Transcriptome analysis of barley (Hordeum vulgare L.) under waterlogging stress and over-expression of HvADH4 gene confers waterlogging tolerance in transgenic Arabidopsis

doi:10.21203/rs.3.rs-2124599/v1

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Research Article

Transcriptome analysis of barley (Hordeum vulgare L.) under waterlogging stress and over-expression of HvADH4 gene confers waterlogging tolerance in transgenic Arabidopsis

https://doi.org/10.21203/rs.3.rs-2124599/v1

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Journal Publication

published 30 Jan, 2023

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Background

Waterlogging is one of the major abiotic stresses in barley and greatly reduces grain yield and quality. To explore the mechanism controlling waterlogging tolerance in barley, physiological, anatomical and transcriptional analysis were performed in two contrasting barley varieties viz. Franklin (susceptible) and TX9425 (tolerant).

Results

Compared to Franklin, more adventitious roots, aerenchymas and higher antioxidant enzymes activities were found in TX9425. 3064 and 5693 differentially expressed genes (DEGs) were identified in TX9425 after 24 h and 72 h waterlogging treatment, respectively, while 2297 and 8462 DEGs were identified in Franklin. The results suggested that TX9425 is less affected by waterlogging stress after 72 h treatment. The DEGs were mainly enriched in energy metabolism, hormone regulation, ROS scavenging, cell wall modifying enzymes and so on. Alcohol dehydrogenase (ADH) plays an important role in response to waterlogging stress. We found that HvADH4 was significantly up-regulated under waterlogging stress in TX9425. The transgenic Arabidopsis overexpressing HvADH4 displayed higher activities of antioxidant enzymes and more tolerant to waterlogging than wild type (WT).

Conclusions

The current results provide valuable information that will be of great value for the exploration of new candidate genes for barley molecular breeding in waterlogging tolerance.

Barley

Waterlogging Stress

Anatomical structure

Transcriptome analysis

ADH

Waterlogging stress, one of the major abiotic stresses affecting crop growth, has become more frequent, severe, and unpredictable due to the excessive water that also decreases the oxygen content in the soil and plant’s ability of nutrients absorption [1, 2]. About 10% of the land area is affected by excessive water on the earth [3]. In general, barley is sensitive to waterlogging, which causes 40%-79% irreversible yield loss, depending on the genotype, growth stage and duration of waterlogging stress [4].

Root is the first organ responding to oxygen shortage, and it is critical for the maintenance of normal physiological activities in plants [5, 6]. As the one of the key features in waterlogging condition, the formation of adventitious roots and aerenchymas exists widely in different plant species [7, 8]. Under waterlogging stress, the seminal root will be replaced by new adventitious roots, subsequently minimizing the distance for oxygen diffusion and improving the nutrient absorption and transport [5]. New adventitious roots contain more aerenchymas, which can help maintain a hypoxia-tolerant pathway, store and exchange of gases within the different tissues, meanwhile reduce the number of oxygen-consuming cells [9]. Flooding-induced formation of aerenchyma involves in selective programmed cell death (PCD), although certain characteristics of apoptosis are lacking. This formation requires ethylene, Ca²⁺ and reactive oxygen species (ROS) signaling and ultimate cell wall degradation [10, 11].

Antioxidant metabolism is one of the fundamental metabolic pathways under waterlogging stress [12]. The production of reactive oxygen species (ROS) is inevitable with plant’s exposure to waterlogging, this includes the superoxide radical (O₂.⁻), hydroxyl radical (•OH) and hydrogen peroxide (H₂O₂), which readily attack leaf chloroplasts and ultimately lead to leaf chlorosis and senescence [13]. To avoid this, a series of antioxidant enzymes will be synthesized/activated to scavenge ROS, such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) [14]. The membrane structure and the activity of the cells would be destroyed by malondialdehyde (MDA), which has been utilized as a reliable indicator for waterlogging tolerance [15]. Thus, high levels of SOD, POD and CAT enzyme activity are critical for the survival of crop under waterlogging conditions [9, 15].

In addition to energy metabolic pathway will be affected by oxygen deficiency. ATP is produced through glycolysis instead of oxidative phosphorylation [16]. Meanwhile, genes associated with ATP and carbohydrate catabolism, such as pyruvate decarboxylase 1 (PDC1), alcohol dehydrogenase 1 (ADH1) and sucrose phosphate synthase (SPS), were significantly up-regulated in the process of anaerobic fermentation [17, 18]. ADH genes of plant play an important role in response to waterlogging. Some studies about the ADH function have been done by transgenic assay, such as in Arabidopsis [19], soybeans [20], kiwifruit [21]. However, the function of ADH genes in response to waterlogging were different in various species.

Even though numerous QTL studies have been conducted on waterlogging in barley, the genes responsible remain unidentified [22]. RNA-sequencing (RNA-seq) technology can identify key genes involved in various biological processes, and has been successfully used to reveal waterlogging responses in cucumber [23], wild soybean [24] and wheat [25]. In this study, the physiology and dynamic RNA-seq analysis on the roots of two barley cultivars exposed to waterlogging stress were conducted. The obtained results provide insights into the physiology and molecular mechanisms underlying the response of barley to waterlogging stress, which will facilitate barley genetic study and breeding applications.

Phenotypic analysis of different barley varieties under the waterlogging treatment

The phenotypes of two genotypes (TX9425 and Franklin) after 21-day waterlogging treatment are shown in Fig. 1. Franklin leaves became more wilted and chlorotic than TX9425 under waterlogging treatment. Plant height, tillers, leaf area, shoot fresh weight and dry weight of Franklin significantly decreased. Comparing with a small decline values were detected in TX9425 leaves (Table 1). No significant difference between Franklin and TX9425 under control condition was observed in adventitious root parameters. However, after three-week waterlogging treatment, the adventitious root length, surface area, volume and number of TX9425 significantly increased and the fold change value ranged from 2.36 to 4.06 compared to the control, while there was no significant difference in Franklin except adventitious root number (Table 2). Therefore, the performance of Franklin and TX9425 displayed significant difference after three-week waterlogging treatment.

Table 1

The effect of waterlogging on agronomic traits between TX9425 and Franklin
Treatment	Leaf Age	Leaf Chlorosis	Plant Height	Tillers	Leaf Area (cm²)	Shoot Fresh	Shoot Dry
Treatment			(cm)		Leaf Area (cm²)	Weight (g)	Weight (g)
Franklin
Control	7.84 ± 0.44a	0.36 ± 0.45a	47.35 ± 2.39a	9.14 ± 3.25a	33.31 ± 4.32a	24 ± 1.13a	2.32 ± 0.15a
Waterlogging	7.97 ± 0.51a	4.96 ± 0.89b	28.38 ± 2.34b	6.75 ± 1.95b	16.67 ± 3.14b	9.97 ± 0.52b	1.35 ± 0.12b
TX9425
Control	7.67 ± 0.4a	0.64 ± 0.49a	52.05 ± 3.48a	6.66 ± 1.54a	41.19 ± 6.15a	23.99 ± 1.4a	2.4 ± 0.2a
Waterlogging	7.86 ± 0.35a	1 ± 0.41b	45.11 ± 3.67b	6.41 ± 1.86a	34.15 ± 7.82b	20.24 ± 1.39b	2.3 ± 0.2a
The different letters within a column for the same line represent significant difference between waterlogging treatment and control.

Table 2

The effect of waterlogging on the adventitious roots between TX9425 and Franklin
Treatment	Total Adventitious Length	Total Adventitious Surface Area	Adventitious Average Diameter	Total Adventitious Root Volume	Adventitious Root Number
Treatment	(cm)	(cm²)	(mm)	(cm³)	Adventitious Root Number
Franklin
Control	63.42 ± 12.36 a	14.8 ± 5.72a	0.75 ± 0.21a	0.29 ± 0.16a	6.17 ± 1.33a
Waterlogging	68.37 ± 12.31a	14.29 ± 3.04a	0.66 ± 0.04a	0.24 ± 0.06a	14.83 ± 2.33b
TX9425
Control	68.41 ± 12.07a	13.93 ± 2.96a	0.62 ± 0.09a	0.22 ± 0.07a	5.83 ± 1.33a
Waterlogging	168.85 ± 13.87b	33.24 ± 8.38b	0.63 ± 0.03a	0.52 ± 0.13b	23.67 ± 3.83b
The different letters within a column for the same line represent significant difference between waterlogging treatment and control.

Physiological And Anatomical Analysis Of Different Barley Varieties Under The Waterlogging Treatment

As shown in Fig. 2A-C, a significant genotype difference of the activities of SOD, CAT and POD in leaf was found. The antioxidant enzyme activities in both varieties all decreased under waterlogging, while the decrease in the tolerant TX9425 was lower. Under waterlogging treatment, the increase of MDA content of Franklin was about 2.1-fold, but that of TX9425 only increased by 1.3-fold (Fig. 2D). The root SOD activity of Franklin and TX9425 increased by 1.2- and 1.5-fold, respectively (Fig. 2E). The CAT activity of Franklin root increased by 1.6-fold, and that of TX9425 increased by 2.1-fold change (Fig. 2G). Moreover, POD enzyme activity of TX9425 increased by 1.4-fold change, and no significant difference was observed in the root of Franklin between waterlogging treatment and control (Fig. 2F). On the contrary, MDA content of Franklin increased by 2.1-fold change compared with the control in root, but the change in TX9425 was not significant (Fig. 2H).

Barley leaf anatomy is a typical monocotyledonous type consisting of epidermis, mesophyll and vascular tissue. Intercellular spaces existed among the mesophyll cells in the control. Under waterlogging, mesophyll cells of Franklin were severely damaged; on the contrary, the leaves of TX9425 developed more lysigenous aerenchyma under waterlogging compared with that of control (Fig. 3A). The adventitious root of barley was composed of epidermis, cortex and cylinder of vascular tissues. Cortex parenchyma cells of adventitious root formed larger number of lysigenous aerenchyma under waterlogging condition, compared with small intercellular space under control condition. Remarkably, the proportion of TX9425 aerenchyma was significantly higher than Franklin after three-week treatment (Fig. 3B). Under waterlogging, the adventitious root was formed in the section of shoot base in both lines, and more adventitious root primordiums were observed in TX9425 than Franklin. Otherwise, in the absence of waterlogging, few adventitious roots were found in both accessions (Fig. 3C, Table 2).

Analysis Of Barley Root Transcriptome Under Waterlogging Stress

In order to reveal molecular mechanisms of barley in response to waterlogging stress, roots were collected from TX9425 and Franklin after 0h, 24 h and 72 h waterlogging treatments. Each sample was subjected to three replicate treatments, and a total of 18 libraries were constructed. A high-throughput Illumina sequencing platform was used to sequence the transcriptome of barley. After removing adaptor sequences, low quality reads, and the reads with more than 10% ambiguous “N” bases, 2.87–7.58 GB data were obtained from each sample. The Q20 values of all transcriptomes were all above 96.42%, and the Q30 values at least 92.41%, indicating the high-quality sequencing data in the RNA-seq experiments (Table S1). Averagely, more than 63% of the valid reads were mapped into the reference barley genome. A principal component analysis (PCA) was conducted on the RNA-seq data set of 18 samples. The control and treatment samples of two genotypes could be clearly separated by the first principal component (PC1), which accounted for 98.53% of the total variation (Fig. 4A).

Identification Of Degs In Two Barley Varieties In Response To Waterlogging Stress

We further compared the DEGs in the two barley varieties subjected to waterlogging stress. We found a total of 3064 DEGs in TX9425 and 2297 DEGs in Franklin after 24 h waterlogging stress compared to the control, by using the parameters of log₂FC ≥ 1 and q value ≤ 0.05. 1335 DEGs were up-regulated and 1729 DEGs were down-regulated in TX9425, while there were 967 up-regulated genes and 1330 down-regulated genes in Franklin (Fig. 4B). By comparing the transcriptome profiles of TX9425 and Franklin, it was observed that total of 2183 DEGs were uniquely expressed in TX9425 only, whereas 1416 DEGs were distinctively found in Franklin under 24 h waterlogging treatment. In addition, 881 DEGs were common between the two genotypes (Fig. 4C).

5693 DEGs and 8462 DEGs were identified under waterlogging treatment (72 h) vs. control in TX9425 and Franklin, respectively. 2012 DEGs were up-regulated and 3681 DEGs were down-regulated in TX9425, while there were 3314 up-regulated genes and 5148 down-regulated genes in Franklin. There were more DEGs after 72 h waterlogging stress than 24 h waterlogging stress. The number of DEGs were significantly different between TX9425 and Franklin (Fig. 4B). 1664 DEGs were uniquely expressed in TX9425 only, whereas a total of 4083 DEGs were distinctively found in Franklin under 72 h waterlogging treatment. Besides, 4029 DEGs were common between the two genotypes (Fig. 4C).

In addition, to verify the reliability of the RNA-seq data, 10 DEGs were randomly selected for the qRT-PCR analysis. Significantly positive correlations were observed between qRT-PCR and RNA sequencing data (r² = 0.82). These results suggested that the RNA-seq data was credible (Fig. 4D).

Functional Annotation Of Waterlogging-responsive Degs

Gene ontology (GO) functional classification analysis was performed to categorize the functions of DEGs during waterlogging stress. The DEGs could be classified into three main ontologies, namely Molecular function, Biological process, and Cellular component (Table S2). As determined through a GO enrichment analysis of these DEGs, the DEGs in TX9425 and Franklin under 24 h waterlogging stress mostly functioned in biological process, metabolic process, transferase activity and catalytic activity (Fig. 5AB). After 72 h waterlogging, the DEGs of TX9425 mainly functioned in metabolic process, biological process, organic cyclic compound binding, heterocyclic compound binding and catalytic activity. However, the DEGs in Franklin mostly showed localization, oxidation-reduction process, protein binding and catalytic activity (Fig. 5CD, Table S2).

For KEGG pathway enrichment analysis, these DEGs were significantly (p < 0.01) enriched into 27 KEGG pathways (Table S3). Under 24 h waterlogging stress, the DEGs of TX9425 were mostly enriched into metabolic pathways and biosynthesis of secondary metabolites. However, the DEGs in Franklin primarily associated with biosynthesis of secondary metabolites and phenylpropanoid biosynthesis. Under 72 h waterlogging stress, the DEGs of TX9425 were mostly enriched into biosynthesis of secondary metabolites, MAPK signaling pathway, toll-like receptor signaling pathway. However, the DEGs in Franklin primarily associated with biosynthesis of secondary metabolites, biosynthesis of antibiotics and phenylpropanoid biosynthesis.

Analysis Of Degs Related To The Energy Metabolism

Energy deprivation is one of the major factors affecting waterlogged plant survival. This is due to mitochondrial respiration in plant roots is limited by hypoxia, and the acceleration of carbohydrate metabolism play more important role for plant survival. The KEGG enrichment analysis showed that many DEGs were involved in the starch and sucrose metabolism, glycolysis/fermentation pathway. As expected, we found that several DEGs, such as sucrose synthase, pyruvate kinase family protein, ATP-dependent 6-phosphofructokinase, alpha-amylase/subtilisin inhibitor, fructose-bisphosphate aldolase 2, were significantly accumulated in both TX9425 and Franklin.

In addition, some DEGs involved in glycolysis/fermentation pathway such as alanine aminotransferase, glyceraldehyde-3-phosphate dehydrogenase C2, alcohol dehydrogenase 1, L-lactate dehydrogenase A, pyruvate decarboxylase-2, were also significantly induced by waterlogging stress in two genotypes. In this study, we found that some genes had different expression levels in two varieties. For example, the pyruvate kinase family protein (HORVU2Hr1G040570) and fructose-bisphosphate aldolase 2 (HORVU3Hr1G088500) were induced at higher levels in TX9425 than Franklin after 24 or 72 h waterlogging treatment. The expression level of ATP-dependent 6-phosphofructokinase (HORVU5Hr1G019030), Alpha-amylase/trypsin inhibitor (HORVU7Hr1G035020), alcohol dehydrogenase 1 (HORVU1Hr1G082250, HORVU4Hr1G016810) were first increased and then decreased in TX9425, while it continuously increased in Franklin. Consequently, TX9425 had a greater energy state than Franklin under waterlogging stress (Table S4).

Hormone Regulation Of Barley Occurrence Under Waterlogging Stress

Hormones play an important role in plant response to environment stress. Here, we identified some DEGs related to hormones which are mainly involved in the biosynthesis of ethylene and auxin. Ethylene is biosynthesized by the activation of 1-aminocyclopropane-1-carboxylicacid synthase (ACS) and ACC oxidase (ACO). 2 ACS and 6 ACO were identified in TX9425 and Franklin. Two ACO genes (HORVU5Hr1G067490, HORVU5Hr1G067530) were significantly accumulated in both varieties, but the genes inductions in TX9425 were greater. 31 DEGs involved in auxin metabolism were identified in two genotypes, including 23 down-regulated and 8 up-regulated genes. After 72 h of waterlogging treatment, the expressions levels of HORVU1Hr1G025670, HORVU3Hr1G064590 and HORVU3Hr1G084840 in TX9425 were significantly higher than that in Franklin (Table S4).

Degs Involved In Ros Scavenging And Cell Wall Modifying Enzymes

Reactive oxygen species (ROS), which are produced when plants experiencing adversity stresses, can damage the normal functions in plant cells. To survive, plants have evolved multiple strategies such as activating antioxidant system remove excess ROS. 124 DEGs involved in ROS scavenging were found in our study, and most of them were down-regulated. These DEGs involves in the synthesis of glutathione S-transferase, peroxidase, catalase, L-ascorbate oxidase, most of which (82 genes, 66.12% of 124) were related to peroxidase. 8 genes related to glutathione S-transferase and 8 genes related to peroxidase were up-regulated in both genotypes, and the fold changes of these genes in TX9425 were significantly higher than that in Franklin (Table S4).

To adapt to waterlogging stress, plants also have evolved many mechanisms, such as the formation of adventitious roots and aerenchyma. The formation of aerenchyma was related to cell wall biosynthesis and loosening. As expected, we found 34 DEGs were involved in cell wall modifying enzymes, such as xyloglucan galactosyltransferase, pectinesterase, respiratory burst oxidase homologue. 8 DEGs were significantly up-regulated in both genotypes. Under waterlogging stress, the genes of HORVU2Hr1G101150 and HORVU4Hr1G081670 in TX9425 had significantly higher expression levels than that in Franklin (Table S4).

Characterization of HvADH4 genes in barley

A total of 44 ADH genes were identified in the barley genome based on the BLAST program. These genes were named HvADH1- HvADH44 according to their order of distribution on the chromosomes (Table S5). In the HvADH genes family, the length of coding sequences ranged from 99 (HvADH17) to 1524 (HvADH37). The size of corresponding amino acids varied between 32 and 507.The theoretical isoelectric point (PI) of these genes ranged from 4.51 to 9.66, and the molecular weight (Mw) varied from 3.47 to 48.11 kDa.

In this study, 17 ADH genes were found to have differential expressions between waterlogging treatment and control (Fig. 6). The highest differential expression was found for the HvADH4 in TX9425, and there was about a 50- fold difference between 24 h and control. We thus performed a standard method to isolate the HvADH4 from TX9425. Sequencing of HvADH4 showed that the full-length gene was 1158 bp in length and encoded 385 amino acids. Multiple amino acids alignment showed that HvADH4 protein shared two highly conserved ADH GroES-like (amino acid 36–156) and Zinc-binding dehydrogenase domains (amino acid 205–336) (Fig. 7A). Phylogenetic tree indicated that the HvADH4 has relatively high homology with protein from Triticum turgidum, and relatively distant sequence homology with the protein from Setaria italica (Fig. 7B).

Overexpression of HvADH4 enhanced waterlogging tolerance

To further verify the function of barley HvADH4 (HORVU1Hr1G082250), transgenic Arabidopsis plants overexpressing the HvADH4 gene from TX9425 were generated. Five week-old plants of the WT and three homozygous T3 transgenic lines were selected for waterlogging stress experiments. As shown in Fig. 8, no obvious changes in morphological and developmental phenotypes appeared between the WT and transgenic lines under normal conditions, while the transgenic lines grew better than WT plants after two weeks of waterlogging (Fig. 8A). Under waterlogging conditions, plant height was reduced by 49.1% in the WT, and 31.2, 36.1 and 40.3% in the transgenic lines (Fig. 8B). Compared to the control, the SPAD value was 61.6% lower in the WT, and 41.4, 51.4, 48.8% lower in the transgenic lines (Fig. 8C). The shoot fresh weights of the transgenic lines were 29.2, 37.2 and 36.5%, respectively, which were lower than in the control, while 65.8% smaller than that in the WT (Fig. 8D). The shoot dry weight decreased by 51.0% in the WT, and by 29.7, 13.3and 22.9% in the transgenic lines (Fig. 8E). In addition, the root lengths of the WT plants reduced greater than that of the transgenic lines during waterlogging stress (Fig. 8F). Furthermore, the average survival rate of the transgenic lines after waterlogging was 81.8%, but that of the WT was only 37.4% (Fig. 8G). Taken together, these data indicate that the overexpression of HvADH4 in Arabidopsis significantly enhances plant waterlogging tolerance.

Overexpression of HvADH4 increased the ROS scavenging capacity

To investigate the difference in physiological response to the waterlogging stress between the WT plants and the transgenic plants, the activities of antioxidant enzymes (SOD, CAT, and POD), ADH activity and MDA content were examined under normal and waterlogging conditions. Transgenic lines showed higher ADH activity than the WT plants even when they were under control conditions, and its remained significantly higher at subsequent times (Fig. 9D). There were no significant differences in the activities of antioxidant enzymes between transgenic lines and WT under normal growth conditions. After waterlogging, the major antioxidant enzymes activities increased markedly in both WT and transgenic plants, reaching peak levels at 6 days of treatment and then decreasing after 9 days of treatment. However, the fold changes were significantly greater in the transgenic lines than in the WT (Fig. 9ABC). MDA content is an important indicator to measure the level of lipid peroxidation. As shown in Fig. 9E, the MDA content in WT plants was significantly higher than that in transgenic lines, and this difference was more pronounced in the 6 d samples. Therefore, these results suggest that the overexpression of HvADH4 enhanced the scavenging ability of ROS in the plants and reduced the oxidative damage of plants under waterlogging stress.

Morpho-anatomical responses to waterlogging stress in barley

Waterlogging tolerance is a complicated trait, both physiologically and genetically [26, 27]. Waterlogging-induced plant growth inhibition is pervasive, including decreased dry weight (DW) in shoots/roots, leaf area, plant height, and chlorophyll content, this inhibition also causes yield penalty [4]. The growth of Franklin was severely affected after 21-day waterlogging treatment compared with TX9425, including plant height, tillers, leaf area, shoot fresh weight and dry weight. The shoot of Franklin appeared wilted and presented more yellow leaves under waterlogging than control. Remarkably, there are more morphological adaptations in waterlogging-tolerant TX9425 than susceptible Franklin.

Root is the first organ affected by waterlogging stress. The formation of new adventitious roots and aerenchyma is one of the most important characteristics that adapt to hypoxia environment [5, 28]. Newly-formed adventitious roots contains more aerenchyma, which not only provided a gas diffusion space with increased O₂ transport from shoots to roots, but also reduced the number of oxygen-consuming cells [29–30]. In the present study, TX9425 had significantly more adventitious roots and aerenchyma than Franklin under waterlogging stress. This phenomenon has been reported in cucumber [23] and maize [31].

Waterlogging Affects Energy Metabolism In Barley

The energy metabolism pathway is critical for plant survival under low-oxygen stress, which is related to starch and sucrose metabolism, glycolysis fermentation. As expected, we found that some DEGs, such as sucrose synthase 4, pyruvate kinase family protein, ATP-dependent 6-phosphofructokinase, fructose-bisphosphate aldolase 2, alcohol dehydrogenase, pyruvate decarboxylase-2, were all up-regulated after waterlogging. Compared to Franklin, the transcription levels of these genes were higher in TX9425 under both control and waterlogging conditions (Table S4).

Sucrose synthase (SUS) and sucrose phosphate synthase are key enzymes for the hydrolysis of sucrose, which play crucial roles in providing adequate sugar supply under waterlogging stress [32]. In low oxygen environments, the genes related to sucrose synthase in Arabidopsis [33], cucumber [23], and phalaris arundinacea [34], were all significantly up-regulated. In addition, knockout of SUS1 and SUS4 induced less tolerant to oxygen deficits compared to wild-type in maize [35] and Arabidopsis [36]. The overexpression of sucrose synthase genes in cucumber confers tolerance against hypoxia stress [37]. Here, the expression of sucrose synthase 4 and sucrose phosphate synthase 1F were higher in TX9425 than Franklin in 24 h waterlogging stress. Pyruvate decarboxylase (PDC) is one of the key enzymes of ethanolic fermentation. PDC1 and PDC2 play an important role in waterlogging stress tolerance in Arabidopsis and Actinidia deliciosa [38]. In this study, only PDC2 was significantly up-regulated under waterlogging stress (Table S4). This result suggest that TX9425 can accumulated more energy by decomposing more carbohydrates and amino acid, making it more readily adaptable to hypoxia stress.

Responses Of Ros And Hormonal To Waterlogging

Ethylene is an important hormone in response to waterlogging stress in plants, which can facilitate adventitious root and aerenchyma formation [5, 39]. In our previous study, we found that the ethylene content distinctly increased in TX9425 after waterlogging stress [40]. The RNA-Seq analysis showed that the expression of two ACO (HORVU5Hr1G067490 and HORVU5Hr1G067530) accumulated in both lines, while the gene expression in TX9425 were more greater. The results wereconsistent with published proteomic studies [41]. Auxin regulates the development of the lateral roots and plays a role in root growth [42]. 31 genes involved in auxin metabolism were identified in this research, and most of them were down-regulated. Interestingly, the auxin storage were negative association with adventitious root initiation in cucumber [23].

Hypoxia stress can cause plants to overproduce ROS, which can cause progressive oxidative damage. To respond to oxidative stress plants have developed antioxidant defense systems, including SOD, CAT, POD, GST [43]. Wang et al. [44] observed the activities of antioxidant enzyme activity increased in soybean under waterlogging conditons. On the contrary, wang et al. [34] found the activities of SOD, CAT, POD were significantly decreased in Phalaris arundinacea response to waterlogging stress. The reason for these two different results may be due to different treatment times and genotype [46]. In this study, we identified 124 DEGs associated with antioxidant system, in which most genes were down-regulated. Similar to POD enzyme activity, 8 genes related to POD were up-regulated in both genotypes and the fold changes of these genes in TX9425 were significantly higher than Franklin. In addition, one CAT gene (CAT2) in both lines were also up-regulated after waterlogging treatment, which indicating that this gene is a key regulator for CAT enzyme activity.

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play a role in the loosening of cell walls and affect cell proliferation. It is involved in plant growth and resistance to stress [47]. We found 24 DEGs were involved in XTH, and XTH 13 was significantly up-regulated in TX9425 under waterlogging stress (Table S4). The present study suggests that the XTH 13 may be closely associated with the formation of adventitious root and aerenchyma.

Importance Of The Adh Gene In The Response To Waterlogging Stress Of Barley

ADH is a major fermentative enzyme for oxidizing ethanol to acetaldehyde, which play a key role in resistance to waterlogging [21]. ADH family genes from tomato [48], rice [49], pyrus bretschneideri [50] and wheat [45] have been detected at the whole genome level. 22 ADH genes have been identified in wheat genome database [45], and we identified 44 ADH genes in barley genome.

It has been observed that the expression of ADH genes is significantly elevated in soybean roots under hypoxia stress [51]. Two genes in kiwifruit roots were also significantly induced after waterlogging treatment. The overexpression of AdADH1 and AdADH2 of kiwifruit enhanced waterlogging tolerance in transgenic Arabidopsis [21]. Shen et al. [45] found that TaADH1/2, TaADH3 and TaADH9 play an important role in waterlogging tolerance of wheat, which were significantly induced by waterlogging. To further validated ADH gene’s function, some transgenic assays were conducted. Overexpression of GmAdh2 gene in transgenic soybean enhanced the glycolysis and alcohol fermentation, and significantly increased the germination of transgenic lines under waterlogging [20]. In contrast, overexpression of the Arabidopsis ADH1 gene and increased ADH activity do not affect ethanol levels and flooding survival tolerance under hypoxic conditions compared to wild type cells [19]. Thus, the function of ADH genes varied with the different of plants and stages. In this study, 17 ADH genes were differentially expressed, of which 7 genes were significantly up-regulated after waterlogging stress. HvADH4 of TX9425 showed the highest level of differential expression. Overexpression of HvADH4 in transgenic Arabidopsis enhanced plant waterlogging tolerance, which could be caused by increasing activities of fermentation and antioxidant enzymes. Therefore, HvADH4 play a key role in waterlogging stress and may be used to enhance waterlogging tolerance in barley breeding.

In this study, two barley varieties with different waterlogging tolerances were subjected to waterlogging treatment. Analyses of the morphologic and physiologic indicators revealed that TX9425 exhibited the stronger waterlogging tolerance than Franklin. To further explore the mechanism controlling waterlogging tolerance in barley, RNA-seq analysis was performed. We identified 3064, 5693 and 2297, 8462 DEGs in TX9425 at 24 h, 72 h and Franklin at 24 h, 72 h, respectively. GO and KEGG analyses showed that the waterlogging tolerance of barley was closely related to energy metabolism, hormone regulation, ROS scavenging, cell wall modifying enzymes. 17 ADH genes were identified to be waterlogging responsive in barley. Among them, the expression level of HvADH4 was significantly different between control and waterlogging. In addition, transgenic Arabidopsis with HvADH4 has improved waterlogging tolerance by deterring ROS accumulation. This work provides important genes for barley breeding with waterlogging tolerance in future.

Plant materials and treatments

TX9425 is waterlogging-tolerant feed barley originating from China, while Franklin is waterlogging-sensitive Australian malting barley [52]. The two accessions were obtained from the National Crop Genebank of China (NCGC, Beijing). The seeds of two genotypes were sown in plastic pots (22 cm×25 cm) filled with the mixture of nutritional substance and vermiculite. Plants were grown in a greenhouse at a temperature of 20°C/day and 15°C/night. Waterlogging treatments started at the four-leaf stage and lasted for three weeks. The waterlogged plants were irrigated with tap water to 2–3 cm above the nutritional substance surface. The control plants were irrigated as needed to avoid drought stress or waterlogging stress. The experiment was carried out with three biological replications. After 21-day treatment, roots and leaves were collected and carefully washed with water for further analysis.

Morphological Characteristic Investigation

The leaf area and physiological traits of leaf were measured on the first fully expanded leaf below the shoot apex. Leaf chlorosis is the proportion of each plant that loses its green color (yellow) [52].

The roots were carefully rinsed with hydropneumatic elutriation device and detached from their nodal bases. Adventitious roots were arranged and floated on shallow water in a glass tray (30 cm × 30 cm), then scanned using Epson Expression 1680 scanner (Seiko Epson Corp, Japan), finally analyzed using WinRHIZO Root Analyzer System (Regent Instruments Inc., Canada) to measure length, diameter, surface area and root volume [53]. The parameters related to adventitious roots were measured with three biological replications, and six plants were selected for each replicate.

Tissue Anatomy

After 21-day waterlogging treatment, samples were obtained from the mature zone of adventitious root (approximately 6 cm from the root apex), shoot base (root node), and leaf (the first fully expanded leaf below the shoot apex). They were cut into 0.5 cm segments, and immediately immersed in 0.1 M glutaraldehyde-phosphate buffer fixative (pH 7.2) at 4°C for at least 3 h. Subsequently, the samples were dehydrated in a graded ethanol series of 20%, 40%, 60%, 80%, 90%, 95%, and 100% (15 min each). Ethanol was replaced with propyleneoxide, and the tissues were infiltrated and embedded in SPI low-viscosity Spurr’s resin [54]. Sections of 1 µm thick were cut with a glass knife on a Leica Ultracut R (Leica Microsystems, Inc., Germany), stained with 0.5% methyl violet for 10 min, and photographed under a light microscope (Leica, Germany). Root aerenchyma area and total root cross-sectional area were measured using the Image-pro plus (IPP) software (Media Cybernetics, USA).

Physiological Trait Analysis

Fresh leaves and roots (0.5 g each) were rinsed thoroughly with distilled water. The crude enzymatic extracts of each line were prepared in 0.05 M phosphate buffer (pH 7.8) after grinding with a pestle and milling to powder in liquid nitrogen. The homogenate was filtered through four layers of muslin cloth and centrifuged at 12 000 g for 10 min at 4°C. The final supernatants were used for physiological and biochemical assays. Chlorophyll content was determined by using the SPAD-502. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the content of malondialdehyde (MDA) and alcohol dehydrogenase (ADH) were measured using the corresponding assay kits (Institute of Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s instructions [40].

Rna-seq And Transcription Analysis

The root of TX9425 and Franklin were collected after waterlogging treatment for 24 h, 72 h, and control without waterlogging. Each treatment was processed with three biological replicates. Total RNA was extracted using the Plant RNA Purification Kit (Tiangen, Beijing, China). Twelve RNA-seq libraries (two accessions × two treatment × three biological replicates) were constructed by Novogene Bioinformatics Technology (Beijing, China) and sequenced by an Illumina HiSeq 2500 platform. Detailed process of transcriptome analysis as described in previous research [55]. The sequencing data were deposited in the NCBI SRA database (Bioproject ID: PRJNA889532). DEGseq was used to identify differentially expressed genes for RNA-seq data between waterlogging treatment and control. And the DEGs were further filtered with P value ≤ 0.05 and normalized fold change ≥ 1.

Quantitative Real-Time RT-PCR

To confirm the reality of candidate genes screened from RNA-seq. 10 candidate genes were selected to further validate by quantitative (qRT-PCR). The method of qRT-PCR was described as previous report [40]. The specific primers used for target were designed using the Primer 6. All the primers are listed in Supplementary Table S5. The Hvactin gene was used as the internal control. Target genes’ relative expression levels were determined as 2^−△△Ct. Three biological replicates and three technical repeats were performed in all the qRT-PCR experiments.

Cloning and Bioinformatic Analysis of HvADH4

Total RNA was extracted from barley leaves of TX9425, and cDNA was synthesized as a template by M-MLV reverse transcriptase (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer’s instructions. Primers of HvADH4 full-length CDS were designed by Primer 6. The PCR products were detected using agarose gel electrophoresis (1.0%). The sequence accuracy of the cloned genes was confirmed by DNA sequencing. The amino acid composition was analyzed with DNAMAN 9.0 software. The molecular weight and pI were examined the online software of ExPASy ProtParam (http://web.expasy.org/protparam/). Homologs of HvADH4 in other plant species were analyzed by the BLAST tool of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). MEGA 7.0 program was used for phylogenetic tree mapping by the neighbor-joining method and 1000 bootstrap replicates.

Candidate gene validation by transgenic Arabidopsis

To further verify the candidate gene, transgenic Arabidopsis plants were generated by floral dipping. The detailed design and methods have been previously described [56]. The Gateway technology (Invitrogen, USA) was used to constructed transgenic lines. Further genetic analysis was performed using the homozygous T₃ generation. Five-week-old Arabidopsis plants (T3 lines) were used for waterlogging treatment. The control plants were kept in normal conditions with regular watering. After the treatment of two weeks, the phenotypic and physiological traits were observed and recorded.

Data Analysis

For phenotypic, physiological parameter, and gene expression analysis was analyzed by Student’s t-test through the SPSS software. *and** represent the significant differences at p < 0.05 and p < 0.01, respectively. All data were presented as mean ± standard deviation (SD) and were measured at least three times. And three biological replicates were set.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors affirm no conflict of interest.

Acknowledgements

Not applicable.

Research involving plants

The authors confirm that all the experimental methods and plants complied with relevant institutional, national, and international guidelines and legislation.

Authors’ Contributions

J. conceived and designed the study, supervised the experiments; H. and S.F. compiled and finalized the article, H.Y. and C.Y. performed the experiments, Y. and H.T. analyzed the data, H.Y. and H.Q. drafted and wrote the manuscript, J. and M. revised the manuscript. All authors read and approved the final manuscript.

Funding

The work was sponsored by Natural Science Foundation of Jiangsu Province (BK20201215), National Barley and Highland Barley Industrial Technology Specially Constructive Foundation of China (CARS-05). Jiangsu (Lianyungang) Modern Agricultural (Science and technology comprehensive demonstration base (JAST[2021]170).

Availability of data and materials

The transcriptome datasets supporting the conclusions of this study are available in the NCBI (BioProject: PRJNA889532).

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No competing interests reported.

TableS1.xlsx
Table S1 The information of transcriptome libraries.
TableS2.xlsx
Table S2 Summary of GO results of the differentially expressed genes.
TableS3.xlsx
Table S3 KEGG enrichment analysis of the differentially expressed genes.
TableS4.xlsx
Table S4 Selected differentially expressed genes with altered expression in roots of waterlogged TX9425 and Franklin that are involved in major metabolism
TableS5.xlsx
Table S5 Properties and locations of the predicted HvADH proteins in barley.

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Transcriptome analysis of barley (Hordeum vulgare L.) under waterlogging stress and over-expression of HvADH4 gene confers waterlogging tolerance in transgenic Arabidopsis

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Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Phenotypic analysis of different barley varieties under the waterlogging treatment

Physiological And Anatomical Analysis Of Different Barley Varieties Under The Waterlogging Treatment

Analysis Of Barley Root Transcriptome Under Waterlogging Stress

Identification Of Degs In Two Barley Varieties In Response To Waterlogging Stress

Functional Annotation Of Waterlogging-responsive Degs

Analysis Of Degs Related To The Energy Metabolism

Hormone Regulation Of Barley Occurrence Under Waterlogging Stress

Degs Involved In Ros Scavenging And Cell Wall Modifying Enzymes

Characterization of HvADH4 genes in barley

Overexpression of HvADH4 enhanced waterlogging tolerance

Overexpression of HvADH4 increased the ROS scavenging capacity

Discussion

Morpho-anatomical responses to waterlogging stress in barley

Waterlogging Affects Energy Metabolism In Barley

Responses Of Ros And Hormonal To Waterlogging

Importance Of The Adh Gene In The Response To Waterlogging Stress Of Barley

Conclusions

Methods

Plant materials and treatments

Morphological Characteristic Investigation

Tissue Anatomy

Physiological Trait Analysis

Rna-seq And Transcription Analysis

Quantitative Real-Time RT-PCR

Cloning and Bioinformatic Analysis of HvADH4

Candidate gene validation by transgenic Arabidopsis

Data Analysis

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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