Phenotype of SGA in triploid wheat hybrids
Causal genes for the abnormal growth phenotypes observed in triploid hybrids with Ldn are widely distributed in A. tauschii (Mizuno et al., 2010). We previously identified five A. tauschii accessions that induce SGA in triploid hybrids with Ldn (Table 1, Fig. 1a,b); three from Azerbaijan, and two were originally collected from Dagestan and Iran (Mizuno et al., 2010). No phenotypic differences were observed among the resulting triploid hybrids exhibiting SGA. In this study, we additionally produced three triploid hybrids that were crossed with two tetraploid wheat accessions, T. turgidum ssp. dicoccoides KU-8736A and T. turgidum ssp. carthlicum KU-138. The three hybrid plants, KU-8736A × A. tauschii KU-2110, KU-8736A × A. tauschii IG47182, and KU-138 × A. tauschii IG47182, exhibited SGA during the winter (January) after the seeds were sown in November (Fig. 1c). All hybrid plants with the SGA phenotype extended two or three leaves without tillering until the spring, and then they died (Fig. 1a,b). Therefore, no offspring were obtained from any hybrid plants displaying SGA. It has previously been reported that high growth temperatures suppress necrotic symptoms more effectively than normal growth temperatures in hybrid necrosis of common wheat, Arabidopsis, and lettuce (Dhaliwal et al., 1986; Bomblies et al., 2007; Jeuken et al., 2009). However, the appearance of the SGA phenotype was also confirmed under high-temperature conditions (30°C) using a plant growth chamber. The SGA phenotype was not alleviated under high-temperature conditions, and the appearance of SGA was temperature-independent.
Figure 1. Photographs of triploid hybrid plants exhibiting severe growth abortion (SGA). (a, b) Photos of SGA triploid plants produced from a cross between Ldn and IG47182, and between Ldn and KU-2110 taken in January and March, respectively. The seeds of these plants were sown during the previous November and grown in a glasshouse. (c) Comparison of wildtype (WT) and SGA triploid plants in February. The white arrow indicates the SGA triploid plant derived from a cross between Triticum turgidum ssp. carthlicum KU-138 and Aegilops tauschii IG47182.
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TEM observation of leaf mesophyll cells
Reactive oxygen species (ROS) generation has been observed in triploid wheat hybrids with type II and III necrosis, as well as wheat and Nicotiana plants undergoing hybrid necrosis (Mino et al., 2002; Sugie et al., 2007; Mizuno et al., 2010, 2011). To study the detailed cytological and physiological changes associated with SGA, the intracellular structures of mesophyll cells were compared among triploid hybrids formed from crosses between Ldn and PI476874 (WT), Ldn and KU-2025 (type II necrosis), and Ldn and IG47182 (SGA) using TEM. For ROS detection under TEM observation, CeCl3 was added to the fixation solution as cerium pretreatment is a highly sensitive procedure for localizing intracellular H2O2. ROS detection is based on the reaction between H2O2 and CeCl3 to produce insoluble precipitates of highly electron-dense cerium perhydroxides (Bestwick et al., 1997; Mizuno et al., 2010).
Living mesophyll cells containing large vacuoles and several chloroplasts were mainly observed in the leaves of WT hybrids and the proximal parts of leaf blades of SGA hybrid plants (Fig. 2a,c). In the living cells of SGA-exhibiting plants, black deposits corresponding to cerium signals were frequently observed within intercellular spaces, indicating the accumulation of H2O2 (Fig. 2c). Numerous fat droplets were also detected within the chloroplasts of these cells (Fig. 2d). In addition, the granum-lamella structure of living cells appeared abnormal, and the linear arrangement of grana was frequently entangled within the chloroplasts of SGA hybrid plants.
Figure 2. Transmission electron microscope observation of mesophyll cells in seedling leaves of triploid hybrid plants. (a) Wildtype (WT) triploid plant. (b) Type II necrosis triploid plant grown at under normal temperature conditions (23°C). (c–e) Severe growth abortion (SGA) triploid plants. Arrows in panel (d) indicate fat droplets. (f) Survival rates of mesophyll cells in the WT and SGA triploid hybrids. For each hybrid, the percentage of living cells was counted in the proximal and distal parts of seedling leaves. Bars, 10 μm (a–c, e); 1 μm (d). Mean values with the same letters in (f) were not significantly different (P > 0.05) (Tukey–Kramer HSD test).
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Plasmolysis, cell membrane disruption, collapse of vacuoles, and organelle degradation were typical characteristics of the dead cells in WT and SGA hybrid plants (Fig. 2b,e). Only a small number of dead cells were detected in the leaf blades of WT plants, and in the proximal parts of leaf blades of SGA plants (Fig. 2f). Although dead mesophyll cells were detected in the distal parts of leaf blades in SGA plants, the color of the leaves remained green (Fig. 2e). The degradation of intracellular structures in the dead cells of SGA plants resembled that seen in type II necrosis plants grown under normal temperature conditions (23°C) (Fig. 2b). Taken together, these observations implied that ROS generation occurred in cells before death.
Alteration of gene expression profiles in crown tissue
To comprehensively compare gene expression profiles between WT (cross between Ldn and PI476874) and SGA (cross between Ldn and IG47182) triploid hybrids, transcriptome analysis was performed using a wheat-specific 38k oligo DNA microarray (Kawaura et al., 2008). For hybridization, total RNA was extracted from crown tissues, consisting of the basal tissue of culms and tillers, shoot apices, and shoot apical meristems (SAMs), of 3-wk-old seedlings grown at normal temperature (23°C). After hybridization with the RNA samples, probes showing at least a threefold difference in signal intensity compared with the WT were defined as either up- or down-regulated genes.
Of the 37 826 probes on the wheat microarray, 3483 (9.2%) and 1914 (5.1%) probes were regarded as up- and down-regulated genes, respectively, in the crown tissues of SGA plants. Based on homology searches of the wheat EST database with probe sequences, 1986 (57.0%) and 1173 (61.3%) of the up- and down-regulated genes, respectively, were categorized into a total of 13 groups based on inferred function (Fig. 3). Of the up-regulated genes, defense-related genes were the most frequently (20.9%) encountered among the 13 groups (Fig. 3; Table S2). Genes related to signal transduction (10.7%), transport (9.7%), and metabolism (8.6%) were also abundantly expressed in the SGA hybrid plants. By contrast, genes related to photosynthesis (35.8%), stress (9.4%), defense (8.8%), and protein synthesis (7.8%) were down-regulated in the SGA plants.
Figure 3. Summary of the microarray analysis for the crown tissues of wildtype (WT) and severe growth abortion (SGA) triploid hybrid plants. A total of 1986 up-regulated (closed bars) and 1173 down- regulated (open bars) genes were classified into 13 functional categories.
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Among the defense-related genes, pathogenesis-related (PR), defensin, chitinase, and peroxidase genes were highly up-regulated in the SGA hybrids (Table S2). In addition, transcripts of a number of disease-resistance genes, including Xa1 homolog and NBS-LRR-type protein genes, accumulated abundantly in the SGA plants. Thus, the up-regulation of a number of defense-related genes was characteristic of triploid hybrids with an SGA phenotype. Moreover, many WRKY-type transcription factor genes were among the up-regulated transcription factor genes in SGA hybrids (Table 2).
Table 2. List of the top 20 up-regulated transcription factor genes in the crown tissue of the severe growth abortion (SGA) triploid hybrid identified by microarray analysis
|Accession no.||Protein||Log2 ratio||E-value|
|EU665440||Triticum aestivum WRKY11 transcription factor||8.11||2.00E−48|
|EU665449||Triticum aestivum WRKY25 transcription factor||8.08||2.00E−75|
|EF488104||Hordeum vulgare WRKY3 transcription factor||6.82||0|
|EF368363||Triticum aestivum WRKY72b transcription factor||5.74||0|
|D38111||Triticum aestivum HBP-1a transcription factor||5.47||8.00E−07|
|AY530950||Zea mays putative zinc finger protein (Z428D03.1)||5.16||3.00E−84|
|AB295664||Triticum aestivum WLHS1-D MADS-box protein||4.58||0|
|EU253554||Triticum aestivum C2H2 zinc finger protein (ZFP2)||4.56||E−163|
|AK107555||Oryza sativa bHLH domain containing protein||4.44||2.00E−62|
|AK073100||Triticum aestivum WRKY66 transcription factor||3.66||2.00E−15|
|EF397613||Triticum aestivum WRKY45 transcription factor||3.51||E−114|
|EU977051||Zea mays CCCH transcription factor||3.50||7.00E−12|
|EU957863||Zea mays nuclear transcription factor Y subunit A-10||3.33||1.00E−11|
|AK106488||Oryza sativa Heat shock transcription factor 29||3.30||4.00E−17|
|AK228110||Arabidopsis thaliana bHLH transcription factor||3.13||7.00E−14|
|AK102203||WUSCHEL-related homeobox 5 protein||3.04||E−104|
|AB028187||Oryza sativa NAC8 protein||2.94||8.00E−89|
|DQ146423||Triticum monococcum VRN1 MADS-box protein||2.93||1.00E−29|
|EU973471||Zea mays myb-related protein Myb4||2.78||3.00E−99|
|AY676928||Oryza sativa WRKY99 transcription factor||2.77||1.00E−12|
Among the 13 gene groups, photosynthesis-related genes showed the highest rate of down-regulation (Fig. 3), particularly in SGA hybrids, with the genes for ribulose-1,5-bisphosphate carboxylase (RuBisCo), chlorophyll a/b binding protein, and RuBisCo activase also included in these groups (Table S3). In addition, numerous transcription factor genes functioning in SAM and leaf primordia were down-regulated in SGA hybrids (Table 3). Particularly, the expression of gene homologs encoding KNOTTED1 (KN1)-homeobox, Myb-domain, and NAC-domain transcription factors, which are significantly associated with SAM function (Fletcher & Meyerowitz, 2000; Veit, 2004), was suppressed in the crown tissues of SGA plants. Abiotic stress-related transcription factor genes, such as wheat LIP19 bZIP and ethylene-responsive transcription factor genes, were also down-regulated in SGA crown tissues.
Table 3. List of the top 20 down-regulated transcription factor genes in the crown tissue of severe growth abortion (SGA) triploid hybrids identified by microarray analysis
|Accession no.||Protein||Log2 ratio||E-value|
|EU963396||Zea mays zinc finger protein CONSTANS-like16||−4.21||8.00E−52|
|EU971309||Zea mays WRKY74 transcription factor||−3.85||6.00E−10|
|AF470059||Sorghum bicolor P-type R2R3 Myb protein (Myb9)||−3.46||4.00E−78|
|AY062179||Oryza sativa AINTEGUMENTA-like protein||−3.20||2.00E−07|
|AY914051||Triticum aestivum leucine zipper protein (zip1)||−2.87||0|
|AY625683||Triticum aestivum NAC2 transcription factor||−2.68||0|
|AB334128||Triticum aestivum WLIP19d transcription factor||−2.61||0|
|CAE53909||Triticum aestivum SWIM Zn-finger protein||−2.60||2.44E−44|
|AJ575665||Triticum aestivum RAFTIN1a anther protein||−2.55||0|
|EU091320||Avicennia marina Myb transcription factor (MYB1)||−2.54||1.00E−68|
|ABA99796||Oryza sativa bZIP transcription factor||−2.46||2.10E−14|
|EU956097||Zea mays nuclear transcription factor Y subunit B-3||−2.41||E−160|
|AP009567||Hordeum vulgare ethylene-responsive transcription factor||−2.32||2.00E−17|
|AJ303355||Hordeum vulgare MCB2 protein||−2.08||0|
|DQ317421||Chasmanthium latifolium KN1 homeodomain protein||−2.05||2.00E−44|
|AB182944||Triticum aestivum WKNOX1b homeodomain protein||−2.03||3.00E−39|
|FJ024049||Zea mays MYB-like protein E1 (MYBE1)||−2.02||1.00E−21|
|AM500853||Hordeum vulgare NAC transcription factor||−2.00||6.00E−06|
|X68600||Hordeum vulgare pZE40||−1.94||E−111|
|Z95771||Arabidopsis thaliana MYB47 R2R3-Myb transcription factor||−1.94||3.00E−14|
To validate the microarray data, RT-PCR and quantitative RT-PCR analyses for 23 selected genes (13 up-regulated and 10 down-regulated genes) were conducted using the two triploid hybrids employed in the microarray analysis. Also, four additional triploid hybrids, consisting of two WT hybrids from crosses between Ldn and KU-2022, and between Ldn and KU-2059, and two SGA hybrids obtained from crossing Ldn with the A. tauschii accessions IG47188 and IG120866, were also included in the RT-PCR and quantitative RT-PCR analyses. Of the 23 genes from the 10 gene categories examined by RT-PCR, the gene expression levels of only two down-regulated genes were inconsistent with the microarray data (Figs S1, S2). For the other 21 genes (91.3%), differences in transcript accumulation levels between the crown tissues of the two triploid hybrids used in the microarray analysis corresponded closely to the microarray results. Moreover, comparison of the 21 gene expression levels among the six triploid hybrids indicated that the differences were consistent with the phenotypic differences between WT and SGA. Therefore, the RT-PCR analyses generally supported the microarray analysis results.
Comparison of gene expression profiles between hybrids displaying SGA and hybrid necrosis
The up-regulation of defense-related genes and down-regulation of photosynthesis-associated genes in SGA hybrids were also commonly observed in hybrid plants displaying types II and III hybrid necrosis, as reported elsewhere (Mizuno et al., 2010, 2011). To characterize the cellular responses associated with SGA in wheat triploid hybrids in greater detail, the expression levels of defense- and photosynthesis-related genes were compared between triploid hybrids with SGA and the two types of hybrid necrosis phenotypes. First, the microarray probes of defense-related genes (n = 409) that were up-regulated in the crown tissues of SGA hybrids were selected and their signal intensities were compared with those obtained from previous comparative studies examining gene expression in the leaves of WT and type III necrosis hybrids and the crown tissues of WT and type II necrosis hybrids under low-temperature conditions (Mizuno et al., 2010, 2011). A significant positive correlation was detected between the gene expression profiles in the leaves of type III necrosis hybrid lines and the crown tissues of SGA hybrids (r = 0.2197, P < 0.001), whereas no positive correlation was observed between the crown tissues of type II necrosis and SGA plants (Table 4). Next, we selected and compared the expression of three defense-related genes, PR1, phenylalanine ammonia lyase (PAL), and WRKY3, among four triploid hybrids using quantitative RT-PCR (Fig. 4). Although type II necrosis plants and SGA hybrid plants accumulated transcripts more abundantly than WT plants, significant differences in expression levels were observed between the crown tissues of SGA hybrids and those of low temperature-treated type II necrosis. Similarly, the 267 and 316 defense-related probes that were up-regulated in types III and II necrosis hybrids, respectively, were compared with the microarray data of the SGA-exhibiting plants, and significant positive correlations were detected in both comparisons (Table 4). In the crown tissues of SGA hybrids, the up-regulation of defense-related genes displayed a similar pattern to the leaves of type III necrosis hybrids.
Table 4. Comparison of gene expression profiles among triploid hybrids with severe growth abortion (SGA), type II necrosis, and type III necrosis phenotypes
|Query||Number of probes||Target expression profile||Correlation coefficient|
|Defense-related genes up-regulated|
| In SGA vs WT||409||Type III necrosis vs WTa||0.2197***|
| In SGA vs WT||409||Type II necrosis vs WTb||0.0783|
| In type III necrosis vs WTa||267||SGA vs WT||0.3339***|
| In type II necrosis vs WTb||316||SGA vs WT||0.2955**|
|Photosynthesis-related genes down-regulated|
| In SGA vs WT||420||Type III necrosis vs WTa||−0.3903***|
| In SGA vs WT||420||Type II necrosis vs WTb||0.2447***|
| In type III necrosis vs WTa||14||SGA vs WT||0.2330|
| In type II necrosis vs WTb||51||SGA vs WT||0.2217|
Figure 4. Comparison of transcript accumulation levels in the crown tissues of wildtype (WT), severe growth abortion (SGA), and type II necrosis triploid hybrids. Quantitative reverse transcriptase (RT)-PCR analyses of three defense-related genes and Wknox1 were conducted. The transcript abundances are shown as mean values relative to the WT triploid, F1 of Ldn and KU-2059. Means ± SD were calculated from the results of quantitative RT-PCR experiments performed in triplicate. The actin gene was used as an internal control. Student’s t-test was used to test for statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001) between SGA and type II necrosis hybrids.
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A markedly larger number of probes (n = 420) for photosynthesis-related genes were down-regulated in the crown tissues of SGA plants than in the leaves of type III necrosis lines (n = 14) and the crown tissues of type II necrosis lines under low-temperature conditions (n = 51). Comparison of the signal intensities between these down-regulated photosynthesis-related probes in the tissues of the SGA, type III necrosis, and type II necrosis hybrids revealed a significant positive correlation between the expression profiles of the crown tissues in the type II necrosis and SGA hybrids (Table 4). However, the correlation between the leaves in the type III necrosis lines and the crown tissues of SGA plants was negative. Moreover, no significant correlation was observed between the 14 and 51 photosynthesis-related probes that were down-regulated in the types III and II necrosis hybrids, respectively. These results imply that the profile of down-regulated photosynthesis-related genes in SGA triploid hybrids was quite different from that in types II and III necrosis plants.
Comparison of photosynthetic activity among triploid hybrids
To examine the effects of down-regulation of photosynthesis-related genes, photosynthetic activity was compared among triploid hybrids produced from crosses of Ldn and KU-2069, Ldn and PI476874, and Ldn and KU-2022 for WT plants, and crosses of Ldn and IG47188, Ldn and IG47182, and Ldn and IG120866 for SGA plants. The photosystem II (PSII) activity of the SGA-exhibiting hybrids was significantly reduced in the first leaves of 3-wk-old seedlings compared with that of the WT hybrid plants (Fig. 5), which is consistent with the down-regulation of photosynthesis-related genes in SGA.
Figure 5. Comparison of chlorophyll fluorescence between wildtype (WT) and severe growth abortion (SGA) triploid hybrids. The first leaves of 3-wk-old seedlings were incubated in the dark and then used to calculate the ratio of variable to maximum fluorescence (Fv/Fm). Means ± SD were calculated from data in 10 experiments. Mean values followed by the same letters are not significantly different (P < 0.05) (Tukey–Kramer’s honestly significant difference (HSD) test).
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Suppression of meristematic activity in SGA
In the microarray analysis, we found that several transcription factor genes associated with SAM function were down-regulated in the crown tissues of SGA plants. One of the identified genes, a wheat KN1-type homeobox gene, Wknox1, has been reported to play a developmentally important role in the SAM of wheat, similar to that of maize KN1 and rice OSH1 (Takumi et al., 2000; Morimoto et al., 2005, 2009). Thus, we compared the accumulation levels of the Wknox1 transcript in the crown tissues of the four triploid hybrids by quantitative RT-PCR. Although no significant difference was observed in Wknox1 expression levels between the WT and type II necrosis plants, expression levels were significantly reduced in the crown tissues of SGA-exhibiting plants (Figs 4, S2).
Dramatic repression of cell cycle-related genes has been reported in the crown tissues of type II necrosis lines, which exhibit growth inhibition at low temperatures (Mizuno et al., 2011). Since we observed that growth inhibition was more severe in plants with an SGA phenotype than in type II necrosis plants, RT-PCR and quantitative RT-PCR analyses were performed to compare the transcript accumulation levels of seven cell cycle-related genes in the crown tissues of three WT and three SGA triploid hybrids. Compared with WT plants, transcript accumulation levels of the seven genes were significantly reduced in SGA plants (Fig. 6a). In particular, transcripts of B-type cyclin, replication protein A1, lethal(2)denticleless-like protein, and histone H4 genes were not detected in the crown tissues of the three SGA hybrids assayed by RT-PCR.
Figure 6. Repression of cell cycle-related gene expression in the shoot apices of wildtype (WT) and severe growth abortion (SGA) triploid hybrids. (a) Reverse transcriptase (RT)-PCR and quantitative RT-PCR analyses of seven cell cycle-related genes. The transcript abundances are shown as mean values relative to the WT triploid (F1 of Ldn and PI476874), and the relative values of transcript accumulation levels are indicated below the electropherograms of RT-PCR products. Means ± SD were calculated from the results of quantitative RT-PCR experiments performed in triplicate. The ubiquitin gene was used as an internal control. Student’s t-test was used to test for statistical significance (*, P < 0.05; **, P < 0.01) between WT and SGA hybrids. (b, c) In situ hybridization of histone H4 mRNA in the longitudinal sections of shoot apices. Bars, 100 μm.
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To examine mitotic cell division activity in the SAM of SGA hybrids, we first prepared longitudinal sections of shoot apices from the seedlings of WT and SGA plants, and then performed in situ mRNA hybridization analysis using a histone H4 antisense probe. The histone H4 gene is specifically expressed in the S phase of the cell cycle, and histone H4 mRNA levels have been used to study cell division activity (Gaudin et al., 2000; Yamaguchi et al., 2010). The size of shoot apices that included SAM from the SGA triploid hybrids (89.63 ± 2.32) was smaller than that of WT triploid plants (120.47 ± 5.93) (Fig. 6b–d), and the difference was statistically significant (Student’s t-test; P = 0.0011). The histone H4 mRNA signals were nonuniformly distributed in the shoot apices of WT hybrid plants derived from a cross between Ldn and PI476874 (Fig. 6b). By contrast, no histone H4-expressing cells were observed in the SAM of SGA-exhibiting hybrid plants produced from a cross between Ldn and IG47182, although a similar nonuniform distribution of the histone H4 signal was found in the developing and elongating leaves of SGA plants (Fig. 6c,d). These results indicated that mitotic cell division activity is strongly suppressed in the SAM of SGA hybrid plants.
In addition, total RNA was extracted from seedlings including the crown tissues at the early developmental stage (Fig. 7a), and expression levels of the cell cycle-related genes were compared between triploid hybrids produced from crosses of Ldn and PI476874 for WT plants, and crosses of Ldn and IG47182 for SGA plants. At the early developmental stage, no SGA symptom was clearly observed, and the seedling phenotype of the SGA plants was similar to that of WT triploid plants. Compared with WT plants, transcript accumulation levels of B-type cyclin and histone H4 genes were significantly reduced in SGA plants, whereas no significant reduction of other gene expression levels was observed (Fig. 7b). These results implied that transcriptional repression of B-type cyclin and histone H4 occurs before appearance of SGA symptom.
Figure 7. Comparison of cell cycle-related gene expression levels in wildtype (WT) and severe growth abortion (SGA) triploid hybrids at the early developmental stage. (a) Overview of the young seedlings of WT and SGA triploid hybrids for the comparative expression analysis. Bar, 1 cm. (b) Reverse transcriptase (RT)-PCR and quantitative RT-PCR analyses of seven selected genes. The transcript abundances are shown as mean values relative to the WT triploid (F1 of Ldn and PI476874), and the relative values of transcript accumulation levels are indicated below the electropherograms of RT-PCR products. Means ± SD were calculated from the results of quantitative RT-PCR experiments performed in triplicate. The Actin gene was used as an internal control. Student’s t-test was used to test for statistical significance (*, P < 0.05; **, P < 0.01) between WT and SGA hybrids.
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