009, Nishiuchi et al., 2012; Takahashi et al., 2014). Aerenchyma consists of longitudinal gas spaces that enable the transport of gases (O2, CO2, ethylene and methane) between and within the aerial and submerged parts (Jackson and Armstrong, 1999; Colmer, 2003; Evans, 2003; Colmer et al., 2006).

Lysigenous aerenchyma is formed by the collapse and lysis of files (rows) of cortical cells via programmed cell death (PCD). Wetland plant species such as rice (Oryza sativa) can constitutively form lysigenous aerenchyma in roots even under well-drained soil conditions, and its formation can be further induced under low oxygen conditions (Shiono et al., 2011). On the other hand, non-wetland plant species such as maize (Zea mays), wheat (Triticum aestivum) and barley (Hordeum vulgare) develop lysigenous aerenchyma under low oxygen conditions but not under well-drained soil conditions (McPherson, 1939; Trought and Drew, 1980; McDonald et al., 2001). The plant hormone ethylene is known to have a role in lysigenous aerenchyma formation (Justin and Armstrong, 1991; Drew et al., 2000). Ethylene is entrapped and accumulated in roots under waterlogged conditions because water surrounding the root prevents its escape (Visser and Voesenek, 2004). Non-wetland plants take several hours to initiate aerenchyma formation under waterlogged conditions (Malik et al., 2003; Haque et al., 2010; Rajhi et al., 2011). Treatments with exogenously supplied ethylene or a precursor of ethylene, 1-aminocyclopropane-l-carboxylic acid (ACC), were found to promote aerenchyma formation even under aerobic conditions in roots of maize (Drew et al., 1979; Konings, 1982) and wheat (Yamauchi et al., 2014). Pre-treating maize roots with 1-methylcyclopropene (1-MCP; an inhibitor of ethylene perception) prevented aerenchyma formation under waterlogging (Rajhi et al., 2011). These results indicate that ethylene is essential for lysigenous aerenchyma formation under waterlogged conditions.

Reactive oxygen species (ROS) are signal molecules that stimulate abiotic stress responses in plants (Suzuki et al., 2011) and are thought to have a role in regulating root aerenchyma formation (Rajhi et al., 2011; Yamauchi et al., 2011). ROS are accumulated during aerenchyma formation in maize root (Bouranis et al., 2003, 2006). ROS accumulation is thought to be mediated by plasma membrane-located NADPH oxidase, which is a key enzyme for the generation of ROS (Torres and Dangl, 2005), during aerenchyma formation (Parlanti et al., 2011; Steffens et al., 2011; Yamauchi et al., 2011) and epidermal cell death at the site of adventitious root emergence (Steffens and Sauter, 2009). Diphenyleneiodonium (DPI), an inhibitor of flavoproteins such as NADPH oxidase, prevented waterlogging-induced aerenchyma formation in maize primary roots (Yamauchi et al., 2011) and ACC-induced aerenchyma formation in wheat seminal roots (Yamauchi et al., 2014). These results suggest that NADPH oxidase-mediated ROS production is responsible for the aerenchyma formation in roots of some gramineous plants under waterlogged conditions.

In the final stage of lysigenous aerenchyma formation, the cell wall is degraded enzymatically (Evans, 2003). During cell wall degradation, cellulase (CEL) hydrolyses cellulose (Cogsgrove, 2005), and polygalacturonase (PG) depolymerizes homogalacturonan, which is a major component of pectin (Niture, 2008). It is thought that the limiting step of CEL action is accessibility of CEL to glucan, and the cell wall-loosening enzyme, expansin (EXP), supports this accessibility to glucan by reducing cell wall tension (Cosgrove, 2000, 2005). Genes encoding CEL, PG and EXP were induced during lysigenous aerenchyma formation under waterlogged conditions (Rajhi et al., 2011). Moreover, CEL activity is increased during aerenchyma formation in hypoxic maize roots (He et al., 1994, 1996a, b).

We previously identified up- and downregulated genes during waterlogging-induced aerenchyma formation in maize primary root by a microarray analysis using laser microdissection (LM)-isolated cortical cells (Rajhi et al., 2011). By the combination of waterlogged conditions and 1-MCP pre-treatment, 239 upregulated and 336 downregulated genes in response to endogenous ethylene under waterlogged conditions were identified (Rajhi et al., 2011). Ethylene can induce PCD in cortical cells for lysigenous aerenchyma formation in maize roots even under aerobic conditions (Drew et al., 1979), suggesting that the expression of genes associated with lysigenous aerenchyma formation is also specifically regulated in root cortical cells in response to ethylene even under aerobic conditions. In this study, to examine this possibility, we identified genes whose transcript levels were increased or decreased in cortical cells of maize primary root by ethylene treatment under aerobic conditions using LM combined with microarray analysis. The transcript profiles of some selected genes in cortical cells during ethylene-induced and the waterlogging-induced aerenchyma formation were analysed by quantitative reverse transcription PCR (qRT-PCR). We also investigated the effects of DPI on ethylene-induced aerenchyma formation, and on the transcript levels of some of the genes identified by the microarray analysis.

MATERIALS AND METHODS

Plant material and growth conditions

Maize (Zea mays, inbred line B73) caryopses were sterilized in a 0·6 % (v/v) sodium hypochlorite solution for 30 min. After washing with deionized water five times, eight seeds were placed on moist chromatography paper (3MM CHR; Whatman, Maidstone, Kent, UK) and rolled up in the paper. The roll of paper was placed vertically in a 2 L grey pot (250 mm height × 90 mm length × 120 mm width) containing 100 mL of deionized water. Only the tip of the paper roll was in the water and none of the root tips of the primary roots of the seedlings reached the water at 4 d after imbibition. The pot was covered with aluminium foil with small holes and incubated at 28 °C in constant light conditions. After 3 d, the root length of each seedling was measured. In this study, we used seedlings whose root lengths were 7·5 ± 1 cm. These seedlings were rolled up in moist chromatography paper and again placed in a pot. The pot was placed in a tightly closed container with or without 1 ppm ethylene gas for 3, 6, 9, 12, 18 and 24 h. For waterlogging treatment, roots of 3-day-old seedlings were submerged in distilled water as described by Rajhi et al. (2011).

Diphenyleneiodonium (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in water, and 500 μm stock solution was prepared. Three-day-old seedlings were transferred to 1 % agar plates containing 0, 20 and 40 μm DPI. The plates were placed in a 2 L grey pot and treated with ethylene for 24 h as described above.

Anatomical observation

Segments of primary root were obtained at 5–10, 15–20, 25–30 and 35–40 mm from the root–shoot junction and at 25–30 mm from the root tip. Transverse sections of primary roots were made by hand sectioning using a razor blade. Aerenchyma formation at 15–20 mm from the root–shoot junction was observed for time-course analysis or DPI treatment. Each section was photographed using a light microscope (BX60; Olympus, Tokyo, Japan) with a CCD camera (DP70; Olympus). Areas occupied by aerenchyma were measured with Image J software (version 1.46r; National Institutes of Health, Bethesda, MD, USA).

Laser microdissection (LM)

The basal part of the primary root (15–20 mm from the root–shoot junction) was fixed in 75 % ethanol:25 % acetic acid. After dehydration in a graded ethanol series, the tissues were embedded in paraffin and sectioned at a thickness of 16 μm. Serial sections were placed onto PEN membrane glass slides (Life Technologies, Gaithersburg, MD, USA) for LM as described by Takahashi et al. (2010). To remove the paraffin, slides were immersed in 100 % Histoclear II (National Diagnostics, Atlanta, GA, USA) for 10 min twice, followed by air-drying at room temperature. Cortical cells, stelar cells and outer cell layers (OCLs) were collected from the root cross-sections using a Veritas Laser Microdissection System LCC1704 (Molecular Devices, Sunnyvale, CA, USA).

RNA extraction

Total RNA was extracted from the LM-isolated tissues using a PicoPure™ RNA isolation kit (Life Technologies) according to the manufacturer’s instructions. The extracted total RNA was quantified with a Quant-iT™ RiboGreen RNA reagent and kit (Life Technologies) according to the manufacturer’s instructions. The quality of total RNA was assessed using a RNA 6000 Pico kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) as described by Takahashi et al. (2010). Total RNA was extracted from root segments using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Microarray experiment

Total RNAs (10 ng each) were labelled with a Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Aliquots of Cy5-labelled and Cy3-labelled cRNA (825 ng each) were used for hybridization in a 4 × 44k Maize Gene Expression Microarray (Agilent Technologies) that contains 42 034 oligo probes to maize genes. Three biological replicates and a colour swap for each replicate were analysed. The hybridized slides were scanned using a DNA microarray scanner G2505C (Agilent Technologies), and signal intensities were extracted by Feature Extraction software (version 10.5.1.1; Agilent Technologies). A complete set of microarray data was deposited in the Gene Expression Omnibus (GEO) repository under accession no. GSE60944.

Microarray data analysis

Microarray signal intensities were digitized, and the log ratio and P-values were obtained by Feature Extraction software version 10.5.1.1 (Agilent Technologies). We selected genes that showed >2·0-fold change in expression between ethylene-treated and -untreated conditions, and P-values < 0·05 in all three replications and in each colour swap [Supplementary Data Tables S1 and S2]. Fold changes in expression were calculated from microarray data sets in root cortical cells of maize primary root treated with waterlogging or 1-MCP that were downloaded from the GEO repository (Rajhi et al., 2011; accession no. GSE22943). The fold change of each probe was calculated using the average of three replications and each colour swap. According to the classification of the Maize Microarray Annotation Database (http://maizearrayannot.bi.up.ac.za/; Coetzer et al., 2011), we removed the probes of ‘inconclusive annotation’ (probes that match many transcripts) and ‘antisense gene model’ (probes that match the antisense direction of a transcript) from the selected gene lists. Maize gene IDs, RAP Os IDs, LOC Os IDs and annotations were obtained from each database as described by Rajhi et al., (2011). Maize gene descriptions were obtained from the GRAMENE database (http://www.gramene.org). Hierarchical clustering analysis was performed using log2 fold change values of the 223 genes selected in this study. Hierarchical clustering of the data set was generated by GenePattern software (Reich et al., 2006).

qRT-PCR analysis

Relative mRNA levels were investigated with qRT-PCR using a StepOnePlus™ real-time PCR system (Life Technologies). First-strand cDNA was synthesized using Superscript III (Life Technologies) from 10 ng of total RNA extracted from LM-isolated tissues and 2 μg from root segments. SYBR® Premix Ex Taq™ II (Takara Bio Inc., Shiga, Japan) was used for subsequent PCR amplification with appropriate primers [Supplementary Data Table S3]: initial denaturation (95 °C for 20 s) and 50 cycles of denaturation (95 °C for 5 s), annealing (60 °C for 20 s) and extension (72 °C for 20 s). Transcript levels of each gene were normalized to the transcript levels of the maize Ubiquitin (ZmUBQ) gene (used as a control gene).

RESULTS

Analysis of aerenchyma formation in maize primary roots under aerobic conditions with ethylene

Three-day-old aerobically grown seedlings were transferred to aerobic conditions with or without 1 ppm ethylene, and the percentage of each cross-section occupied by aerenchyma was determined along primary roots after 24 h of treatment (Fig. 1A). In our experimental conditions, lysigenous aerenchyma formation was significantly induced by ethylene treatment for 24 h in the basal part of roots [at 5–10, 15–20, 25–30 and 35–40 mm from the root–shoot junction (B10, B20, B30 and B40, respectively; Fig. 1B)] but hardly induced without ethylene treatment. Lysigenous aerenchyma did not form at 25–30 mm from the root tips (T30; Fig. 1B) under either condition. The percentage of aerenchyma was largest at 15–20 mm from the root–shoot junction (Fig. 1B). Subsequently, the time-course of aerenchyma formation was analysed at 15–20 mm from the root–shoot junction of primary roots of maize seedlings grown under aerobic conditions with or without ethylene treatment (Fig. 1C). Aerenchyma formation was first observed at 12 h after the initiation of ethylene treatments, and gradually increased during ethylene treatment (Fig. 1C).

Fig. 1.

Fig. 1.

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Induction of aerenchyma formation by ethylene treatment in maize primary root. Three-day-old maize seedlings were grown under aerobic conditions with or without 1 ppm ethylene during 24 h. (A) Cross-sections of ethylene-treated or untreated maize primary root. Asterisks shows aerenchyma in cortical cells. (B) Rate of aerenchyma formation of maize primary root measured at the basal [5–10 (B10), 15–20 (B20), 25–30 (B30) and 35–40 (B40) mm from the root–shoot junction] and the apical part of the root [25–30 (T30) mm from the root tip]. (C) The ratio of aerenchyma, expressed as a percentage of the cross-section at 15–20 mm from the root–shoot junction every 6 h after treatment.

Microarray analysis in cortical cells of maize primary roots during ethylene-induced aerenchyma formation

Aerenchyma formation at 15–20 mm from the root–shoot junction was first observed at 12 h after the initiation of ethylene treatment (Fig. 1C). To identify genes whose transcript levels were increased or decreased during the ethylene-induced aerenchyma formation, 3-day-old maize seedlings were treated with ethylene for 6 h or left untreated. The segment at 15–20 mm from the root–shoot junction was then excised from the whole root, and the cortical cells were isolated by LM for microarray analysis. In cortical cells of maize primary roots under aerobic conditions with ethylene treatment, 109 genes were upregulated and 114 genes were downregulated (Fig. 2; Supplementary Data Tables S1 and Table S2). Previously, Rajhi et al. (2011) identified genes whose transcript levels were increased or decreased during lysigenous aerenchyma formation under waterlogged conditions, and whose transcript profile changes were suppressed by an ethylene perception inhibitor, 1-MCP. As a result, 239 upregulated genes and 336 downregulated genes were identified in response to endogenous ethylene under waterlogged conditions (i.e. under the conditions inducing aerenchyma formation; Rajhi et al., 2011). Comparison of the transcript profiles obtained in this study with the profiles obtained in the previous study (Rajhi et al., 2011) showed that of 109 genes upregulated by ethylene treatment under aerobic conditions in the present study, only 28 (26 %) were upregulated in response to endogenous ethylene under waterlogged conditions in the previous study, and of 114 genes downregulated by ethylene treatment under aerobic conditions in the present study, only 59 (52 %) were downregulated in response to endogenous ethylene under waterlogged conditions in the previous study (Fig. 2A, B). Hierarchical clustering analysis of genes selected in this study and the previous study (Rajhi et al., 2011) showed that the changes in gene expression in response to ethylene treatment were similar to the changes in response to 1-MCP treatment, but not similar to the changes in response to waterlogging (Fig. 2C).

Fig. 2.

Fig. 2.

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Venn diagram and hierarchical clustering analyses of up- or downregulated genes during aerenchyma formation. Three-day-old maize seedlings were treated with 1 ppm ethylene for 6 h. Cortical cells were isolated from cross-sections of tissues at 15–20 mm from the root–shoot junction of ethylene-treated maize root by laser microdissection and were used for the microarray analysis. Microarray data of waterlogging with or without 1-MCP treatment are reproduced from Rajhi et al. (2011). The seedlings were grown under waterlogged conditions for 12 h. For 1-MCP treatment, 2·5-day-old seedlings were pre-treated with 1 ppm 1-MCP for 12 h, and then were grown under waterlogged conditions for 12 h (Rajhi et al., 2011). (A) Comparison of genes that were upregulated by ethylene treatment and genes that were upregulated by 12 h waterlogging treatment and downregulated under waterlogged conditions by 12 h of 1-MCP pre-treatment. (B) Comparison of genes that were downregulated by ethylene treatment and genes that were downregulated by waterlogged conditions and upregulated under waterlogged conditions by 12 h of 1-MCP pre-treatment. (C) Hierarchical clustering analysis of 223 up- or downregulated genes by ethylene treatment. Analysis was performed using log2 fold change values by GenePattern software. Rep1, 2 and 3 indicate samples #1, #2 and #3 of three biological replicates. ‘Rep1-1 and Rep1-2’, ‘Rep2-1 and Rep2-2’ or ‘Rep3-1 and Rep3-2’ indicate two colour swaps for each replicate. (+) with treatment; (−) without treatment. C2H4, ethylene treatment; MCP, 1-MCP treatment. WL, waterlogged conditions; Aer, aerobic conditions.

The microarray results identified 15 genes (11 upregulated and 4 downregulated) related to transcriptional regulation (Table 1). Five of the upregulated genes were also upregulated in response to endogenous ethylene under waterlogged conditions (Table 1; Rajhi et al., 2011). The microarray data also identified genes related to cell wall modification (Table 2) and proteolysis (Table 3). During the ethylene-induced aerenchyma formation in this study, 12 cell wall modification-related genes were upregulated (Table 2). Five of the upregulated genes [including CEL (GRMZM2G147221) and PG (GRMZM2G037431)] were also upregulated in response to endogenous ethylene under waterlogged conditions (Table 2). Seven proteolysis-related genes were upregulated by ethylene treatment under aerobic conditions (Table 3). Only three of these genes (GRMZM2G091578, GRMZM2G133394 and GRMZM2G354373) were also upregulated in response to endogenous ethylene under waterlogged conditions (Table 3).

Table 1.

Transcription factor-related genes that were up- or downregulated in LM-isolated cortical cells in ethylene-treated roots (at 15–20 mm from the root–shoot junction) under aerobic conditions

Maize gene IDs* GRAMENE description +C2H4 vs. −C2H4† WL vs. Aer‡ −MCP vs. +MCP‡ RAP Os IDs§ RAP gene annotation LOC Os IDs
AC233899.1 Putative homeobox DNA-binding and leucine zipper domain family protein 5·10 3·79 2·21 Os03g0188400 Helix–loop–helix DNA-binding domain containing protein. LOC_Os03g08930.1
GRMZM2G002131 NA 3·01 0·70 13·84 Os09g0456800 Similar to Heat stress transcription factor Spl7 (Heat shock transcription factor) (Heat shock factor RHSF10). LOC_Os09g28354.1
GRMZM2G055180 Ethylene-responsive transcription factor 2 (ERF2) 8·44 2·30 11·38 Os04g0546800 Pathogenesis-related transcriptional factor and ERF domain containing protein. LOC_Os04g46220.1
GRMZM2G080516 AP2-EREBP transcription factor 3·20 1·36 3·27 Os04g0546800 Pathogenesis-related transcriptional factor and ERF domain containing protein. LOC_Os04g46220.1
GRMZM2G106673 Uncharacterized protein 3·36 4·69 10·36 Os06g0194400 Transcriptional factor B3 family protein. LOC_Os06g09420.1
GRMZM2G110057 NA 10·94 1·08 31·52 Os01g0876300 F-box associated type 1 domain containing protein. LOC_Os01g65510.1
GRMZM2G131281 NA 4·09 1·57 7·41 Os08g0537900 Similar to predicted protein. Hypothetical conserved gene. LOC_Os08g42550.2
GRMZM2G134717 Putative NAC domain transcription factor superfamily protein 4·72 1·23 7·48 Os01g0672100 No apical meristem (NAM) protein domain containing protein. LOC_Os01g48130.1
GRMZM2G137582 EIN3-binding F-box protein 1 (EBF1) 10·95 4·86 3·24 Os06g0605900 Leucine-rich repeat, cysteine-containing sub-type containing protein. LOC_Os06g40360.1
GRMZM2G161435 Uncharacterized protein 6·66 2·12 31 Os02g0226600 Homeodomain-related domain containing protein. LOC_Os02g13310.1
GRMZM2G312201 NA 7·23 1·06 0·47 Os01g0816100 Similar to NAC domain protein. LOC_Os01g60020.1
AC232238.2** NA 0·30 0·35 0·02 Os01g0859500 Similar to Basic leucine zipper protein (Liguleless2). LOC_Os01g64020.1
GRMZM2G168002** G2-like transcription factor 0·20 2·48 0·14 Os08g0434700 Homeodomain-like containing protein. LOC_Os08g33750.1
GRMZM2G172657** Uncharacterized protein 0·08 0·09 0·05 Os07g0586900 GRAS transcription factor domain containing protein. LOC_Os07g39820.1
GRMZM5G842961** NA 0·11 0·50 0·05 Os02g0656600 Similar to Dehydration responsive element binding protein 2B (DREB2B protein). LOC_Os02g43940.1
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+C2H4, aerobic conditions with ethylene; −C2H4, aerobic conditions without ethylene; WL, waterlogged conditions; Aer, aerobic conditions; −MCP, waterlogged conditions without 1-methylcyclopropene (1-MCP) pre-treatment; +MCP, waterlogged conditions with 1-MCP pre-treatment; NA, not available

*IDs were obtained from the MaizeSequence Database.

†Values are fold change and were the average of three biological replications.

‡Data are from Rajhi et al. (2011). Values are the average fold change of three biological replications.

§IDs were obtained from RAP DB Build 5.

IDs were obtained from the MSU.

**Genes downregulated under ethylene-treated conditions; all other genes are upregulated.

Table 2.

Cell wall modification-related genes that were up- or downregulated in LM-isolated cortical cells in ethylene-treated roots (at 15–20 mm from the root–shoot junction) under aerobic conditions

Maize gene IDs* GRAMENE description +C2H4 vs. −C2H4† WL vs. Aer‡ −MCP vs. +MCP‡ RAP Os IDs§ RAP gene annotation LOC Os IDs
AC204604.3 NA 2·63 1·45 4·07 Os02g0702400 Pectinacetylesterase family protein. LOC_Os02g47400.1
GRMZM2G018416 Uncharacterized protein 6·34 1·24 11·99 Os02g0588550 Hypothetical gene.
GRMZM2G026980 Xyloglucan endotransglucosylase/hydrolase protein 23 8·61 0·90 1·60 Os06g0696600 Similar to Xyloglucan endo-transglycosylase homolog. LOC_Os06g48180.1
GRMZM2G037431 Polygalacturonase 4·52 5·56 2·55 Os01g0636500 Similar to Polygalacturonase PG2. LOC_Os01g44970.1
GRMZM2G048430 Ripening protein 3·18 2·43 4·16 Os06g0711800 Pectinesterase inhibitor domain containing protein. LOC_Os06g49760.1
GRMZM2G079263 Polygalacturonase 3·33 0·18 14·98 Os05g0542800 Pectin lyase fold/virulence factor domain containing protein. Similar to polygalacturonase. LOC_Os05g46510.1
GRMZM2G082520 Beta-expansin 1a 19·43 0·46 64·12 Os10g0555900 Similar to Beta-expansin. Beta-expansin precursor. LOC_Os10g40720.1
GRMZM2G119471 50S ribosomal protein L5 14·65 1·50 2·69 Os03g0124900 Pectin lyase fold/virulence factor domain containing protein. LOC_Os03g03350.1
GRMZM2G147221 Cellulase containing protein 4·06 2·12 3·53 Os05g0244500 Glycoside hydrolase, family 5 protein. Similar to cellulase containing protein. LOC_Os05g15510.1
GRMZM2G149422 Uncharacterized protein 7·71 1·09 75·95 Os02g0756850 Conserved hypothetical protein.
GRMZM2G342246 Beta-expansin 7 15·14 2·11 15·12 Os10g0555900 Similar to Beta-expansin. Beta-expansin precursor. (EXPB7) LOC_Os10g40720.1
GRMZM2G474194 Beta-expansin 3 (EXPB3) 4·91 3·01 2·92 Os03g0102700 Similar to Expansin-B LOC_Os03g01270.1
GRMZM2G103672** Beta-expansin 4 0·20 0·35 0·36 Os05g0246300 Barwin-related endoglucanase domain containing protein. LOC_Os05g15690.1
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+C2H4, aerobic conditions with ethylene; −C2H4, aerobic conditions without ethylene; WL, waterlogged conditions; Aer, aerobic conditions; −MCP, waterlogged conditions without 1-methylcyclopropene (1-MCP) pre-treatment; +MCP, waterlogged conditions with 1-MCP pre-treatment; NA, not available.

*IDs were obtained from the MaizeSequence Database.

†Values are fold change and were the average of three biological replications.

‡Data are from Rajhi et al. (2011). Values are the average fold change of three biological replications.

§IDs were obtained from RAP DB Build 5.

IDs were obtained from the MSU.

**This gene is downregulated under ethylene-treated conditions; all other genes are upregulated.

Table 3.

Protolysis-related genes that were up- or downregulated in LM-isolated cortical cells in ethylene-treated roots (at 15–20 mm from the root–shoot junction) under aerobic conditions

Maize gene IDs* GRAMENE description +C2H4 vs. −C2H4† WL vs. Aer‡ -MCP vs +MCP‡ RAP Os IDs§ RAP gene annotation LOC Os IDs
GRMZM2G020146 Uncharacterized protein 10·17 0·40 95·43 Os02g0114200 Serine carboxypeptidase III precursor (EC 3.4.16.5). Similar to Serine carboxypeptidase 3. LOC_Os02g02320.1
GRMZM2G022799 Putative metacaspase family protein 19·00 1·19 152·01 Os11g0134700 Similar to Metacaspase 7 (Metacaspase 4). LOC_Os11g04010.1
GRMZM2G091578 Subtilisin-like protease 11·34 5·12 17·05 Os03g0761500 Similar to Subtilisin-like protease (Fragment). LOC_Os03g55350.1
GRMZM2G133394 Uncharacterized protein 45·53 16·57 10·37 Os08g0267300 Peptidase A1 domain containing protein. LOC_Os08g16660.1
GRMZM2G153977 Uncharacterized protein 2·99 0·49 5·84 Os05g0567100 Aspartic proteinase oryzasin 1 precursor (EC 3.4.23.–). Similar to Phytepsin. LOC_Os05g49200.1
GRMZM2G354373 Putative subtilase family protein 5·14 2·59 1·83 Os08g0452100 Peptidase S8, subtilisin-related domain containing protein. LOC_Os08g35090.1
GRMZM2G456217 Vignain 21·36 1·05 351·35 Os08g0556900 Similar to Cysteine proteinase (EC 3.4.22.–). Similar to vignain. LOC_Os08g44270.1
GRMZM2G159016** Uncharacterized protein 0·22 0·20 0·05 Os05g0582800 Peptidase S10, serine carboxypeptidase family protein.
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+C2H4, aerobic conditions with ethylene; −C2H4, aerobic conditions without ethylene; WL, waterlogged conditions; Aer, aerobic conditions; −MCP, waterlogged conditions without 1-methylcyclopropene (1-MCP) pre-treatment; +MCP, waterlogged conditions with 1-MCP pre-treatment.

*IDs were obtained from the MaizeSequence Database.

†Values are fold change and were the average of three biological replications.

‡Data are from Rajhi et al. (2011). Values are the average fold change of three biological replications.

§IDs were obtained from RAP DB Build 5.

IDs were obtained from the MSU.

**This gene is downregulated under ethylene-treated conditions; all other genes are upregulated.

Tissue-specific transcript profile analysis using LM

Stelar cells, cortical cells and OCLs were isolated from paraffin-embedded sections of maize primary roots using LM for qRT-PCR. Two genes related to transcriptional regulation were selected for qRT-PCR analysis. One of them (GRMZM2G137582) encodes an F-box protein and is highly homologous to an ethylene-inducible EIN3-binding F-box protein 1 (EBF1) gene in arabidopsis. In this study, we designated this gene as EBF1. The other gene (ERF2; GRMZM2G055180) encodes a transcription factor that has an ethylene response factor (ERF) domain. The transcript levels of the EBF1 gene were significantly increased in all of the LM-isolated tissues in response to ethylene under aerobic conditions (Fig. 3A). The transcript levels of the ERF2 gene were significantly increased in all of the LM-isolated tissues, but the increase was greatest in the cortical cells (Fig. 3A).

Fig. 3.

Fig. 3.

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Transcript profiles of selected genes in LM-isolated stelar cells, cortical cells and outer cell layers. Three-day-old maize seedlings were treated with 1 ppm ethylene for 6 h (see key in first graph). Stellar cells (Ste), cortical cells (Cor) and outer cell layers (OCLs) were isolated from 15–20 mm from the root–shoot junction of ethylene-treated maize root using LM. LM-isolated tissues were used for RNA extraction and subsequent qRT-PCR. (A) Relative transcript levels of ethylene signalling-related genes (EBF1 and ERF2). (B) Relative transcript levels of cell wall modification-related genes (CEL, PG, EXPB3 and EXPB1A). (C) Relative transcript levels of proteolysis-related genes (Vignain and MC). Values are means (n = 3) ± s.d. Asterisks indicate a significant difference between samples with and without ethylene treatment (P < 0·05, two-sample t-test).

Cell wall modification enzymes, CEL, PG and EXP, are encoded by gene families (Knowles et al., 1987; Hadfield and Bennett, 1998; Li et al., 2002). Among them, we selected four genes for tissue-specific gene expression analysis: CEL (GRMZM2G147221), PG (GRMZM2G037431) and two EXP genes [GRMZM2G474194 and GRMZM2G082520 (EXPB1A)]. GRMZM2G474194 was described as an uncharacterized protein in the GRAMENE database, but we designated it as EXPB3 according to Wu et al. (2001). The expression of CEL, PG and EXPB3 was confined to the cortical cells under aerobic conditions with ethylene treatment (Fig. 3B). The transcript level of EXPB1A (GRMZM2G082520) was increased in cortical cells and in cells of the OCLs under aerobic conditions with ethylene treatment (Fig. 3B).

Among the proteolysis-related genes, the cysteine proteinase Vignain gene (GRMZM2G456217) and Metacaspase gene (MC; GRMZM2G022799) were selected for qRT-PCR analysis because these genes are thought to be involved in cell death (Gietl and Schmid, 2001; Suarez et al., 2004). Ethylene treatment increased the transcript levels of Vignain and MC specifically in cortical cells under aerobic conditions, but not in stelar cells or cells of the OCLs (Fig. 3C).

Time-course transcript profile analysis in LM-isolated root cortical cells under aerobic conditions with ethylene or under waterlogged conditions

The transcript levels of the EBF1 and ERF2 genes were significantly increased, and peaked within 3 h after the initiation of growth under aerobic conditions with ethylene treatment (Fig. 4A) and at 6 h under waterlogged conditions (Fig. 5A).

Fig. 4.

Fig. 4.

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Time-course transcript profiles of selected genes in LM-isolated maize cortical cells under aerated conditions with ethylene (see key in first graph). Maize primary roots with or without ethylene treatment were collected at 0, 3, 6 and 12 h after treatment. Cortical cells were isolated from the paraffin-embedded sections at 15–20 mm from the root–shoot junction of ethylene-treated maize root using LM. LM-isolated tissues were used for RNA extraction and subsequent qRT-PCR. (A) Relative transcript levels of ethylene signalling-related genes (EBF1 and ERF2). (B) Relative transcript levels of cell wall modification-related genes (CEL, PG, EXPB3 and EXPB1A). (C) Relative transcript levels of proteolysis-related genes (Vignain and MC). Values are means (n = 3) ± s.d. Asterisks indicate a significant difference between samples with and without ethylene treatment (P < 0·05, two-sample, t-test).

Fig. 5.

Fig. 5.

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Time-course transcript profiles of selected genes in LM-isolated maize cortical cells under waterlogged conditions (see key in first graph). Maize primary roots under waterlogged or aerobic conditions were collected at 0, 3, 6 and 12 h after treatment. Cortical cells were isolated from the paraffin-embedded sections at 15–20 mm from the root–shoot junction of waterlogging-treated maize root using LM. LM-isolated tissues were used for RNA extraction and subsequent qRT-PCR. (A) Relative transcript levels of ethylene signalling-related genes (EBF1 and ERF2). (B) Relative transcript levels of cell wall modification-related genes (CEL, PG, EXPB3 and EXPB1A). (C) Relative transcript levels of proteolysis-related genes (Vignain and MC). Values are means (n = 3) ± s.d. Asterisks indicate a significant difference between samples with and without ethylene treatment (P < 0·05, two-sample t-test).

The transcript abundances of CEL and EXPB3 peaked at 6 h under aerobic conditions with ethylene treatment (Fig. 4B), and at 12 h under waterlogged conditions (Fig. 5B). The transcript abundance of PG peaked at 6 h under both conditions (Figs 4B and 5B). The transcript level of EXPB1A significantly increased at 6 h after initiation of ethylene treatment under aerobic conditions (Fig. 4B) but it did not significantly increase at 12 h after the initiation of growth under waterlogged conditions (Fig. 5B).

The increase of Vignain and MC transcripts was first observed at 6 h after the initiation of ethylene treatment under aerobic conditions (Fig. 4C). Their transcript levels were not significantly increased at 12 h after growth under waterlogged conditions (Fig. 5C).

Effect of DPI treatment on ethylene-induced aerenchyma formation and gene expression at 15–20 mm from the root–shoot junction

We investigated the effect of DPI treatment on ethylene-induced aerenchyma formation to examine whether NADPH oxidase-mediated ROS production was involved in the ethylene-induced lysigenous aerenchyma formation in maize primary roots. Three-day-old aerobically grown seedlings were transferred to aerobic conditions with 1 ppm ethylene together with 0, 20 and 40 μm DPI (Fig. 6). Root elongation was significantly inhibited by ethylene or DPI treatment (Supplementary Data Fig. S1). Aerenchyma formation was significantly inhibited by DPI treatment in a dose-dependent manner (Fig. 6). The percentage of aerenchyma formation was almost the same in roots with 1 ppm ethylene and 40 μm DPI treatments as in roots without either treatment (Fig. 6).

Fig. 6.

Fig. 6.

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Inhibition of ethylene-induced aerenchyma formation by DPI treatment. Three-day-old maize seedlings were transferred to agar plates containing 0, 20 and 40 μm DPI, and grown for 24 h under 1 ppm ethylene. (A) Cross-sections of primary root at 15–20 mm from the root–shoot junction. Asterisks shows aerenchyma in cortical cells. Scale bar = 200 μm. (B) The ratio of aerenchyma was expressed as a percentage of the cross-section at 15–20 mm from the root–shoot junction. Values are means (n = 8) ± s.d. Different lower case letters indicate a significant difference among all the conditions (P < 0·05, one-way ANOVA and then Tukey’s test for multiple comparisons).

To determine whether DPI affects the expression of the ethylene-inducible genes during lysigenous aerenchyma formation in maize primary roots, total RNA was extracted from root segments at 15–20 mm from the root–shoot junction. The transcript levels of these genes were determined by qRT-PCR. The transcript levels of PG, EXPB3 and EXPB1A were increased by 1 ppm ethylene, and the increase was suppressed by 40 μm DPI (Fig. 7B). On the other hand, the transcript levels of EBF1, ERF, CEL, Vignain and MC were increased by ethylene treatment, but the increase was not suppressed by DPI treatment (Fig. 7A–C).

Fig. 7.

Fig. 7.

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Effect of DPI treatment on the transcript abundance of selected genes. Three-day-old maize seedlings were transferred to agar plates containing 0 or 40 μm DPI. Seedlings were grown for 6 h with or without ethylene. RNA for qRT-PCR was extracted from root segments from 15–20 mm below the root–shoot junction in maize primary roots. (A) Relative transcript levels of ethylene signalling-related genes (EBF1 and ERF2). (B) Relative transcript levels of cell wall modification-related genes (CEL, PG, EXPB3 and EXPB1A). (C) Relative transcript levels of proteolysis-related genes (Vignain and MC). Values are means (n = 4) ± s.d. Different lower case letters indicate a significant difference among all the conditions (P < 0·05, one-way ANOVA and then Tukey’s test for multiple comparisons).

DISCUSSION

Aerenchyma formation at the basal part of the root

Ethylene-induced aerenchyma formation was observed at 10–40 mm from the root–shoot junction, but not at 25–30 mm from the root tip in maize primary root (Fig. 1B), even though all positions of the root were evenly exposed to ethylene. This suggests that the basal region of the root (at 10–40 mm from the root–shoot junction) is more responsive to ethylene than the apical region (at 25–30 mm from the root tip).

A small amount of aerenchyma was formed at 5–10, 15–20 and 25–30 mm from the root–shoot junction of the root without ethylene treatment (Fig. 1B). For this experiment, the maize seedlings were placed in a tightly closed container with or without ethylene treatment. Ethylene-induced aerenchyma formation might have occurred even in the absence of ethylene treatment, because putting the plants in the container may have slightly increased the endogenous ethylene contents by reducing the rate of ethylene loss.

Aerenchyma formation and lateral root emergence in maize roots were increased in response to a lower phosphorus status in soil (Postma and Lynch, 2011), suggesting that there is a relationship between aerenchyma formation and lateral root emergence (Postma and Lynch, 2011; York et al., 2013). Ethylene is involved not only in aerenchyma formation, but also in lateral root initiation and emergence (reviewed by Jung and McCouch, 2013). High concentrations of ethylene inhibit lateral root initiation, whereas low concentrations promote lateral root initiation. On the other hand, high concentrations of ethylene promote lateral root emergence (reviewed by Jung and McCouch, 2013). Indeed, in the tropical forage grass Brachiaria humidicola, waterlogging (which increases endogenous ethylene concentrations in plant roots) reduced the number of lateral roots, whereas it increased the lengths of the lateral roots at the basal parts of nodal roots (Cardoso et al., 2014). Thus, differences in quantities of ethylene and/or responsiveness to ethylene between air-grown and waterlogged roots or between the basal and apical parts of roots may affect aerenchyma formation as well as lateral root initiation and emergence.

Effects of aerobic conditions with ethylene treatment and waterlogged conditions on the timing of aerenchyma formation

The initiation of aerenchyma formation was earlier under aerobic conditions with ethylene treatment than under waterlogged conditions, as the initiation of aerenchyma formation was first observed at 12 h after the start of ethylene treatment (Fig. 1C) and at 24 h after the start of growth under waterlogged conditions (Rajhi et al., 2011). The ethylene treatment suddenly exposed maize seedlings to a high concentration of ethylene. On the other hand, under waterlogged conditions, ethylene may gradually accumulate in roots through the prevention of gas diffusion (Visser and Voesenek, 2004), and through the activation of ethylene biosynthesis (Atwell et al., 1988; He et al., 1996a). Therefore, transmission of the ethylene signal to the cortical cells might be faster under aerobic conditions with ethylene treatment than under waterlogged conditions. This may explain why induction of lysigenous aerenchyma formation in maize primary roots takes longer under waterlogged conditions than under aerobic conditions with ethylene treatment.

Rajhi et al. (2011) identified genes whose transcript levels were increased or decreased during lysigenous aerenchyma formation under waterlogged conditions, and whose transcript profile changes were suppressed by an ethylene perception inhibitor, 1-MCP. In this study, we identified additional genes that responded to ethylene under aerobic conditions. The hierarchical clustering analysis of the selected genes showed that the expression patterns of the genes that responded to ethylene treatment were similar to those of the genes that responded to 1-MCP treatment, but not similar to those of the genes that responded to the waterlogging treatment (Fig. 2C). These results are in agreement with the finding that only 26 % of upregulated and 52 % of downregulated genes identified in this study were regulated similarly in response to endogenous ethylene under waterlogged conditions (Fig. 2A, B). This result suggests that the ethylene-responsive genes are not necessarily responsive to waterlogging treatment. Alternatively, the difference in some transcript profiles between this study and those found in a previous study may also be due to the difference in the timing of initiation of aerenchyma formation between under aerobic conditions with ethylene treatment and under waterlogged conditions (Fig. 1C; Rajhi et al., 2011). Indeed, the increases in the EXPB1A, Vignain and MC transcripts under waterlogged conditions were delayed when compared with the increases under aerobic conditions with ethylene treatment (Figs 4B, C and 5B, C). The present results, together with the previous results (Rajhi et al., 2011), help to narrow down of the number of candidate genes involved in ethylene-induced lysigenous aerenchyma formation in maize primary root.

Control of cortical cell-specific aerenchyma formation

The transcript level of EBF1 increased under waterlogged conditions, and the increase was suppressed by 1-MCP pre-treatment (Fig. 5A; Table 1). Moreover, its transcript levels were increased by ethylene treatment, even under aerobic conditions (Fig. 4A; Table 1), suggesting that expression of EBF1 is an indicator of ethylene perception. The expression of EBF1 was stimulated by ethylene in all of the LM-isolated tissues (Fig. 3A), indicating that stelar cells, cortical cells and OCLs in roots were evenly exposed to ethylene. The transcript levels of ERF2 were significantly increased by ethylene in all of the LM-isolated tissues, but the level was the highest in the cortical cells (Fig. 3A). On the other hand, the transcript levels of genes encoding cell wall modification- and proteolysis-related genes were specifically increased in cortical cells during lysigenous aerenchyma formation in maize primary root under aerobic conditions with ethylene treatment (Fig. 3B, C). As ethylene can easily diffuse to the adjacent cells, these findings suggest that all types of tissues/cells are evenly exposed to ethylene, but that downstream regulation of gene expression through ethylene signalling is dependent on the states of each tissue/cell type. Indeed, only the cortical cells expressed PCD-related genes and formed lysigenous aerenchyma in response to ethylene.

Possible roles of cell wall degradation- or cell lysis-related gene products in lysigenous aerenchyma formation

In the final stage of aerenchyma formation, the cell wall is degraded enzymatically (Evans, 2003). Ethylene increased the transcript abundance of CEL and PG (Figs 3B and 4B). These results are consistent with previous finding that the increase of CEL activity was preceded by increases in the activities of ethylene biosynthesis enzymes (He et al., 1996a). In addition, the transcript level of EXPB3 was increased in cortical cells by ethylene (Figs 3B and 4B) and waterlogging treatment (Fig. 5B), and EXP is thought to be involved in CEL activity against glucan (Cosgrove, 2000, 2005). The finding that ethylene and waterlogging induced ethylene signalling genes earlier than cell wall modification enzymes in cortical cells of maize roots (Figs 4A, B and 5A, B, respectively) suggests that the genes encoding cell wall modification enzymes are regulated by transcription factors involved in ethylene signalling during lysigenous aerenchyma formation.

As in the case with cell wall degradation, cell lysis is observed in the final stage of lysigenous aerenchyma formation (Evans, 2003). Vignain encodes a papain-type cysteine endopeptidase (CysEP) that has an endoplasmic reticulum (ER) retention signal (HDEL) at the C-terminus. CycEP is synthesized in senescing cells in various organs, such as the endosperm during seed germination, and flower petals (Schmid et al., 1999). CycEP is thought to be involved in the degradation of cytosolic components during PCD in Ricinus communis (Gietl and Schmid, 2001). Similarly, the gene encoding vignain identified in this study may play a role in the lysis of cellular components during lysigenous aerenchyma formation. Plant metacaspases are classified into two types based on their structures (Tsiatsiani et al., 2011). Type II metacaspase in Picea abies (mcII-Pa) is responsible for PCD during embryogenesis, and the downregulation of the expression or treatment with mcII-PA inhibitor suppressed PCD (Suarez et al., 2004; Bozhkov et al., 2005). AtMC8 and AtMC9 of arabidopsis are type II metacaspases (Vercammen et al., 2004; He et al., 2008). AtMC8 is involved in UV- and H2O2-induced cell death (He et al., 2008), and AtMC9 is involved in cell death during xylem formation (Bollhöner et al., 2013). Interestingly, the MC gene identified in this study is homologous to AtMC9 (48 % identity, 59 % similarity). These observations suggest that Vignain and MC are involved in ethylene-induced lysigenous aerenchyma formation.

Involvement of ROS in ethylene-induced aerenchyma formation

During lysigenous aerenchyma formation in rice under submerged conditions, NADPH oxidase plays a role in the accumulation of H2O2 and subsequent cell death in leaf sheaths (Parlanti et al., 2011) and internodes (Steffens et al., 2011). In both of these studies, these changes were prevented by the treatment with the NADPH oxidase inhibitor DPI. DPI also reduces lysigenous aerenchyma formation under waterlogged conditions in maize primary root (Yamauchi et al., 2011) and wheat seminal root (Yamauchi et al., 2014). In this study, aerenchyma formation was significantly inhibited by DPI treatment in a dose-dependent manner (Fig. 6), indicating that NADPH oxidase-mediated ROS production may also contribute to the ethylene-induced aerenchyma formation in maize primary root even under aerobic conditions. DPI treatment suppressed the ethylene-induced increase in PG, EXPB3 and EXPB1A transcripts, but did not affect the transcript abundance of other genes (EBF1, ERF2, CEL, Vignain and MC), during lysigenous aerenchyma formation (Fig. 7B), suggesting that there are both ROS signalling-dependent and independent regulatory pathways for ethylene-induced aerenchyma formation.

Previously, we demonstrated that the gene encoding ZmRBOH was significantly upregulated and that the gene encoding ZmMT, a cysteine-rich metallothionein that has ROS-scavenging activity, was downregulated in cortical cells of maize primary roots at 12 h after the initiation of growth under waterlogged conditions (Rajhi et al., 2011; Yamauchi et al., 2011). However, in this study, we did not find any RBOH and MT gene homologues among the genes up- or downregulated by ethylene under aerobic conditions, although we cannot rule out the possibility that their expression changes are regulated transiently. In seminal roots of wheat seedlings, the transcript levels of three copies of TaRBOH genes were not drastically affected by pre-treatment with the ethylene precursor ACC under aerobic conditions (Yamauchi et al., 2014). However, their transcript levels were strongly increased in seminal roots of wheat seedlings with ACC pre-treatment during subsequent growth under waterlogged conditions (Yamauchi et al., 2014). Moreover, waterlogging increased the transcript level of ZmRBOH approx. 100-fold, but this upregulation was not completely inhibited by pre-treatment with 1-MCP (it dropped to one-tenth the level observed in waterlogging) (Rajhi et al., 2011). The expression of ZmRBOH might be induced by oxygen deficiency and might be further enhanced by the effect of gradually accumulated ethylene under waterlogged conditions.

CONCLUSIONS

Several genes related to cell wall modification and proteolysis are specifically up- or downregulated in cortical cells during lysigenous aerenchyma formation under aerobic conditions with ethylene treatment. However, the molecular mechanisms underlying the tissue-specific occurrence of the PCD events during lysigenous aerenchyma formation remain unclear. Although stelar cells, cortical cells and OCLs in root were exposed to and perceived ethylene evenly, the expression of genes related to cell wall modification and proteolysis was most strongly increased in cortical cells. Thus, cortical cell-specific factors downstream of ethylene perception may control the ethylene-induced aerenchyma formation through the subsequent gene expression in cortical cells.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Table S1: genes upregulated by ethylene. Table S2: genes downregulated by ethylene. Table S3: primer sequences for quantitative RT-PCR. Figure S1: effect of DPI treatment on root elongation.

Supplementary Data
supp_115_6_879__index.html (1.2KB, html)

ACKNOWLEDGEMENTS

We thank Drs A. Oyanagi, K. Kawaguchi, Y. Mano, F. Abe, M. Obara, T. Abiko, H. Mori and S. Nishiuchi for stimulating discussions, and Dr Y. Nagamura and Ms T, Motoyama for their support with the microarray experiments. This work was partly supported by a grant from the Bio-oriented Technology Research Advancement Institution (Promotion of Basic Research Activities for Innovative Biosciences), a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan, and grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Supplementary Materials

Supplementary Data
supp_115_6_879__index.html (1.2KB, html)
supp_mcv018_aob-14730-s01.pdf (73.4KB, pdf)
supp_mcv018_aob-14730-s03.pdf (51.8KB, pdf)
supp_mcv018_aob-14730-s02.xlsx (33.7KB, xlsx)
supp_mcv018_aob-14730-s04.xlsx (35KB, xlsx)

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