Accelerated Senescence and Enhanced Disease Resistance in Hybrid Chlorosis Lines Derived from Interspecific Crosses between Tetraploid Wheat and Aegilops tauschii

Hybrid chlorosis, a type of hybrid incompatibility, has frequently been reported in inter- and intraspecific crosses of allopolyploid wheat. In a previous study, we reported some types of growth abnormalities such as hybrid necrosis and observed hybrid chlorosis with mild or severe abnormalities in wheat triploids obtained in crosses between tetraploid wheat cultivar Langdon and four Ae. tauschii accessions and in their derived synthetic hexaploids. However, the molecular mechanisms underlying hybrid chlorosis are not well understood. Here, we compared cytology and gene expression in leaves to characterize the abnormal growth in wheat synthetics showing mild and severe chlorosis. In addition, we compared disease resistance to wheat blast fungus. In total 55 and 105 genes related to carbohydrate metabolism and 53 and 89 genes for defense responses were markedly up-regulated in the mild and severe chlorosis lines, respectively. Abnormal chloroplasts formed in the mesophyll cells before the leaves yellowed in the hybrid chlorosis lines. The plants with mild chlorosis showed increased resistance to wheat blast and powdery mildew fungi, although significant differences only in two, third internode length and maturation time, out of the examined agricultural traits were found between the wild type and plants showing mild chlorosis. These observations suggest that senescence might be accelerated in hybrid chlorosis lines of wheat synthetics. Moreover, in wheat synthetics showing mild chlorosis, the negative effects on biomass can be minimized, and they may show substantial fitness under pathogen-polluted conditions.


Introduction
Allopolyploid speciation, which is one of the major evolutionary processes in higher plants [1,2], is achieved through various processes including interspecific hybrid formation and endoreduplication.In interspecific crosses, the hybrid plants sometimes break down, a phenomenon called hybrid incompatibility.Hybrid incompatibility inhibits allopolyploid speciation.Common wheat (Triticum aestivum L., 2n = 6x = 42, genome constitution AABBDD) is an allohexaploid species derived through allohexaploidization between cultivated tetraploid wheat (Triticum turgidum L., 2n = 4x = 28, AABB genome) and wild diploid progenitor Aegilops tauschii Coss.(2n = 2x = 14, DD genome) [3].Hexaploid wheat plants with the AABBDD genome can be obtained through artificial hybrids (2n = 3x = 21, ABD genome) between tetraploid wheat and Ae.tauschii, called synthetic hexaploid wheat [4].Growth incompatibilities were first reported half a century ago in ABD triploids [5][6][7].Recently, the tetraploid wheat cultivar Langdon (Ldn) was found to be an efficient AB genome parent for the production of synthetic hexaploid wheat [8].Numerous synthetic wheat hexaploids have been produced from ABD hybrids between Ldn and various Ae.tauschii accessions [9], whereas several types of hybrid growth abnormalities were observed in many cross combinations [10,11].The incompatibilities between the wheat AB and D genomes include hybrid chlorosis, severe growth abortion, and two types of hybrid necrosis (type II and type III necrosis), which function as postzygotic reproductive barriers preventing the production of synthetic hexaploid wheat [11].Cell death occurs gradually beginning with older tissues in hybrid lines showing type III necrosis, whereas type II necrosis lines show a necrotic phenotype under low temperature conditions [11].
Most hybrid incompatibilities are caused by interaction of at least two loci or alleles, a basis of the Dobzhansky-Muller model [12].Hybrid necrosis is generally triggered by autoimmune response, and the causal genes encode disease resistance-related proteins in higher plants such as Arabidopsis and lettuce [13][14][15][16].The up-regulation of defense-related genes such as pathogen-related genes is observed in plants exhibiting hybrid weakness as well as hybrid necrosis, and the causal genes for hybrid weakness are also disease resistance genes in rice [17,18].In ABD triploids, the programmed cell death of severe growth abortion and two types of hybrid necrosis, type II and III necrosis, is accompanied by a significant increase in expression of defense-related genes [11,19,20].Up to now, no gene expression profiles of ABD hybrid plants showing hybrid chlorosis have been reported.Leaves of the hybrid plants exhibiting chlorosis gradually turn yellowish with age.Four Ae. tauschii accessions reportedly induce hybrid chlorosis in ABD triploids crossed with Ldn [11].The chlorosis phenotype is different from that of the hybrid chlorosis observed in interspecific crosses between a few accessions of cultivated emmer wheat (T.turgidum ssp.dicoccon (Schrank) Thell., AABB genome) and many accessions of cultivated and wild timopheevii wheat (Triticum timopheevii Zhuk., AAGG genome); hybrid chlorosis in tetraploid wheat is lethal [21].Hybrid chlorosis between T. turgidum and T. timopheevii is controlled by two dominant complementary genes, Cs1 on chromosome 5A and Cs2 on chromosome 4G [22].In tetraploid and hexaploid wheat, interaction between Ch1 on 2A and Ch2 on 3D induces hybrid chlorosis with lethality [23][24][25].In addition, three chlorophyll-related abnormalities, striato-virescence, delayed virescence, and albino, are triggered by complementary gene interactions on chromosomes 2A, 3A, or 2B in tetraploid wheat [26].Wheat plants showing striato-virescence and delayed virescence are viable and fertile, although albinos are not.
Intraspecific hybrids with severe growth defects show enhanced disease resistance, and in Arabidopsis, the enhanced resistance is related to activation of the salicylic acid (SA) stress signaling pathway [14].Most plant lesion mimic mutants with either necrotic or chlorotic lesions have activated defense responses and show increased resistance to virulent pathogens compared with wild-type plants [27].Reduced growth in the increased-resistance plants is assumed to be due to the high cost for maintenance of defense response-related pathways [14,28,29].In the ABD triploids with growth abnormalities, phenotypic effects of hybrid chlorosis seem milder than hybrid necrosis and severe growth abortion [10,11].The objectives of this study were: 1) to clarify transcriptional and physiological characteristics of hybrid chlorosis of the ABD triploids, 2) to map one of the causal genes for hybrid chlorosis on the wheat D-genome chromosome, and 3) to evaluate the level of disease resistance.Based on these results, we aimed to elucidate the mechanism inducing hybrid chlorosis, and discussed the relationship between growth defects due to hybrid incompatibility and enhanced disease resistance in ABD triploid wheat.

Phenotype of hybrid chlorosis in triploid wheat hybrids and wheat synthetics
Triploid plants between Ldn and Ae.tauschii that showed hybrid chlorosis were fertile, and produced selfed seeds, from which synthetic hexaploids with 42 chromosomes germinated.Two synthetic hexaploid lines, Ldn/KU-2111 and Ldn/KU-20-1, showed an identical chlorosis phenotype, whereas the chlorosis phenotype of another Ldn/IG47202 hexaploid line was milder (Fig. 1A).In the two former chlorosis lines (severe chlorosis), yellowish lesions or sectors appeared on the leaf surface gradually starting with older tissues, and then the leaves turned brown.The yellowish sectors appeared similarly, in an age-dependent manner, and the color change to brown was not observed until senescence began in the distal part of leaves in Ldn/IG47202 (mild chlorosis) (Fig. 1B).The phenotype of these hybrid chlorosis lines was not lethal, and selfed seed progeny could be obtained.

Chromosomal location of the causal gene for hybrid chlorosis
The severe chlorosis line Ldn/KU-2111 was crossed with the mild chlorosis line Ldn/IG47202 for allelism test, and 156 F 2 plants obtained from the cross were grown in the 2011-2012 season.The F 1 plants seemed to show the severe chlorosis phenotype and no F 2 progeny without any chlorosis symptoms was observed, indicating that the causal gene for severe chlorosis was closely linked or overlapped with that for mild chlorosis in the D genome.
To assign the D-genome causal gene for severe chlorosis, 96 F 2 plants from a cross of Ldn/ PI476874, showing normal growth phenotype without any chlorosis symptoms (wild type, WT), and Ldn/KU-2111 were used as a mapping population.In the F 2 mapping population, 70 plants with chlorosis symptoms and 26 WT plants were observed.The chlorosis-inducing allele was dominant, and the segregation ratio statistically fitted a 3:1 ratio (χ 2 value = 0.637, P > 0.05), which was consistent with Mendelian segregation of alleles of a single gene.
A single locus that controlled hybrid chlorosis in Ae. tauschii was assigned to the short arm of chromosome 7D in the Ldn/PI476874//Ldn/KU-2111 population (Fig. 2).The locus related to induction of chlorosis was mapped together with 11 simple sequence repeat (SSR) markers, 4 cleaved amplified polymorphic sequence (CAPS) markers, and Lr34.The four CAPS and three SSR markers were respectively developed based on reference to single nucleotide polymorphism (SNP) data for Ae.tauschii and survey sequence data for 7DS [30,31].The hybrid chlorosis-related locus, named Hybrid chlorosis1 (Hch1), was within a 5.3 cM interval between the two linked SSR marker loci Xkupg4 and Xkupg28 (Fig. 2).

TEM observation of leaf mesophyll cells
The organelle structures of mesophyll cells were compared for green and yellowish leaves of the Ldn/PI476874 and Ldn/KU-2111 synthetic hexaploids based on transmission electron microscopy (TEM) observation (Fig. 3A).In the cells of green leaves of the Ldn/KU-2111 plants, chloroplasts were slightly shrunken compared with those of the Ldn/PI476874 plants (Fig. 3B).The area of chloroplasts photographed at random was significantly larger in Ldn/PI476874 than in green or yellowish leaves of Ldn/KU-2111 (Fig. 3C).The stromal area of chloroplasts was less in the mesophyll cells of Ldn/KU-2111 than Ldn/PI476874.In yellowish leaves, the grana were severely collapsed (Fig. 3B).The proportion of grana to chloroplast was determined for the Ldn/PI476874 and Ldn/KU-2111 plants (Fig. 3D); the area ratio was significantly larger in green leaves of Ldn/KU-2111 than in those of Ldn/PI476874.
Plastoglobuli, globular masses of lipoprotein within chloroplasts [32], were more conspicuous in chloroplasts of leaves in Ldn/KU-2111 than in Ldn/PI476874 (Fig. 3B).In fact, the number of plastoglobuli per chloroplast was significantly higher in Ldn/KU-2111 than in Ldn/ PI476874, whereas no significant difference in the number of plastoglobuli was observed between the green and yellowish leaves of Ldn/KU-2111 (Fig. 3E).

Alteration of gene expression profiles in hybrid chlorosis
To comprehensively compare gene expression profiles among WT (Ldn/PI476874) and the two hexaploid synthetics showing hybrid chlorosis (Ldn/IG47202 and Ldn/KU-2111), transcriptome analysis was performed using a wheat-specific 38k oligo DNA microarray [33].For hybridization, total RNA was extracted from the oldest leaves of 3-week-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 to the WT were defined as either upor down-regulated genes.
Of the 37,826 probes on the wheat microarray, 1,663 (4.4%) and 3,864 (10.2%) probes were regarded as genes up-and down-regulated, respectively, in leaves of the mild chlorosis line (Ldn/IG47202) compared with the WT line (Fig. 4A).In the severe chlorosis line (Ldn/KU-2111), 2,421 (6.4%) and 2,130 (5.6%) probes were found to be up-and down-regulated, respectively.Based on homology searches of the wheat expressed sequence tag (EST) database with probe sequences, 625 (37.6%) and 1,760 (45.5%) of up-and down-regulated genes, respectively, were categorized into a total of 17 groups based on inferred function (Fig. 4B).Of the up-regulated genes, carbohydrate metabolism-related genes were the most frequently encountered (Fig. 4B).Genes related to defense, stress, transcription factors, and transport were also abundantly expressed in the hybrid plants showing mild chlorosis.In contrast, genes related to transport, DNA binding, photosynthesis, and cell maintenance were down-regulated in these plants.Similarly, 947 (39.1%) and 786 (36.9%) genes were up-and down-regulated in the severe chlorosis line, respectively.Carbohydrate metabolism-related genes were the most frequently found to be up-regulated, whereas photosynthesis-related genes were the most dramatically down-regulated in the severe chlorosis plants.
Among the carbohydrate metabolism-related genes, beta-glucosidase, beta-1,3-glucanase precursor genes and beta-fructofuranosidase were highly up-regulated in the chlorotic plants (Tables 1 and 2).In addition, transcripts of a number of defense-related genes, including chitinase and sulfur-rich/thionin-like protein genes, accumulated abundantly in the chlorotic plants.Moreover, many WRKY-type transcription factor genes, which play important roles in immune responses, senescence, and various other processes of plants [34,35], were found among the up-regulated transcription factor genes in both chlorosis lines (Tables 3 and 4).In the severe hybrid chlorosis line, three NAC transcription factor genes including NAM-B1, which is associated with senescence and nutrient remobilization from leaves to developing grains [36], were up-regulated (Table 3).In both hybrid chlorosis lines, photosynthesis-related genes showed a high rate of down-regulation, including the genes for photosystem I and photosystem II proteins, chlorophyll a/b binding protein, and ribulose-1,5-bisphosphate carboxylase activase.

Comparison of gene expression profiles between wheat synthetics displaying chlorosis and necrosis
We compared gene expression profiles among the hybrid chlorosis lines and a type III necrosis line, which was a synthetic wheat line of Ldn/KU-2828 [11].The signal-intensity differences of all probes relative to WT signals were compared among leaves showing mild and severe hybrid chlorosis and type III hybrid necrosis.Significantly strong positive correlations were detected between the mild and severe hybrid chlorosis lines and between the severe hybrid chlorosis and type III necrosis lines (Fig. 5A-B).However, no correlation of signal intensity was observed between leaves showing mild chlorosis and type III necrosis (Fig. 5C).Moreover, the expression profiles of the severe and mild hybrid chlorosis and type III necrosis lines were compared using defense-, carbohydrate metabolism-, and photosynthesis-related probes in which expression levels were changed for each growth abnormality relative to WT (Table 5, S1-S3 Tables).Significant positive correlations were observed between the profiles of the defense-related genes in mild and severe chlorosis and between the profiles of these genes in hybrid chlorosis and type III necrosis.Similar observations were found in the expression profiles of carbohydrate metabolism-related genes between mild and severe chlorosis.In photosynthesis-related genes, however, significant positive correlations were detected in some of the combinations.

Expression analysis of senescence-and defense-related genes
To validate the microarray data, quantitative reverse transcription (RT)-PCR was conducted for six up-regulated genes.Two senescence-associated genes, TaSAG5 and TaSAG7 [37], were significantly up-regulated in leaves of the two hybrid chlorosis lines compared with the WT synthetic wheat line, and transcript levels were higher in the severe chlorosis line than in the mild chlorosis line (Fig. 6A-B).Similarly, a defense-related gene, PR1, was dramatically activated in hybrid chlorosis, and its transcript levels correlated well with the severity of chlorosis (Fig. 6C).Up-regulation of a gene encoding the alpha chain of nascent-polypeptide-associated complex, which can reversibly bind to ribosomes and regulate translocation of newly synthesized proteins [38,39], was observed in hybrid chlorosis (Fig. 6D), but not in type III necrosis [11].Two WRKY transcription factor genes, WRKY11 and WRKY35, were significantly upregulated in hybrid chlorosis, and their transcript levels were higher in 3-week-old seedlings of the severe chlorosis line than the mild chlorosis line (Fig. 6E-F).
Next, RT-PCR analysis of four selected genes up-regulated in hybrid chlorosis was conducted using 2-, 3-, and 4-week-old seedling leaves of three WT and three hybrid chlorosis  lines.This time-course study confirmed the results from microarrays and quantitative RT-PCR.Transcript accumulation of the four genes was more abundant in the three hybrid chlorosis lines than in the three WT lines of synthetic wheat (Fig. 6G).

Comparison of photosynthetic activity among synthetic wheat lines
To examine the effects of down-regulation of photosynthesis-related genes, photosynthetic activity was compared among three synthetic wheat lines, WT Ldn/PI476874 and hybrid necrotic lines Ldn/IG47202 and Ldn/KU-2111.The photosystem II activities of the chlorosis-exhibiting lines were significantly reduced in the first leaves of 3-and 4-week-old seedlings compared to those of the WT line (Fig. 7), which is consistent with the down-regulation of photosynthesisrelated genes in hybrid chlorosis.The reduction of photosystem II activity appeared more remarkable in the severe chlorosis line than in the mild chlorosis line, and the Fv/Fm value was significantly different in severe chlorosis plants grown for 2 weeks.

Estimation of agricultural traits in hybrid chlorosis lines
To assess the effects of hybrid chlorosis on various agricultural traits, we grew two F 2 populations derived from crosses of WT to chlorosis synthetic wheat lines, Ldn/CGN10768//Ldn/ IG47202 (N = 103) and Ldn/PI476874//Ldn/KU-2111 (N = 108).The former population was from a cross between the WT and mild chlorosis lines, and the latter between the WT and  6 were compared.
In the Ldn/PI476874//Ldn/KU-2111 population, significant differences between the WT and chlorosis F 2 individuals were found in all examined traits except for spike length (Table 6).The severe chlorosis plants showed a phenotype of reduced height, low seed fertility, small seed weight, and late flowering compared with the WT plants.On the other hand, no significant differences were observed in most examined traits between the WT and mild chlorosis plants.Significant reductions in third internode length and delay of maturation time were detected in the mild chlorosis plants.

Disease resistance in hybrid chlorosis lines
A number of defense-related genes were up-regulated in the hybrid chlorosis lines, as revealed by microarray analyses (Fig. 4).For evaluation of disease resistance to a Triticum isolate, Br48, of the blast fungus (Magnaporthe oryzae, syn.Pyricularia oryzae) [40], two WT synthetic wheat lines, Ldn/CGN10768 and Ldn/PI476874, and two showing chlorosis, Ldn/IG47202 and Ldn/KU-2111, were inoculated with Br48.As previously reported [41], green and brown infection types are respectively considered to be susceptible (or virulent) and resistant (or avirulent).In all examined lines, 1-and 2-week-old seedlings showed infection types 4G and 5G [41], meaning that these seedlings were susceptible to Br48 (Table 7).However, the infection types of 3-week-old seedlings were 1B, 2B, and 4B in both chlorosis lines but not in the WT synthetic lines.These observations clearly indicated that the hybrid chlorosis-showing plants were more resistant to the wheat blast fungus than the WT plants at the 3-week-old seedling stage.
To examine the effects of hybrid chlorosis on disease resistance, we compared cellular responses to the wheat blast fungus in the first leaves of mild chlorosis and WT lines of synthetic wheat.The inoculated leaves were fixed 48 h after inoculation with Br48, and observed under a fluorescent microscope.Appressoria of Br48 formed on epidermal cells of the leaves (Fig. 8).In some attacked cells, the hypersensitive reaction (HR) occurred, which was accompanied by  strong yellow fluorescence as previously observed [40].In other cells with no fluorescence, no resistance reaction to the appressorial penetration was observed, and infection hyphae were well developed (Fig. 8).
In most attacked epidermal cells of the highly susceptible barley cultivar Nigrate, HR was rarely observed and the infection hyphae were successfully developed (Table 8).The WT and hybrid chlorosis lines of wheat synthetics showed partial resistance to Br48, and HR was observed in the first leaves of 1-, 2-, 3-week-old seedlings.HR was more frequently observed in 2and 3-week-old seedlings of the mild chlorosis line than in those of the WT synthetic wheat line (Table 8).The HR frequency was dramatically increased from one to two weeks after seed sowing in the mild chlorosis line.The significant differences in cellular responses to Br48 infection indicated that seedlings of the mild chlorosis line are more resistant to Br48 than the WT.
Moreover, we examined the defense response to an isolate, Th-1, of the wheat powdery mildew fungus (Blumeria graminis f. sp.tritici) in the first leaves of mild chlorosis and WT lines of synthetic wheat.The WT synthetic wheat line Ldn/PI476874 showed clear powdery mildew resistance to Th-1 (S1 Fig. ).Since Ldn was completely sensitive to Th-1, the resistance of Ldn/ PI476874 would be transmitted from the parental Ae. tauschii accession PI476874 which revealed also clear resistance to Th-1.The mild chlorosis line Ldn/IG47202 showed the necrotic cell death on leaves.The area and number of the necrotic lesions decreased in the three-weekold plants of Ldn/IG47202 in which the yellow lesions started to appear.No necrotic lesion appeared on non-infected leaves in Ldn/IG47202, and thus the necrotic lesions were generated by the infection of Th-1.These observations suggested that the area and number of the necrotic lesions were negatively correlated with appearance of the yellow lesions in the mild chlorosis line of synthetic wheat.

Discussion
Allopolyploidy provides advantages to establishment of new plant species, including fixation of heterosis [1].However, hybrid growth abnormalities, observed frequently in interspecific crosses, inhibit allopolyploid speciation.Hybrid chlorosis, observed in some cross combinations between tetraploid wheat and Ae.tauschii, is recognized as one such growth abnormality in allohexaploid wheat speciation [11].In hybrid chlorosis lines of synthetic allohexaploid wheat, yellowish lesions and sectors on the leaf surface appear gradually in older tissues.In type III necrosis, necrotic cell death also gradually begins from older tissues, whereas the lesion color is clearly different from that in hybrid chlorosis.In type III necrosis, necrotic cell death is triggered by an HR-like reaction and accompanied by generation of reactive oxygen species [11].Within the chloroplasts of mesophyll cells in leaves undergoing type III necrosis, oil droplets, meaning plastoglobuli, are more frequently found than in leaves of WT.In addition, plasmolysis, collapse of vacuoles, and organelle degradation are observed as typical features of dead cells in type III necrosis.Unlike type III necrosis, in leaves developing chlorosis, abnormal chloroplasts were found within mesophyll cells (Fig. 3).Plastoglobuli appeared before the yellowish sectors formed on the leaf surfaces of the plants showing hybrid chlorosis.Shrinkage of the chloroplasts occurred, with appearance of many plastoglobuli.This ultrastructural modification of chloroplasts is compatible with the typical features of hybrid chlorosis in the wheat synthetics, which is different from the features of type III necrosis.
It was previously reported that an increased number of plastoglobuli accompanies thylakoid breakdown during senescence [32].In addition, oxidative stress to the photosynthetic apparatus increases the number of plastoglobuli [32].Thus, an increase in the number of plastoglobuli seems to be due to generation of reactive oxygen species in chloroplasts of leaves with type III necrosis [11].In comparative transcriptome analyses, the gene expression profile of plants showing hybrid chlorosis, especially severe chlorosis, resembled that of type III necrosis (Fig. 5, Table 5).Up-regulation of defense-and carbohydrate metabolism-related genes was commonly observed in severe and mild hybrid chlorosis and type III necrosis, which suggested that these hybrid incompatibilities could at least partly share common mechanisms to induce their symptoms.The number of genes detected with defense-related probes showing expression upregulated at least 3 times more than in WT was more abundant for type III necrosis than hybrid chlorosis, whereas the number of up-regulated probes related to carbohydrate metabolism was more abundant for hybrid chlorosis than type III necrosis (Tables 1, 2 and 5).In the hybrid chlorosis lines, senescence-associated genes and NAM-B1 were greatly up-regulated.NAM-B1, encoding a NAC transcription factor, functions to control leaf senescence and is associated with grain protein, zinc, and iron contents [36].The rice NAM-B1 homologs also act as regulators of leaf senescence [39,42].Therefore, these expression profiles showed that hybrid chlorosis resulting from interaction between the AB and D wheat genomes has unique features distinct from hybrid necrosis, and suggests that senescence might be accelerated in the hybrid chlorosis lines of wheat synthetics.Abnormal chloroplasts are accompanied by an increase in the number of plastoglobuli generated in green leaves of the hybrid chlorosis line, and chloroplast degradation was observed in the yellowish leaves (Fig. 3).These TEM observations also supported the idea that accelerated senescence occurs in leaves of the hybrid chlorosis lines.
Hybrid incompatibility between two diverging lineages is generally explained by interaction of at least two loci or alleles [12].The hybrid chlorosis observed in some crosses between tetraploid wheat and Ae.tauschii appears to be due to an epistatic interaction of two loci from the AB and D wheat genomes, considered to be a Dobzhansky-Muller-type hybrid barrier [11].Several complementary genes for chlorophyll abnormalities have been studied in wheat.The Ch1 hybrid chlorosis gene has been assigned to chromosome 2A in tetraploid and hexaploid wheat, and Ch2 to chromosome 3D in hexaploid wheat [23][24][25].Hybrid chlorosis in some interspecific crosses of tetraploid wheat is respectively controlled by Cs1 and Cs2 on chromosomes 5A and 4G [22].Three additional chlorophyll abnormalities are caused by the following complementary gene interactions: sv1 (3A) and sv2 (2A), dv1 (2B) and dv2 (supposedly 2A), and abn1 (2A) and abn2 (2B) in T. turgidum [26].The D genome-located causal gene, Hch1, for hybrid chlorosis in ABD triploids and wheat synthetics was assigned to the short arm of chromosome 7D (Fig. 2).Thus, Hch1 is a novel gene inducing hybrid chlorosis in Triticum species.We propose the presence of a gene, Hch2, in the AB genome of tetraploid wheat, which acts as a gene complementary to Hch1 for hybrid chlorosis in ABD triploids.Although many complementary pairs of hybrid chlorosis genes have been reported, no locus has been successfully cloned in wheat.To understand the molecular basis of and physiological changes in hybrid chlorosis, map-based cloning of these causal genes would be required in future studies.In our previous study, a causal gene for type III necrosis in the D genome, Nec1, was also assigned to 7DS [11].Hch1 was located at a similar chromosomal region to Nec1.Because Ae. tauschii accessions inducing type III necrosis are genealogically distinct from those inducing hybrid chlorosis [11], the Hch1 and Nec1 alleles could have independently arisen in the Ae.tauschii population.Many disease resistance genes such as Lr34 are concentrated around the Hch1 and Nec1 region of 7DS [30,43].Most causal genes that have been isolated to date are related to disease resistance [13-16], which suggests that Hch1 and Nec1 may be any of the disease resistance genes located in the 7DS region.
In the hybrid chlorosis lines examined in the present study, a lot of WRKY transcription factor genes were up-regulated (Tables 3 and 4).The putative Hch1-Hch2 interaction should activate multiple WRKY transcription factors in the hybrid chlorosis plants.WRKY transcription factors play important roles in biotic and abiotic stress responses and senescence in higher plants [34,35].In particular, many WRKY genes have been identified to act as positive regulators of SA-dependent immune responses [44].In rice, for example, OsWRKY45 functions to regulate disease resistance against the rice blast fungus [45].A number of defense-related genes, such as PR1, were also up-regulated in the hybrid chlorosis plants (Fig. 4), which could be at least partly controlled through activation of WRKY genes in these plants.Many WRKY genes are activated not only in hybrid chlorosis lines but also in wheat ABD triploids showing hybrid necrosis and severe growth abortion [11,19,20].The direct targets of the epistatic interaction of causal genes for these types of hybrid incompatibility should be studied to clarify the signaling pathways for transcriptional activation of the WRKY genes in the abnormal ABD triploid plants.
Few significant differences in agricultural traits were found in WT synthetic wheat plants and those showing mild chlorosis, although remarkable growth inhibition was observed in plants showing severe chlorosis (Table 6).Transcripts of defense-related and WRKY genes accumulated abundantly in leaves of the plants showing mild chlorosis, which in fact showed enhanced resistance to the wheat blast fungus through HR (Tables 7 and 8).The enhanced levels of wheat blast disease resistance were correlated with progress of chlorosis symptoms (Tables 7  and 8).In addition, we confirmed disease resistance to wheat powdery mildew fungus.The WT synthetic wheat line used in the disease resistance assay unfortunately showed clear wheat powdery mildew resistance because the powdery mildew resistance gene was transmitted from the parental Ae. tauschii accession PI476874, whereas the powdery mildew resistance also appeared to be enhanced in the mild chlorosis line of synthetic wheat (S1 Fig. ).Such enhanced disease resistance to wheat blast and powdery mildew fungi was visibly confirmed in threeweek-old plants of the mild chlorosis line in which the yellow lesions started to appear.These observations strongly suggest that the level of disease resistance is greatly strengthened in the mild chlorosis plants through up-regulation of defense-related genes.Generally, hybrid necrosis extensively inhibits plant growth and dramatically reduces yield, even if SA-dependent immune responses are activated in plants showing these symptoms [11,14,18], which is an undesirable effect for crop breeding.Tight association of cell death with defense responses and processes involved in senescence has been observed in some cell death-related mutants of Arabidopsis.A loss-of-function mutant analysis of ACCELERATED-CELL-DEATH11 (ACD11) in Arabidopsis, for example, implied that both defense-and senescence-related signal pathways may lead to programmed cell death in plant leaves [46].A recent study of allelic variation at the ACD6 locus in Arabidopsis revealed a negative correlation between the amount of vegetative growth and level of resistance to pathogens [47].The hyperactive ACD6 allele, inducing delayed growth and reduced biomass, strongly enhances disease resistance.This result indicates that some lesion-mimic mutant alleles can provide significant advantages for pathogen attack despite their negative effects on biomass.In the case of hybrid chlorosis triggered by the Hch1-Hch2 interaction, a similar trade-off between disease resistance and reduced biomass could be assumed.In particular, it might be expected that the negative effects on biomass of mild chlorosis-showing plants could be minimized and substantial fitness conveyed under pathogen-polluted environments.Activation of SA-related immune responses surely reduces fitness in the absence of pathogens [48], but increases it in the presence of pathogens [49].Fine-tuning of the trade-offs of the Hch1-Hch2 interaction would be required for substantial use of mild chlorosis in wheat breeding under inferior environments, such as fields with frequent occurrence of disease.Production of near-isogenic lines for Hch1 and Hch2 should be effective for precise evaluation of the trade-offs between disease resistance and reduced biomass in further studies.

Mapping of the D-genome causal gene for hybrid chlorosis
For the mapping population, 96 F 2 plants from a cross between WT (synthetic hexaploid wheat line Ldn/PI476874) and the severe hybrid chlorosis line (synthetic hexaploid wheat line Ldn/ KU-2111) were used.Total DNA was extracted from leaves of the parental strains and F 2 individuals, and SSR genotyping was performed.For SSR genotyping, 40 cycles of PCR were performed using 2x Quick Taq HS DyeMix (TOYOBO, Osaka, Japan) and the following conditions: 10 s at 94°C, 30 s at the annealing temperature, and 30 s at 68°C.Information on the SSR markers and their annealing temperature was obtained from the National BioResource Project (NBRP) KOMUGI web site (http://www.shigen.nig.ac.jp/wheat/komugi/strains/aboutNbrpMarker.jsp) and the GrainGenes web site (http://wheat.pw.usda.gov/GG2/maps.shtml).The PCR products were separated in 2% agarose or 13% non-denaturing polyacrylamide gels and visualized under UV light after staining with ethidium bromide.Genetic mapping was performed using the MAP-MAKER/EXP version 3.0b package, and the threshold for log-likelihood scores was set at 3.0; genetic distances were calculated with the Kosambi function [51].
For mapping of leaf rust-resistance locus Lr34 [43], the primer set 5'-TGCGGCGATTCTA TACTACT-3' and 5'-CCGACATCAAGAACCTCC-3' was designed.The PCR-amplified products were digested by the 4-bp cutting restriction enzyme TaqI, and the digests were separated in a 13% non-denaturing polyacrylamide gel.In addition, seven markers were newly developed (S4 Table ).Three SSR markers were designed based on survey sequence data for the short arm of chromosome 7D [30], searching for SSR motifs using SciRoKo version 3.4 software [52].Additional SNPs were identified in target gene regions by sequencing approximately 700 bp of amplified DNA of Ae. tauschii accessions PI476874 and KU-2111.The target gene regions were selected based on reference sequences of Brachypodium and RNAseq data for Ae.tauschii leaves [31,53].The nucleotide sequences were determined using an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA), and SNPs were found through sequence alignment using GENETYX-MAC version 12.00 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA).The identified SNPs were converted to CAPS markers.For these CAPS markers, the PCR annealing temperatures were 54 or 58°C.The PCR fragments digested by the indicated restriction enzymes were separated in 13% nondenaturing polyacrylamide gels and stained with ethidium bromide (S4 Table ).

TEM observation
Seedling leaves of two synthetic hexaploid wheat lines, Ldn/PI476874 for WT and Ldn/KU-2111 for severe chlorosis, grown at 23°C for 3 weeks, were used for TEM.The leaf blades were cut into 1 mm 2 pieces and then incubated in a freshly prepared solution of 5 mM CeCl 3 buffered with 50 mM 3-(N-morpholino)propanesulfonic acid at pH 7.2 for 1 h.The fixation of samples and the sectioning of ultrathin sections 90 nm thick were conducted according to our previous report [20].Sections were examined with a Hitachi-7100 electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV.Three sections cut from leaf resin blocks of two plants were used for ultrastructural analysis of chloroplasts.More than 25 mesophyll cells were examined in each of the sections.The areas of three different structures, such as chloroplasts, grana and plastoglobuli, were measured by Image J software (National Institutes of Health, USA; http://imagej.nih.gov/ij/).

Transcriptome analysis
Three synthetic hexaploid wheat lines, Ldn/PI476874 as WT, Ldn/KU-2111 with severe hybrid chlorosis and Ldn/IG47202 with mild hybrid chlorosis, were used for microarray analysis.Total RNA was extracted from leaves of WT and hybrid chlorosis lines grown at 23°C for 3 weeks using an RNeasy Plant Mini kit (Qiagen, Hilden, Germany).A KOMUGI 38k oligonucleotide DNA microarray (Agilent Technologies, Santa Clara, CA) was supplied by the National BioResource Project (NBRP)-Wheat, Japan (https://www.nbrp.jp)for analysis.Detailed information on the 38k microarray platform can be found in Kawaura et al. [33] and the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) website under GPL9805.Hybridization of Cy3-labeled cRNA, washing and image scanning were performed according to our previous report [20].Two independent experiments were conducted for each sample.All microarray data were deposited as GSE59640 in the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/),including supplementary files, GSM1441345-1441350.
The functions of probes and genes were predicted by both BLAST and BLASTx searches (E value < 1e -10 ) against the DNA Data Bank of Japan (DDBJ) database.The NBRP KOMUGI website (http://www.shigen.nig.ac.jp/wheat/komugi/array/index.jsp) was also used for functional identification of the proteins encoded by the probes.The UniProtKB Protein Knowledgebase (http://www.uniprot.org)was used for functional categorization of identified genes.

RT-PCR and quantitative RT-PCR analyses
RT-PCR and quantitative RT-PCR were performed using RNA isolated from leaves of the three WT (Ldn/PI476874, Ldn/KU-2059, and Ldn/KU-2159) and three chlorosis (Ldn/ IG47202, Ldn/KU-2111, and Ldn/KU-20-1) lines.Each plant was grown at 23°C for 3 weeks, and total RNA was extracted using Sepasol-RNA I (Nacalai Tesque, Kyoto, Japan).First-strand cDNA was synthesized from DNase I-treated RNA samples using ReverTra Ace Reverse Transcriptase (Toyobo, Osaka, Japan) and an oligo(dT) 20 primer.The gene-specific primer sets for RT-PCR and qRT-PCR are listed in S5 Table.PCR-amplified products were separated by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide.The transcript accumulation of each gene was detected by quantitative RT-PCR using a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany) with THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primer sets.The Actin gene was used as an internal control, and relative expression was calculated as 2 -ΔΔCt , representing the value relative to the transcript levels in leaves of WT (Ldn/PI476874).

Photosynthetic activity
A portable JUNIOR-PAM fluorometer (Heinz Walz GmbH, Effeltrich, Germany) was used for measurement of the maximum photochemical quantum yield of photosystem II (Fv/Fm).The first leaves of 2-, 3-and 4-week-old WT and hybrid chlorosis plants were incubated under dark conditions for 1 h and Fv/Fm was measured at ten independent locations on each leaf blade according to the instructions in the operating manual for the fluorometer.

Measurement of agricultural traits
Seeds of F 2 individuals from two crosses of the synthetic hexaploid lines, between Ldn/ CGN10768 (WT) and Ldn/IG47202 (mild chlorosis) and between Ldn/PI476874 (WT) and Ldn/KU-2111 (severe chlorosis), were sown in November 2010, and plants were grown individually in pots arranged randomly.Eleven traits, column and spike length, length of the first, second and third internodes, selfed seed fertility, seed number per spike, hundred grain weight, and days to heading, flowering and maturation, were measured on the earliest tiller of each F 2 plant.

Evaluation of disease resistance
A field isolate, Br48, of the wheat blast fungus was used to assay disease resistance.Br48 is virulent on most accessions of hexaploid and tetraploid wheat [41].Twelve seeds of each line were sown, and the adaxial surface of leaves of 1-, 2-and 3-week-old seedlings was sprayed with a conidial suspension (1-2 x 10 5 conidia/mL) of Br48.The preparation of the suspension and incubation of inoculated plants in humid boxes were performed according to Chuma et al. [41].The inoculated plants were then transferred to growth chambers at 22°C and 26°C, and disease symptoms were rated 5 d after inoculation.The size and rate of disease symptoms were evaluated according to Chuma et al. [41].The size was rated using six progressive grades from 0 to 5, and the disease lesions were classified into two categories on the basis of their color, brown (B) or green (G).The infection types were represented as the combination of the size and color [41].In addition, 48 h after inoculation at 22°C, the inoculated leaves were harvested and fixed for microscopic observation according to Murakami et al. [40].HR and hyphal growth were evaluated under bright and fluorescent fields of a fluorescence microscope with excitation filter B (Olympus, Tokyo, Japan).The barley cultivar Nigrate, used as a control, is highly susceptible to Br48 [54].
An isolate, Th-1, of wheat powdery mildew fungus was also used to assay disease resistance.For preparation of inoculum, 10-d-old seedlings of a wheat cultivar 'Norin 4' were inoculated with conidia of Th-1 and maintained in growth chambers at 22°C.Twelve seeds of each line were sown, and the adaxial surface of leaves of 1-, 2-and 3-week-old seedlings was inoculated with Th-1 by blowing conidia from the infected seedlings of Norin 4. The inoculated plants were then transferred to the growth chambers, and disease symptoms were observed 8 d after inoculation.The entire experiments for the powdery mildew resistance assay were repeated two times.

Fig 1 .
Fig 1. Synthetic hexaploid wheat plants exhibiting hybrid chlorosis.(A) Phenotypes of the normal growth (WT, Ldn/KU-2126), severe chlorosis (Ldn/ KU-2111) and mild chlorosis (Ldn/IG47202) lines.The plants with mild chlorosis were grown for two months after sowing and showed lighter yellowish symptoms than those with severe chlorosis.(B) The first leaf phenotypes of plants with WT and with hybrid chlorosis.Each plant was grown at 23°C for 2 to 4 weeks.doi:10.1371/journal.pone.0121583.g001

Fig 2 .Fig 3 .
Fig 2. Genetic mapping of Hch1 to the short arm of chromosome 7D.Genetic distances (cM) are shown on the left and markers on the right.doi:10.1371/journal.pone.0121583.g002

Fig 4 .
Fig 4. Summary of results from microarray analysis.(A) Venn diagram of genes differentially expressed between plants with severe chlorosis, mild chlorosis and type III necrosis.(B) Number of up-and down-regulated genes in leaves of the Ldn/KU-2111 and Ldn/IG47202 lines.Genes up-and downregulated in the chlorosis line (Ldn/KU-2111) and (Ldn/IG47202) relative to WT were categorized into 18 functional groups.doi:10.1371/journal.pone.0121583.g004

Fig 5 .
Fig 5. Correlation between the log 2 ratios of genes with altered expression among plants with severe and mild chlorosis and type III necrosis.(A) Scatter diagram of the genes with altered expression in mild and severe chlorosis.(B) Scatter diagram of the genes with altered expression in severe chlorosis and type III necrosis.(C) Scatter diagram of the genes with altered expression in type III necrosis and mild chlorosis.doi:10.1371/journal.pone.0121583.g005

Fig 6 .
Fig 6.Transcript accumulation of the SAGs and other genes identified in microarray analysis, as revealed by RT-PCR and quantitative RT-PCR.(A, B) Two wheat SAGs.Student's t-test was used for statistical significance (**P < 0.01) of line differences.Total RNA was extracted from the first leaves of the 3-week-old seedlings.Results represent mean ± standard errors (n = 3).The Actin gene was used as an internal control.(C-F) Four genes identified in microarray analysis.Mean values with the same letters were not significantly (P < 0.05) different (Tukey-Kramer's HSD test).Results represent mean ± standard errors (n = 3).The Actin gene was used as an internal control.(G) RT-PCR analysis of four genes up-regulated in hybrid chlorosis.doi:10.1371/journal.pone.0121583.g006

Fig 7 .
Fig 7. Comparison of photosynthetic activity of WT and chlorosis lines based on chlorophyll fluorescence.Each plant was grown at 23°C for 2 to 4 weeks.Mean values with the same letters were not significantly different (P < 0.05) (Tukey-Kramer's HSD test).doi:10.1371/journal.pone.0121583.g007

Table 1 .
List of the top 20 up-regulated carbohydrate metabolism genes in leaves of the severe chlorosis line Ldn/KU-2111 identified by microarray analysis. doi:10.1371/journal.pone.0121583.t001

Table 2 .
List of the top 20 up-regulated carbohydrate metabolism genes in leaves of the mild chlorosis line Ldn/IG47202 identified by microarray analysis. doi:10.1371/journal.pone.0121583.t002

Table 3 .
The top 20 up-regulated transcription factor genes in leaves of the severe chlorosis line Ldn/KU-2111, as identified by microarray analysis. doi:10.1371/journal.pone.0121583.t003

Table 4 .
The top 19 up-regulated transcription factor genes in leaves of the mild chlorosis line Ldn/IG47202, as identified by microarray analysis. doi:10.1371/journal.pone.0121583.t004

Table 5 .
Comparison of gene expression profiles among leaves of synthetic wheat with severe chlorosis, mild chlorosis and type III necrosis phenotypes.

Table 6 .
Comparison of agricultural characters in F 2 individuals between WT and hybrid chlorosis lines.

Table 7 .
Responses of four synthetic wheat lines to the wheat blast fungus Br48.

Table 8 .
Cellular responses of the first leaves in mild chlorosis and WT lines to the wheat blast fungus Br48.