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Physiology and Molecular Biology of Plants logoLink to Physiology and Molecular Biology of Plants
. 2019 Nov 30;26(1):143–162. doi: 10.1007/s12298-019-00727-8

Impact of arbuscular mycorrhizal fungi (AMF) on gene expression of some cell wall and membrane elements of wheat (Triticum aestivum L.) under water deficit using transcriptome analysis

Zahra Moradi Tarnabi 1, Alireza Iranbakhsh 1,, Iraj Mehregan 1, Rahim Ahmadvand 2
PMCID: PMC7036378  PMID: 32153322

Abstract

Mycorrhizal symbiotic relationship is one of the most common collaborations between plant roots and the arbuscular mycorrhizal fungi (AMF). The first barrier for establishing this symbiosis is plant cell wall which strongly provides protection against biotic and abiotic stresses. The aim of this study was to investigate the gene expression changes in cell wall of wheat root cv. Chamran after inoculation with AMF, Funneliformis mosseae under two different irrigation regimes. To carry out this investigation, total RNA was extracted from the roots of mycorrhizal and non-mycorrhizal plants, and analyzed using RNA-Seq in an Illumina Next-Seq 500 platform. The results showed that symbiotic association between wheat and AMF and irrigation not only affect transcription profile of the plant growth, but also cell wall and membrane components. Of the 114428 genes expressed in wheat roots, the most differentially expressed genes were related to symbiotic plants under water stress. The most differentially expressed genes were observed in carbohydrate metabolic process, lipid metabolic process, cellulose synthase activity, membrane transports, nitrogen compound metabolic process and chitinase activity related genes. Our results indicated alteration in cell wall and membrane composition due to mycorrhization and irrigation regimes might have a noteworthy effect on the plant tolerance to water deficit.

Keywords: Arbuscular mycorrhizae, Plant cell wall, RNA-Seq, Triticum aestivum, Water deficit

Introduction

Bread wheat (Triticum aestivum L.) is one of the main crops containing high carbohydrate content that provides more than one-third of the world population’s food (Gill et al. 2004). Water deficiency is considered as one of the most significant limiting factors of wheat crop production and plant growth parameters in Iran (Nezhadahmadi et al. 2013), and other arid and semi-arid areas all around the world (Li et al. 2019). One of the most beneficial cooperation in the environment that can decrease adverse effects of abiotic stresses is arbuscular mycorrhizal symbiosis, in which mutualistic confrontation is beneficial for both plant and fungi (Ye et al. 2019). To colonize and establish a relationship, root cell wall is the first and basic border that must be passed by AMF, and initiate an interaction and nutrient exchange (Rich et al. 2014). Cell wall as the outermost part of the plant cells plays crucial roles in growth, development, preservation of the cell shape, as well as protection against variety of external and internal factors for plant (Essahibi et al. 2018). As apoplastic compartments of plant cell, cell wall and membrane are directly involved in establishment and development of the mycorrhizal symbiosis. For constructing the symbiotic structure in roots of host plant, it needs to access cell wall and membrane materials to make a conservative envelope which keeps and improves symbiotic relationship, and also plant cells integrity (Essahibi et al. 2018). Guether et al. (2009) have reported the induced genes related to plasma membrane and cell wall activities affected by AMF as plant response to water stress. Nanjareddy et al. (2017) performed comparative transcriptome analysis of Phaseoulus vulgaris symbiotic-roots with AMF and identified differentially expressed genes associated with defense responses and cell wall. Recently, next generation sequencing (NGS) based on transcriptome (TC) has provided rapid genome wide transcript profiling. Transcriptome based analysis of genes helps to better understand biological processes including development, organogenesis and responses to biotic and abiotic stresses (Zhu et al. 2012; Brenchley et al. 2012; Jayasena et al. 2014; Lucas et al. 2014). Dong et al. (2012) used Illumina (NGS) for studying gene expression changes in Sinapis alba under water stress. However, identification of all genes associated with AMF symbiosis demands extensive further research (Garcia et al. 2017; Sugimura and Saito 2017; An et al. 2018; Nanjareddy et al. 2017; Li et al. 2018; Jacott et al. 2017). The experimental hypothesis of the present study was that the mycorrhization could improve the wheat tolerance to water stress by changing cell wall and membrane genes expression pattern in addition to the other common factors that influence the plant tolerance. To test the hypothesis, the contribution of an AM fungal species (Funneliformis mosseae) to a drought semi-tolerant cultivar, “Chamran”, was studied in both normal irrigation and water deficit conditions. Moreover, gene expression changes in cell membrane and cell wall were investigated using transcriptome analysis. This can be the first study on bread wheat plants, in which we exposed plants to four different growth conditions using AMF alleviation and different irrigation regimes, and our concentration has been on some genes related to cell wall and membrane and their relation with enhancement of plant tolerance to water shortage.

Materials and methods

Plant material and growth conditions

The wheat cultivar Chamran, a drought semi-tolerant cultivar, was used in this study. Seeds were surface-sterilized in 70% ethanol for 2 min, rinsed in sterile distilled water for three times, and soaked in 40% sodium hypochlorite for 10 min. Finally, seeds were rinsed six times in sterile distilled water. To germinate seeds, they were transferred into sterile Petri dishes with wet filter papers at 26 °C. After 3 days, 10 germinated seeds were planted into each 0.5 L plastic autoclaved pots containing sterile field soil, sand and perlite (2:1:1, V:V:V). The air-dried soil samples which collected from Tehran, Iran, were passed through a 2 mm sieve, and diluted with sand and perlite, sterilized for 1 h using 100 °C steaming on 3 consecutive days. The pH of soil was 8; the texture of soil before mixing was composed of 35.5% sand, 15.4% clay, 48.7% silt, and almost 0.5% organic matters.

AMF and water deficit treatments

In order to evaluate the effect of AMF and water deficit on transcript level of Chamran cultivar, four combinations of treatments were performed in a factorial experiment design in two levels for both AMF (inoculated and non-inoculated) and irrigation (normal and water deficit) with three replications and 10 seedlings per replicate. For AMF inoculation, Funneliformis mosseae, isolate Gmb17, was provided by Department of Plant Protection, Faculty of Agriculture, Rafsanjan University, Iran.

Inoculum was prepared in the pots containing 100–120 mycorrhizal infection unit/g of a mixture of soil and sand and the germinated seeds were planted.

The pots of each level of AMF treatment, inoculated and non-inoculated plants, were irrigated in two levels including normal irrigation (each pot three times weekly, 50 ml each time) and water deficit condition (each pot three times weekly irrigating, 25 ml per time) for 8 weeks.

Mycorrhizal colonization assessment

For colonization assessment, four 8-week old plants and 15 segments of roots of both AMF treatments were removed and washed in tap water. After washing, the roots were cleared in 10% KOH solution, and stained with 0.1% trypan blue according to Phillips and Hayman method (1970). For evaluating of colonization, roots were cut down into 1 cm fragments and located on microscopic slides in lactoglycerol. Analysis of colonization percentage was performed based on McGonigle et al. (1990) and under a light microscope.

Assessment of plant growth parameters

After 8 weeks, plants were harvested and some growth related parameters such as root and shoot lengths, root and shoot fresh weights, and spike fresh weight were measured.

RNA extraction and NGS sequencing

Eight weeks after transplanting germinated seeds into the pots, two biological replicates of root samples were prepared from each treatment, including mycorrhizal plants in normal irrigation and water deficit conditions and also non-mycorrhizal plants in normal irrigation and water deficit condition. Each biological replicate contained a pooled root sample of 10 seedlings. Then, total RNA was extracted from 30 mg of root tissues of each pooled sample using Qiagen kit according to the manufacturer’s instruction and immediately stored at − 80 °C. RNA integrity and concentration was measured using gel electrophoresis and Agilent 2100 Bio-analyzer. An Illumina TruSeq stranded total RNA HT sample preparation kit was used to convert total RNA into library based on manufacturers instruction with an average library size of 360 bp. The reads were de-multiplexed with one allowed mismatch and “no lane splitting”. Libraries were sequenced using an Illumina Next Seq 500/High Output Chemistry with 150 bp paired-end reads. Estimated output was approximately 120 Gb and 800 Million reads, paired-end sequencing with 150 bp read length generated from eight samples.

Paired-end RNA-seq reads were mapped to wheat reference genome (http://plants.ensemble.org/Triticum_aestivum; Clavijo et al. 2017) using TopHat v2.1.1 (http://ccb.jhu.edu/software/tophat/index.shtml; Kim et al.; licensee BioMed Central Ltd. 2013) and Bowtie2 v2.2.9 (Langmead and Salzberg 2012). Assembling of transcripts and determining their expression level were performed using Cufflinks (v2.2.1) based on fragments per kb of exon per million mapped reads (FPKM). A q value of 0.01 and Log twofold change (LOG2FC) ≥ 2 and ≤ − 2 was considered as significant threshold for gene differential expression.

Quantitative real-time-PCR analysis

The quantitative RT-PCR was performed to validate the RNA-Seq data in four randomly selected transcripts. Gene specific primers were designed using Oligo 7 software. Total RNA from control and treatment of each condition was treated with 1.5 µl DNAaseI to remove DNA contamination. cDNA was synthesized by mixing 2 µg of total RNA, 0.2 µl specific primers, 0.5 µl Oligo dT and 10 µl DEPC water using Thermo-scientific kit according to manufactures’ instructions. Then, the mixture was incubated at 65 °C for 10 min; 10 µl of thermo-scientific master mix 2X was added, and the samples were incubated at 42 °C for 90 min.

Real-time PCR was carried out using Step-one RT-PCR and 1 µl of cDNA template, 0.3 µl of primers, 3 µl SYBR green mix 5X Takara mix, 0.25 µl ROX and 15 µl DEPC water. The PCR cycling program included of: 94 °C for 120 s, 94 °C for 15 s, 60 °C for 30 s and 72 °C for 20 s, and melting curve point was tested in the range of 60 and 95 °C with a heating rate of 5 °C for every 15 s. Treatment and control samples were assessed in two independent biological replicates.

The relative expression was calculated using 2−∆∆CT method (Rasmussen 2001). Elongation factor-1-alpha (EF-1α) gene (AF475129), a housekeeping gene, was used as endogenous gene to normalize the expression level of the target genes (Balestrini and Lanfranco 2006).

Statistical analysis

Statistical analysis of data was carried out using SPSS program. The mean differences were estimated based on the Duncan test at a probability of 5%.

Result

Detection of mycorrhizal colonization percentage

Non-inoculated roots did not show any colonization with AMF in roots. The colonization rate in inoculated roots under water deficiency and normal irrigated plants was 55% and 72.5% respectively (Fig. 1).

Fig. 1.

Fig. 1

Colonization rate of two inoculated treatments: D + M: mycorrhizal plants under water deficiency, N + M: non-mycorrhizal plants with normal irrigation

Analysis of plant growth related traits

The individual application of AMF significantly increased root fresh weight by 23.8% when compared to the untreated control (Fig. 2a). Water deficit treatment led to the significant decrease in root fresh weight by 72.5% relative to the control. Interestingly, the mycorrhiza treatment mitigated the growth inhibition associated with water deficit by 15.5% (Fig. 2a). Showing similar trend, AMF inoculation enhanced shoot fresh weight by 15.6% than the control. Water deficit significantly reduced shoot fresh weight by 56.7% which was relieved by AMF supplementation and reached to 32% in the AMF + Water deficit treatment group (Fig. 2b). Similarly, the water deficit was associated with significant decrease in both root and shoot length (Fig. 2c, d). Moreover, water deficit-associated reduction in spike fresh weight was significantly alleviated by AMF utilization (Fig. 2e).

Fig. 2.

Fig. 2

Water deficit and/or mycorrhiza-mediated changes in growth-related parameters. a root fresh weight, b shoot fresh weight; c root length, d shoot length; e spike fresh weight. Bars indicate the standard errors

Bioinformatic analysis of NGS RNA-Seq data

To perform bioinformatics analysis, TopHat Alignment software v 2.1.1 was used, and reads trimmed to 125 bp; also, read mapping and FRKM estimation of reference genes and transcripts were performed using TopHat 2 aligner. Assembly of novel transcripts, and differential expression of novel and reference transcripts were implemented with Cufflinks v 2.2.1 software. The summary of the bioinformatics data obtained in this study is shown in Table 1.

Table 1.

Read numbers achieved from bioinformatics analysis

Sample name Sequenced reads Overall read mapping rate (%) Aligned pairs Multiple alignments Discordant alignments Concordant alignment (%)
A (D − M) 59651235 56.1 31304199 2720387 (8.7%) 757957 (2.4%) 51.2
E (D − M) 63094284 55.1 32499992 3114223 (9.6%) 898573 (2.8%) 50.1
B (D + M) 68576329 51.2 32762588 5609760 (17.1%) 1951342 (6.0%) 44.9
D (D + M) 81053620 49.9 37989411 5144110 (13.5%) 1626126 (4.3%) 44.9
G (N − M) 72618555 44.6 29503597 2708038 (9.2%) 642998 (2.2%) 39.7
K (N − M) 63905579 38.7 22643132 4092368 (18.1%) 1217924 (5.4%) 33.5
H (N + M) 64067079 49.4 29488910 2548789 (8.6%) 656539 (2.2%) 45.0
I (N + M) 59966731 51.2 28647390 2643137 (9.2%) 742274 (2.6%) 46.5

D − M: non-mycorrhizal roots under water deficiency, N − M: non-mycorrhizal roots with normal irrigation, D + M: mycorrhizal roots under water deficiency, N + M: mycorrhizal roots with normal irrigation

Paired-end RNA-Seq reads were obtained from each library aligned to Triticum aestivum genome sequence accessible at http://plants.ensemble.org/Triticum_aestivum for distinguishing per gene molecular function or biological processes. Log 2 fold change (LOG2FC) ≥ 2 and ≤ − 2 were considered for dividing genes to two up-regulated and down-regulated genes, subsequently.

Differentially expressed genes

In the present study, we generated four libraries including non-mycorrhizal roots with normal irrigation (N − M), mycorrhizal roots with normal irrigation (N + M), non-mycorrhizal roots under water deficiency (D − M) and mycorrhizal root under water deficiency (D + M).

While the published “Triticum aestivum L.” transcriptome includes 174639 gene transcripts, from among, 104390 are coding genes and 18087 are non-coding genes (http://plants.ensemble.org/Triticum_aestivum), we totally found 114428 genes expressed in wheat roots from four libraries of which, 12774 transcript were differentially expressed, 8754 transcripts had known biological process and molecular function; 1022 transcripts were non-coding and others were still unknown for their biological and molecular functions.

The number of identified genes in each library and treatment is summarized in Table 2.

Table 2.

Summary of identified genes in four different libraries

Data N − M N + M D − M D + M
Total annotated genes 83691 84513 84418 85454
Up-regulated genes 10966 11825 10329 13405
Down-regulated genes 1709 2608 3226 2081
Significant genes based on expression difference 1759 4226 2701 4808
Genes with known function 1156 2535 1680 3049
Genes with unknown function 603 1691 1021 1759
ncRNA (in significant genes) 27 149 135 132

N − M: non-mycorrhizal roots with normal irrigation, N + M: mycorrhizal roots with normal irrigation, D − M: non-mycorrhizal roots under water stress D + M: mycorrhizal root under water stress

Transcriptome in each condition was screened for differentially expressed genes (DEGs) associated with cell components namely cell wall and plasma membrane. Some genes were only induced or suppressed in one special treatment and considered as specific responsive genes.

DEGs associated with cell wall and plasma membrane under water deficiency (D − M vs N − M)

Considering the cutoff threshold of log2 (≥ 2 and ≤ − 2) and q value of ≤ 0.01, a total number of 30 genes, associated with cell wall and plasma membrane showed significantly differential expression under water deficit. Among the up-regulated genes, chitinase activity, lipid metabolic process, lignin catabolic process and malate dehydrogenase activity genes were only induced in plants under water deficit condition but not in normal irrigation regime suggesting water deficit responsive genes (see Tables 3, 4 and Fig. 3). Compared to normal irrigation in our experiments, water deficit could also up-regulate the expression of genes related to carbohydrate metabolic process, lipid catabolic process, oxidation/reduction process, hydrolase activity and plant-type cell wall organization, and down-regulate the expression of genes associated with carbohydrate metabolic process, lipid metabolic process, transmembrane transport and cell wall macromolecule catabolic process (13 genes).

Table 3.

Induced and suppressed transcripts identified in wheat cv. Chamran under water-deficit

Gene ID Protein name Value_1 (D-M) Value_2 (N-M) Accession ID in NCBI Source organism
TRIAE_CS42_4BL_TGACv1_321131_AA1055910 Anti-sigma-I factor RsgI6-like 0 0.67093 XM_020311775.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_320705_AA1046770 Xyloglucan endotransglucosylase/hydrolase protein 31-like 0 0.41745 XM_020338007.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_U_TGACv1_642890_AA2124500 Chi gene for endochitinase 1.97046 0 X76041.1 T.aestivum (Chinese spring)
TRIAE_CS42_7DS_TGACv1_622542_AA2041460 Patatin-like protein 2 3.80167 0 XM_020293126.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_6DL_TGACv1_526537_AA1686380 Putative laccase-9 19.6026 0 XM_020320013.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_344866_AA1150230 Malate dehydrogenase [NADP] 1 12.0765 0 XM_020325953.1 Aegilops tauschii subsp. tauschii
Table 4.

DEGs identified in wheat cv. Chamran showing down-regulation and up-regulation under water deficit

Gene ID Protein name Log2 (fold change) Accession ID in NCBI Source organism
TRIAE_CS42_4BS_TGACv1_327875_AA1077360 Protein ZINC INDUCED FACILITATOR-LIKE 1-like − 7.0352 XM_020334708.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129932_AA0399780 Beta-glucosidase BoGH3B-like − 5.3941 XM_020296045.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_133066_AA0441180 WT003_M11 − 3.9284 AK332352.1 Triticum aestivum L.
TRIAE_CS42_3DL_TGACv1_250209_AA0864280 cht4 chitinase − 3.6586 KR049250.1 Triticum aestivum L.
TRIAE_CS42_4DS_TGACv1_361542_AA1169880 Putative clathrin assembly protein At1g33340 − 3.1668 XM_020300262.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AL_TGACv1_095187_AA0308020 Beta-glucosidase BoGH3B-like − 3.1291 XM_020296045.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129576_AA0389360 Putative xyloglucan endotransglucosylase/hydrolase protein 13 − 2.8189 XM_020307537.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_130022_AA0401820 WT003_N09 − 2.6079 AK332377.1 Triticum aestivum L.
TRIAE_CS42_2AL_TGACv1_093126_AA0272780 Fructan 6-exohydrolase − 2.5755 AB196524.1 Triticum aestivum L.
TRIAE_CS42_U_TGACv1_643084_AA2127090 Lipase-like − 2.5461 XM_020344369.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DL_TGACv1_158029_AA0506490 Xyloglucan endotransglucosylase/hydrolase protein 24-like − 2.2207 XM_020307539.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_328000_AA1080810 HIPL1 protein-like − 2.1827 XM_020300864.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_328633_AA1091330 sn1-specific diacylglycerol lipase beta − 2.0071 XM_020326437.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7DL_TGACv1_604135_AA1994270 Patatin-like protein 2 2.01008 XM_020306914.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_132929_AA0440510 Beta-glucosidase 2.02 AB236423.1 Triticum aestivum L.
TRIAE_CS42_1BS_TGACv1_049914_AA0164140 tplb0043f07 2.04793 AK450976.1 Triticum aestivum L.
TRIAE_CS42_4BL_TGACv1_320333_AA1035610 B12 alpha expansin 6 (EXPA6) 2.12293 KC441067.1 Triticum aestivum L.
TRIAE_CS42_1BS_TGACv1_049965_AA0164870 Phenylalanine ammonia-lyase-like 2.18288 XM_020317649.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129561_AA0388630 Beta-glucosidase 2.25564 AB100035.1 Triticum aestivum L.
TRIAE_CS42_4DL_TGACv1_343409_AA1134090 Omega-3 fatty acid desaturase, endoplasmic reticulum-like 2.38774 XM_020337300.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_328921_AA1095710 Beta-tubulin (TUB) 2.71602 MG852134.1 Triticum aestivum L.
TRIAE_CS42_1BS_TGACv1_050729_AA0174830 Phenylalanine ammonia-lyase-like 2.81184 XM_020319266.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_246242_AA0834280 Expansin-A24-like 3.38153 XM_020322455.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AL_TGACv1_094425_AA0297430 Beta-glucosidase 5.74049 AB548284.1 Triticum aestivum L.
Fig. 3.

Fig. 3

Log2 (fold change) of down and up-regulated genes under water stress. 1: transmembrane transport, 2, 3, 6, 8, 9, 12, 15, 19: carbohydrate metabolic process, 4: chitinase activity, 5: clathrin coat assembly, 7: xyloglucan metabolic process, 10, 13, 20: lipid metabolic process, 11: cellular glucan metabolic process, 14: lipid catabolic process, 16, 18, 22: l-phenylalanine catabolic process, 17, 23: plant-type cell wall organization, 21: microtubule-based process, 24: beta-glucosidase activity

DEGs associated with cell wall and plasma membrane in AMF-colonized wheat root (N − M vs N + M)

This experiment was performed to identify the expressed genes in the symbiosis between mycorrhiza and wheat in normal irrigation. Generally, 23 and 24 up- as well as down-regulated genes related to cell wall and plasma membrane were identified, respectively (Table 5, Fig. 4).

Table 5.

DEGs identified in wheat cv. Chamran showing down-regulation and up-regulation in AMF colonized roots under normal irrigation

Gene ID Protein name Log2 (fold change) Accession ID in NCBI Source organism
TRIAE_CS42_2AL_TGACv1_094425_AA0297430 Beta-glucosidas − 5.0471 AB548284.1 Triticum aestivum L.
TRIAE_CS42_2BL_TGACv1_129561_AA0388630 Beta-glucosidase − 3.7674 AB100035.1 Triticum aestivum L.
TRIAE_CS42_2AL_TGACv1_093096_AA0271920 Probable xyloglucan endotransglucosylase/hydrolase protein 12 − 3.7085 XM_020317472.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7AS_TGACv1_569995_AA1828380 Expansin-A17-like − 3.3671 XM_020293675.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3AS_TGACv1_212195_AA0698660 Xyloglucan endotransglycosylase/hydrolase protein 8-like − 3.3353 XM_020316160.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4AS_TGACv1_306275_AA1005550 Phytosulfokines 4-like − 3.3329 XM_020303724.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_131489_AA0428390 Xyloglucan endotransglucosylase/hydrolase protein 12 − 3.1424 XM_020317472.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_329606_AA1102700 Xylanase inhibitor − 3.0417 AB302973.1 Triticum aestivum L.
TRIAE_CS42_7DS_TGACv1_622353_AA2038220 Expansin-A17-like − 2.9 XM_020293675.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_342392_AA1112380 Phytosulfokines 4-like − 2.6674 XM_020303724.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1DL_TGACv1_062431_AA0214390 Patatin-like protein 1 − 2.4622 XM_020344309.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BL_TGACv1_033545_AA0140280 Patatin-like protein 1 − 2.4214 XM_020344309.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_328921_AA1095710 Beta-tubulin (TUB) − 2.3301 MG852134.1 Triticum aestivum L.
TRIAE_CS42_4BS_TGACv1_327950_AA1079560 WT005_P03 − 2.299 AK333221.1 Triticum aestivum L.
TRIAE_CS42_5AL_TGACv1_374195_AA1193240 Beta-glucosidase 30-like − 2.2883 XM_020332410.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7BL_TGACv1_578733_AA1899350 Agglutinin isolectin 3-like − 2.2651 XM_020325269.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3DS_TGACv1_271881_AA0910150 Xyloglucan endotransglycosylase/hydrolase protein 8-like − 2.2407 XM_020316162.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_320919_AA1051690 Isoform GSr1 (GS) − 2.2254 AY491968.1 Triticum aestivum L.
TRIAE_CS42_4DL_TGACv1_345130_AA1151850 Laccase-3-like − 2.149 XM_020292685.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DS_TGACv1_361309_AA1165460 Beta-3 chain − 2.1292 XM_020308075.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BS_TGACv1_051414_AA0179560 tplb0012l18 − 2.0773 AK456462.1 Triticum aestivum L.
TRIAE_CS42_4DS_TGACv1_363201_AA1183500 tplb0005g02 − 2.0566 AK454358.1 Triticum aestivum L.
TRIAE_CS42_4BL_TGACv1_320618_AA1044770 Laccase-3-like − 2.0072 XM_020292685.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3DL_TGACv1_250306_AA0865810 Expansin-A8-like − 2.0057 XM_020317488.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_321498_AA1061390 Beta-glucosidase 6 2.05694 XM_020290811.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BS_TGACv1_049914_AA0164150 Phenylalanine ammonia-lyase-like 2.08107 XM_020317638.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DS_TGACv1_177268_AA0571460 Uncharacterized 2.12878 XM_020322743.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DL_TGACv1_158049_AA0507280 Aldose 1-epimerase-like 2.13367 XM_020290960.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_114151_AA0364450 Gamma gliadin-A1 2.30073 MG560140.1 Triticum aestivum L.
TRIAE_CS42_2AS_TGACv1_113897_AA0362110 Putative amidase C869.01 2.309 XM_020311367.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AL_TGACv1_001256_AA0027880 Endo-1,3-beta-glucosidase 13-like 2.43686 XM_020325014.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BL_TGACv1_033482_AA0139800 Glucan endo-1,3-beta-glucosidase 13-like 2.44328 XM_020325006.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DS_TGACv1_360999_AA1158040 SET4_M23 2.52346 AK330584.1 Triticum aestivum L.
TRIAE_CS42_1BL_TGACv1_030467_AA0091630 ABC transporter B family member 11-like 2.60003 XM_009391731.2 Musa acuminata subsp. malaccensis
TRIAE_CS42_1DS_TGACv1_080643_AA0251500 Laccase-25-like 2.68528 XM_020300631.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4AL_TGACv1_290204_AA0982950 tplb0045l10 2.78117 AK451279.1 Triticum aestivum L.
TRIAE_CS42_2BL_TGACv1_129780_AA0395640 Triacylglycerol lipase 2-like 2.87066 XM_020303075.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BS_TGACv1_147826_AA0487990 GT75-3 3.1223 KM670460.1 Triticum aestivum L.
TRIAE_CS42_2BS_TGACv1_146914_AA0475330 3.24964
TRIAE_CS42_2DS_TGACv1_180213_AA0610440 UDP-arabinopyranose mutase 3 3.27702 XM_020345004.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_113522_AA0357250 UDP-arabinopyranose mutase 3 3.42686 XM_020345004.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BS_TGACv1_145994_AA0452200 WT004_F09 3.68528 AK332578.1 Triticum aestivum L.
TRIAE_CS42_2DL_TGACv1_157981_AA0504650 Beta-glucosidase 12-like 3.70993 XM_020329910.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_113828_AA0361200 Syntaxin-132-like 3.71212 XM_020290887.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_331402_AA1109940 Heparan-alpha-glucosaminide N-acetyltransferase-like 4.87088 XM_020314296.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BS_TGACv1_327875_AA1077360 Protein ZINC INDUCED FACILITATOR-LIKE 1-like 5.5069 XM_020334708.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BS_TGACv1_051503_AA0180180 Alpha-mannosidase-like 6.50912 XM_020334225.1 Aegilops tauschii subsp. tauschii
Fig. 4.

Fig. 4

Log2 (fold change) of down- and up-regulated genes in symbiotic normal-irrigated plants. 1: scopolin beta-glucosidase activity, 2, 8, 14, 15, 22, 25, 31, 32, 43: carbohydrate metabolic process, 3: xyloglucan metabolic process, 4, 9, 24, 33: plant-type cell wall organization, 5, 7, 17: cellular glucan metabolic process, 6, 10: growth factor activity, 11, 12, 21, 37: lipid metabolic process, 13, 20: microtubule-based process, 16: chitin binding, 18: nitrogen compound metabolic process, 19, 23, 35: lignin catabolic process, 26: l-phenylalanine catabolic process, 27, 29, 39: d-alanine ligase activity, 28: hexose metabolic process, 30: carbon–nitrogen ligase activity, 34: transmembrane transport, 36: long-chain fatty acid-CoA ligase activity, 38, 41: cellulose biosynthetic process, 40: UDP-arabinopyranose mutase activity, 42, 44: SNAP receptor activity, 45, 46: integral component of membrane, 47: mannose metabolic process

Among DEGs, 13 significant genes classified as a group of carbohydrate metabolic process; 7 showed up-regulation, and 6 were down-regulated due to mycorrhizal symbiosis. Genes related to hexose metabolic process, UDP-arabinopyranose mutase activity and alpha mannosidase activity were up-regulated. 5 genes were associated with lipid metabolic process; 2 appeared as up-regulated, and three were down-regulated. 2 cellulose biosynthetic process genes indicated increase in their expression influenced by mycorrhization. Among down-regulated genes, 3 of them were related to plant-type cell wall organization, 2 were linked to cell proliferation, 2 genes were involved in microtubule based process; 4 genes were annotated as transmembrane transports.

Apart from normal irrigation condition in this comparison, AMF led to the expression of 19 genes that were not expressed in control plants (Table 6). The most of these induced genes were associated with transporter activity and cell growth, and this can be attributed to the effect of AMF on improving plant behavior.

Table 6.

Induced transcripts identified in wheat cv. Chamran AMF colonized roots in normal irrigation

Gene ID Protein name Value 1 (N − M) Value 2 (N + M) Accession ID in NCBI Source organism
TRIAE_CS42_6DL_TGACv1_527547_AA1705600 Laccase-21-like 0 0.54396 XM_020336472.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AL_TGACv1_000750_AA0018290 Glucan endo-1,3-beta-glucosidase 13-like 0 1.56382 XM_020325012.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_132538_AA0438240 Acidic endochitinase-like 0 19.0282 XM_020321162.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_130318_AA0408760 Acidic endochitinase-like 0 19.645 XM_020321162.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7DL_TGACv1_603253_AA1979180 Putative cellulose synthase A catalytic subunit 11 0 1.02689 XM_020331230.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AL_TGACv1_001368_AA0029420 S-type anion channel SLAH2-like 0 0.38224 XM_020295652.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AS_TGACv1_021078_AA0080890 CASP-like protein 4D1 0 2.29829 XM_020308584.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_130367_AA0409580 WAT1-related protein At1g68170-like 0 1.79156 XM_020322988.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3AS_TGACv1_211406_AA0689890 Phenylalanine ammonia-lyase-like 0 0.66478 XM_020341608.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_226641_AA0818320 Ammonium transporter 3 member 1-like 0 6.40827 XM_020310974.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3DL_TGACv1_249337_AA0845800 Sugar transport protein 1-like 0 1.14417 XM_020334105.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4AS_TGACv1_306829_AA1013910 WAT1-related protein At1g68170-like 0 0.7281 XM_020338332.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_321771_AA1065240 Heptahelical transmembrane protein 4-like 0 0.85227 XM_020290761.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_344866_AA1150230 Malate dehydrogenase [NADP] 1 0 5.60577 XM_020325953.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BL_TGACv1_031679_AA0118650 Beta-glucosidase 22-like 0 6.93309 XM_020330200.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129438_AA0383660 COBRA-like protein 7 0 0.4556 XM_020312442.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_130858_AA0418950 Beta-glucosidase 12-like 0 8.15626 XM_020329910.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DS_TGACv1_180706_AA0611240 COBRA-like protein 7 0 0.72135 XM_020322746.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_112645_AA0342840 Copper transporter 4-like 0 0.59084 XM_020325425.1 Aegilops tauschii subsp. tauschii

DEGs associated with cell wall and plasma membrane in AMF- roots under water deficit (D − M vs D + M)

Colonization of wheat roots with AMF changed the transcriptome profile of water-deficit plants. Compared to non-AMF roots, we observed 6 and 28 down-regulated and up-regulated genes in AMF-roots, respectively, which were associated with cell wall and plasma membrane (Table 7).

Table 7.

DEGs identified in wheat cv. Chamran showing down-regulation and up-regulation in AMF colonized roots under water deficiency

Gene Id Protein name Log2(fold change) Accession ID in NCBI Source organism
TRIAE_CS42_3B_TGACv1_224369_AA0795760 HIPL1 protein-like − 3.9997 XM_020331354.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_320919_AA1051690 Isoform GSr1 (GS) − 2.5829 AY491968.1 Triticum aestivum L.
TRIAE_CS42_4AS_TGACv1_307728_AA1023060 Cereale cytosolic glutamine synthetase isoform (GS1-4) − 2.2636 JN188394.1 Triticum turgidum subsp. durum
TRIAE_CS42_2DL_TGACv1_158029_AA0506490 Xyloglucan endotransglucosylase/hydrolase protein 24-like − 2.2546 XM_020307539.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_343321_AA1133210 Glutamine synthetase isoform GSr2 (GS) gene − 2.1786 AY491969.1 Triticum aestivum
TRIAE_CS42_2AL_TGACv1_093096_AA0271920 Probable xyloglucan endotransglucosylase/hydrolase protein 12 − 2.1437 XM_020317472.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AL_TGACv1_002286_AA0040530 Fatty acid amide hydrolase-like 2.0061 XM_020323605.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_112829_AA0345860 Plastid acetyl-CoA carboxylase (Acc-1) gene 2.02936 EU660900.1 Triticum aestivum
TRIAE_CS42_2AL_TGACv1_093900_AA0288950 Beta-glucosidase 16-like 2.07524 XM_020327283.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_5BS_TGACv1_423370_AA1375340 PI-PLC X domain-containing protein At5g67130-like 2.16637 XM_020312303.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DL_TGACv1_157992_AA0505150 Beta-glucosidase 16-like 2.17812 XM_020327283.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_321498_AA1061390 Beta-glucosidase 6 2.28425 XM_020290811.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BL_TGACv1_030467_AA0091630 ABC transporter B family member 11-like 2.31482 XM_009391731.2 Musa acuminata subsp. malaccensis
TRIAE_CS42_1BL_TGACv1_032327_AA0128440 WAT1-related protein At5g07050-like 2.509 XM_020338654.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DL_TGACv1_158049_AA0507280 Aldose 1-epimerase-like 2.51875 XM_020290960.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BS_TGACv1_146482_AA0466120 ABC transporter B family member 4-like 2.56886 XM_020293040.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_131851_AA0432880 Aldose 1-epimerase-like 2.60357 XM_020290960.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_343670_AA1138050 Protein transport protein Sec61 subunit alpha-like 2.61792 XM_020290809.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_112362_AA0336420 ABC transporter B family member 4-like 2.69261 XM_020293040.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129780_AA0395640 Triacylglycerol lipase 2-like 3.34377 XM_020303075.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_113028_AA0350040 ABC transporter C family member 14-like 3.34819 XM_020332672.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_6AL_TGACv1_474105_AA1534120 2-Alpha-l-fucosyltransferase-like 3.42443 XM_020309974.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DS_TGACv1_360999_AA1158040 SET4_M23 3.58926 AK330584.1 Triticum aestivum L.
TRIAE_CS42_4AL_TGACv1_290204_AA0982950 tplb0045l10 3.70217 AK451279.1 Triticum aestivum L.
TRIAE_CS42_2AL_TGACv1_094425_AA0297430 Beta-glucosidas 3.80462 AB548284.1 Triticum aestivum L.
TRIAE_CS42_2DL_TGACv1_157981_AA0504650 Beta-glucosidase 12-like 3.89686 XM_020329910.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_113828_AA0361200 Syntaxin-132-like 3.91589 XM_020290887.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BS_TGACv1_145994_AA0452200 WT004_F09 4.11817 AK332578.1 Triticum aestivum L.
TRIAE_CS42_2AS_TGACv1_113522_AA0357250 UDP-arabinopyranose mutase 3 4.12231 XM_020345004.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_221856_AA0751810 Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A-like 4.43885 XM_020315658.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DS_TGACv1_180213_AA0610440 UDP-arabinopyranose mutase 3 4.50941 XM_020345004.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BS_TGACv1_147826_AA0487990 GT75-3 4.56522 KM670460.1 Triticum aestivum L.
TRIAE_CS42_4BS_TGACv1_331402_AA1109940 Heparan-alpha-glucosaminide N-acetyltransferase-like 5.66096 XM_020314296.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BS_TGACv1_051503_AA0180180 Alpha-mannosidase-like 7.44879 XM_020334225.1 Aegilops tauschii subsp. tauschii

The interaction between mycorrhiza and water-deficit, caused up-regulation of 28 genes related to carbohydrate metabolic process, fatty acid metabolic process, lipid metabolic process, transmembrane transport activity, hydrolase activity, cellulose biosynthetic process, mannose metabolic process and ligase activity. 11 of these genes (see Table 7 and Fig. 5) were classified as a group of carbohydrate metabolic process of which some of them had role in cell wall biogenesis, consisting of genes related to mannosidase activity, galactoside-2-alpha-L-fucosylteransferase activity, hexose metabolic process, β-glucosidase activity, UDP-arabinopyranose mutase and hydrolase activity. Two other genes were associated with cellulose biosynthetic process (KM670460.1, XM_020345004.1). In AMF plants, ten membrane-related genes as transporters and compartments were up-regulated in wheat plants under water deficit.

Fig. 5.

Fig. 5

Log2 (fold change) of down and up-regulated genes under mycorrhizal water-stress plants. 1, 9, 11, 12, 26: carbohydrate metabolic process, 2, 3, 5: nitrogen compound metabolic process, 4: cellular glucan metabolic process, 6: xyloglucan metabolic process, 7: carbon–nitrogen ligase activity, 8: fatty acid biosynthetic process, 10, 20: lipid metabolic process, 13, 14, 16, 19, 21, 30: transmembrane transport, 15, 17: hexose metabolic process, 18, 33: integral component of membrane, 22: galactoside 2-alpha-l-fucosyltransferase activity, 23: plant-type cell wall, 24: long-chain fatty acid-CoA ligase activity, 25: beta-glucosidase activity, 27, 28: SNAP receptor activity, 29, 32: cellulose biosynthetic process, 31: UDP-arabinopyranose mutase activity, 34: mannose metabolic process

In addition to up-regulated transcripts associated with cell wall and membrane, 25 genes were recognized only in AMF inoculated roots but not in the control samples suggesting AMF specific genes under water deficit.

However, only 1 gene was suppressed in the treated samples (Table 8).

Table 8.

Induced and suppressed transcripts identified in wheat cv. Chamran in AMF roots under water deficiency

Gene ID Protein name Value 1 (D − M) Value 2 (D + M) Accession ID in NCBI Source organism
TRIAE_CS42_6DL_TGACv1_527547_AA1705600 Laccase-21-like 0 0.94653 XM_020336472.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_132538_AA0438240 Acidic endochitinase-like 0 34.2003 XM_020321162.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_130318_AA0408760 Acidic endochitinase-like 0 37.2347 XM_020321162.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AL_TGACv1_096263_AA0318280 COBRA-like protein 7 0 0.57753 XM_020312442.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7DL_TGACv1_603253_AA1979180 Putative cellulose synthase A catalytic subunit 11 0 1.39848 XM_020331230.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_6AL_TGACv1_472962_AA1527570 Chitinase 6-like 0 3.36273 XM_020317430.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AL_TGACv1_001368_AA0029420 S-type anion channel SLAH2-like 0 1.48604 XM_020295652.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AS_TGACv1_021078_AA0080890 CASP-like protein 4D1 0 2.3401 XM_020308584.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_130367_AA0409580 WAT1-related protein At1g68170-like 0 7.59737 XM_020322988.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_131444_AA0427790 Probable purine permease 11 0 1.81115 XM_020344559.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3AS_TGACv1_210868_AA0680480 Protein NRT1/PTR FAMILY 8.2-like 0 1.02073 XM_020335824.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3AS_TGACv1_211406_AA0689890 Phenylalanine ammonia-lyase-like 0 1.25925 XM_020341608.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_221895_AA0752610 protein NRT1/PTR FAMILY 4.5-like 0 36.8762 XM_020341045.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_226641_AA0818320 Ammonium transporter 3 member 1-like 0 6.44725 XM_020310974.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_227304_AA0822670 Protein NRT1/PTR FAMILY 1.2-like 0 3.71422 XM_020312497.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3DL_TGACv1_249337_AA0845800 Sugar transport protein 1-like 0 0.63211 XM_020334105.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4AS_TGACv1_306829_AA1013910 WAT1-related protein At1g68170-like 0 2.20539 XM_020338332.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4BL_TGACv1_321310_AA1058840 Nitrate transporter (TaNPF6.1 gene) 0 1.14233 HF544988.1 Triticum aestivum cv. Paragon
TRIAE_CS42_4BL_TGACv1_321771_AA1065240 Heptahelical transmembrane protein 4-like 0 1.55394 XM_020290761.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_5AL_TGACv1_373992_AA1186890 Nitrate transporter (TaNPF6.1 gene) 0 1.86885 HF544988.1 Triticum aestivum cv. Paragon
TRIAE_CS42_2AL_TGACv1_094209_AA0294360 Beta-glucosidase 12-like 0 0.61419 XM_020329909.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2DL_TGACv1_161025_AA0556070 COBRA-like protein 7 0 0.69365 XM_020312442.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7AL_TGACv1_559082_AA1798000 Class II chitinase gene 0 1.06682 AY973229.1 Triticum aestivum cultivar Gamenya
TRIAE_CS42_2AL_TGACv1_093541_AA0282250 HKT1 0 0.41727 KF443078.1 Triticum durum
TRIAE_CS42_3DL_TGACv1_251611_AA0883070 UDP-glucuronate:xylan alpha-glucuronosyltransferase 1-like 0 0.49347 XM_020310973.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_6DL_TGACv1_526537_AA1686380 Putative laccase-9 19.5213 0 XM_020320013.1 Aegilops tauschii subsp. tauschii

DEGs related to cell wall and plasma membrane in AMF roots under water deficit and normal irrigation

In this experiment, the transcripts of AMF colonized roots under water deficit were compared to normal irrigation.

By evaluating the difference between their gene expression patterns, we identified 25 significantly DEGs associated with cell wall and plasma membrane. Among these, 12 and 13 genes down-regulated as well as up-regulated in D + M (Table 9 and Fig. 6). Regarding the slight difference between these two treatments, it could be concluded that the effect of mycorrhiza on the amount of expression was greater than that of irrigation regimes. 10 significantly expressed genes were categorized as a group of carbohydrate metabolic process of which half were down-regulated and the others were up-regulated. 2 up-regulated genes were involved in lipid metabolic process, and the remaining two had role in cell division and plant-type cell wall organization. Considering the specific genes, three were expressed only in D + M samples. Cellulose synthase activity, plant-type cell wall organization and carbohydrate metabolic process genes were found specific related to N + M, and the genes associated with lipid metabolic process, chitin binding and plant-type hypersensitive response only expressed in D + M (Table 10).

Table 9.

DEGs identified in wheat cv. Chamran showing down-regulation and up-regulation in AMF colonized roots under water deficit and normal irrigation

Gene Id Protein name Log2(fold change) Accession ID in NCBI Source organism
TRIAE_CS42_4BS_TGACv1_328907_AA1095330 Cytosolic glutamine synthetase (GSe-B4) gene − 4.6836 KF242513.1 Triticum turgidum
TRIAE_CS42_4DS_TGACv1_361179_AA1162880 Glutamine synthetase cytosolic isozyme 1-3 − 3.4703 XM_020323172.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129932_AA0399780 Beta-glucosidase BoGH3B-like − 3.3528 XM_020296045.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7BL_TGACv1_578733_AA1899350 Agglutinin isolectin 3-like − 3.1321 XM_020325269.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AL_TGACv1_094425_AA0297430 Beta-glucosidas − 3.1248 AB548284.1 Triticum aestivum L.
TRIAE_CS42_5AL_TGACv1_374243_AA1194960 Beta-glucosidase BoGH3B-like − 2.6959 XM_020293489.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_343897_AA1141200 Glucan endo-1,3-beta-glucosidase 1-like − 2.5333 XM_020343229.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7DL_TGACv1_602790_AA1968680 Agglutinin isolectin 3-like − 2.4325 XM_020325269.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4AL_TGACv1_288277_AA0943450 Cytosolic glutamine synthetase (GSe-A4) gene − 2.3568 KF242512.1 Triticum turgidum
TRIAE_CS42_2AL_TGACv1_095187_AA0308020 Beta-glucosidase BoGH3B-like − 2.288 XM_020296045.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2BL_TGACv1_129561_AA0388630 Beta-glucosidase − 2.1882 AB100035.1 Triticum aestivum L.
TRIAE_CS42_2BL_TGACv1_130367_AA0409580 WAT1-related protein At1g68170-like − 2.0993 XM_020322988.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1DS_TGACv1_080861_AA0254810 Phenylalanine ammonia-lyase-like 2.00132 XM_020319266.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_2AS_TGACv1_113168_AA0352430 Phenylalanine ammonia-lyase-like 2.05164 XM_020310911.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_3B_TGACv1_224369_AA0795760 HIPL1 protein-like 2.11947 XM_020331354.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BS_TGACv1_050729_AA0174830 Phenylalanine ammonia-lyase-like 2.24533 XM_020319266.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_5AS_TGACv1_392939_AA1266520 2.27591 CT009586.1 Triticum aestivum
TRIAE_CS42_1AL_TGACv1_001387_AA0029810 Chromosome 3B-specific BAC library 2.29746 FN564431.1 Triticum aestivum
TRIAE_CS42_1AL_TGACv1_001256_AA0027880 Endo-1,3-beta-glucosidase 13-like 2.30594 XM_020325014.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4DL_TGACv1_343409_AA1134090 Omega-3 fatty acid desaturase, endoplasmic reticulum-like 2.37468 XM_020337300.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_4AS_TGACv1_308797_AA1029560 Expansin-A12-like 2.50807 XM_020306411.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1BS_TGACv1_049914_AA0164140 tplb0043f07 2.57319 AK450976.1 Triticum aestivum L.
TRIAE_CS42_1DL_TGACv1_061987_AA0206950 Glucan endo-1,3-beta-glucosidase 13-like 2.63057 XM_020325014.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_5AL_TGACv1_374914_AA1211820 Uncharacterized LOC109761629 2.67513 XM_020320447.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1DL_TGACv1_061159_AA0187430 Glucan endo-1,3-beta-glucosidase 13-like 3.07913 XM_020325013.1 Aegilops tauschii subsp. tauschii
Fig. 6.

Fig. 6

Log2 (fold change) of down- and up-regulated genes in symbiotic normal and low-irrigated plants. 1, 2, 9: nitrogen compound metabolic process, 3, 6, 7, 10, 11, 15, 19, 23, 24, 25: carbohydrate metabolic process, 4, 8: chitin binding, 5: scopolin beta-glucosidase activity, 12: transmembrane transporter activity, 13, 14, 16: l-phenylalanine catabolic process, 17: cell division, 18, 20: lipid metabolic process, 21, 22: plant-type cell wall organization

Table 10.

Induced transcripts identified in wheat cv. Chamran AMF colonized roots in normal and deficit conditions

Gene ID Protein name Value_1 (D + M) Value_2 (N + M) Accession ID in NCBI Source organism
TRIAE_CS42_7AL_TGACv1_557532_AA1782680 Mixed-linked glucan synthase 2-like 0 0.38386 XM_020308861.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1DL_TGACv1_061987_AA0206890 Glucan endo-1,3-beta-glucosidase 13-like 0 0.70549 XM_020325007.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_5BL_TGACv1_405270_AA1323650 Expansin-A31-like 0 0.41256 XM_020334430.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_7DS_TGACv1_622542_AA2041460 Patatin-like protein 2 3.75019 0 XM_020293126.1 Aegilops tauschii subsp. tauschii
TRIAE_CS42_1AL_TGACv1_003048_AA0047290 Germ agglutinin isolectin A 1.21392 0 M25536.1 Wheat (T.aestivum)
TRIAE_CS42_3DL_TGACv1_251714_AA0884080 WPR4b gene 0.545005 0 AJ006099.1 Triticum aestivum

Validation of DEGs by q-RT PCR

To confirm the accuracy of the result of transcriptome analysis, the expression of three randomly selected DEGs, in RNA-Seq analysis were examined by qRT-PCR (Fig. 7).

Fig. 7.

Fig. 7

Log 2 (fold change) of three selected genes, Z1: cellulose biosynthetic process, Z2: beta-glucosidase BoGH3B-like Z3: chitinase activity, analyzed by q-RT PCR

The genes examined with q-RT PCR represented the close expression level similar to RNA-Seq data. The selected genes were associated to cellulose biosynthetic process (XM_020345004.1), beta-glucosidase BoGH3B-like (XM_020296045.1), and chitinase activity (AY973229.1), respectively.

Discussion

Since severe soil drought significantly decrease root AMF colonization and hyphal growth in the soil, we applied a mild water stress in our experiments. The beneficial role of the symbiosis relationship concerning the tolerance to abiotic stresses is clearly revealed (Bernardo et al. 2019; Ren et al. 2019; Ouledali et al. 2019). AMF inoculation not only enhanced plant growth and yield, but also improved plant resistance against water deficit. In line with our findings, AMF improved plant growth and productivity under water stress situation (Li et al. 2019). The improvement of plant growth can be related to root system development and recuperating of water and minerals accessibility. This conclusion is in corroboration with previous findings (Nezhadahmadi et al. 2013; Li et al. 2019; Ye et al. 2019).

To discover novel genes involved in plant-AMF interaction in different conditions, whole-genome Transcriptome analysis technique was used. Moreover, applying two biological replicates per condition with 10 pooled roots per replicate for sequencing can strengthen our results. We have also considered precise criteria in order to separate differential expressed genes which have resulted in the omission of genes with low expression differences.

Interaction with arbuscular micorrhizal fungus Funneliformis mosseae, cell wall of plant root is the first line of defense which can highly protect the plant. On the other hand, in mycorrhizal symbiosis, plant root cell wall opens a way for symbiotic establishment, development and nutrient exchange (Rich et al. 2014). In the present study, we tried to connect some cell wall and membrane genes generated from RNA-Seq data set to plants growth and tolerance against water deficit in the presence or absence of AMF; since the most up- and down- regulated genes related to transferase activity and biosynthetic processes are cell membrane proteins (An et al. 2018; Vangelisti et al. 2018). Here we focused on several DEGs that were directly or indirectly involved in cell wall and membrane modulation and synthesis, and tried to make a connection between these DEGs and plant tolerance. It seems that the plant cell wall plays a significant role in stress perception through changing and remodeling the growth strategies in response to stress (Kesten et al. 2017). Furthermore, different molecules of cell wall can play a role as signaling factors to warn the plant immune system under adverse situations (Malinovsky et al. 2014). Cellulose as the major component of cell wall has considerably shown up-regulation in mycorrhizal plants. Comparing D − M/D + M, we found three genes (XM_020312442.1, XM_020331230.1, XM_020312442.1) to be only expressed in mycorrhizal treatment as mycorrhizal-induced gene (Table 8); analogously, there were three specific induced cellulose-related genes (Table 6) in N − M/N + M comparison (XM_020331230.1, XM_020312442.1, XM_020322746.1). Considering these data, it seems that mycorrhizal symbiosis can effectively increase cellulose biosynthetic process and cell growth. Interestingly, cellulose synthases and also linked microtubules can perceive stress signals directly or indirectly to trigger reproducing and remodeling the cellulose microfibrils and proteins like expansin as the best response (Kesten et al. 2017). Wang et al. (2016) have reported that cellulose deficient mutant plants are more sensitive to abiotic stress than wild-type ones (Wang et al. 2016); therefore, a stress-response related role can be considered for cellulose, since cellulose microfibrils and the other factors that lead the direction of cell growth can be regulated by water availability (Wang et al. 2016). Based on our results, increased cellulose synthesis occurs in plants in normal irrigation, and this data is content with plant growth analysis (Fig. 2); and as we observed a gene (XM_020308861.1) only expressed in N + M treatment compared to D + M (Table 10), it can be perceived that probably the better irrigation condition leads to more cellulose microfibril biosynthesis in plant cell wall, and consequently, cell growth and symbiosis establishment; thus, we can introduce this gene (XM_020308861.1) as a N + M-specific gene. Therefore, cellulose biosynthesis related genes recognized as significant expressed in mycorrhizal-alleviated plants. Our results suggest that a defense-related role can be considered for cellulose in plant cell wall, since cellulose is the most abundant component of that, and cell wall appears as a very complex network for providing protection and perception to confront critical situations (Kesten et al. 2017; Vangelisti et al. 2018; Ren et al. 2019). A possible explanation for increasing of cellulose biosynthesis might be a relevance between cellulose content increase toward raising alleviated wheat plants tolerance; referring previous studies (Balestrini and Bonfante 2014; Kesten et al. 2017; Wang et al. 2016; Zhang et al. 2014), disordering the cellulose microfibriles organization occurred by biotic or abiotic factors, water deficit or AMF in this case, can trigger defense response in plants; Although discovering the exact mechanism requires more investigation.

Deducing from our results, lipid metabolic process associated genes such as fatty acid biosynthetic process, were up-regulated in AMF plants in comparison to non-mycorrhizal plants, especially under water deficit; comparing D − M/D + M conditions, there were three up-regulated genes related to lipid biosynthesis, and two up-regulated (FN564431.1, XM_020337300.1) and one specific gene (XM_020293126.1) related to D + M treatment in comparison with N + M. Since lipids are very important component of cell membrane, water deficiency may affect the composition and amount of lipids due to stress (Ivanov and Harrison. 2018). Increasing the lipid metabolic process due to AMF and well watering may suggest the role of lipids in maintaining the normal structure and function of cell membrane (Ivanov et al. 2019); to this aim, since AMF increases plant defense in stress situation, specially water deficit condition, it endeavors to enhance the cell membrane thickness by increased biosynthesis of lipid compartments either for forming arbuscular structures or counteracting abiotic stresses. Considering the increased lipid-related gene expression in D + M plants, we propose the role of AMF to increase the plant tolerance by targeting the cell membrane composition, which is consistent with previous reports (López-Ráez et al. 2010; Zhang et al. 2014).

In addition, we identified genes related to microfibril organization and cytoskeleton structure as mycorrhizal-responsive genes. As the cytoskeletal restructuring is related to cell membrane integrity, different stresses such as water shortage can change this arrangement, membrane fluidity and transmembrane transporters activity (Vangelisti et al. 2018). It could be suggested that up-regulation of the cytoskeletal compartments related genes in symbiotic plants protect the membrane integrity and transports in response to water stress; as it has been suggested that de-polymerization and re-organization of the cytoskeletal microtubules can be important for plant tolerance to salt stress as an abiotic stress (Wang et al. 2013). It is demonstrated that mycorrhizal symbiosis can enhance the absorption of minerals in host plant (Smith et al. 2011). In agreement with these results, we found the up-regulation of genes involved in membrane transferase compartments because of mycorrhizal relationship. Genes such as ammonium transporters and integral components of membrane are presented as mycorrhizal-responsive genes (Tables 6, 8). Up-regulation of ammonium transporters in AMF roots of host plants has already been reported in some plant species (Breuillin-Sessoms et al. 2015; Hong et al. 2012; Pérez-Tienda et al. 2014). It is proposed that the presence of transporters in pre-arbuscular membrane can help arbusculated cells survival and maintenance of symbiotic relationship in root cells (Breuillin-Sessoms et al. 2015).

Moreover, several carbohydrate metabolic process related genes were identified as significant differentially expressed genes in this study and mainly related to root cell wall degradation and remodeling (Vangelisti et al. 2018); which some such as mannosidase, hexose metabolic process and β-glucosidase activity with roles in cell wall biogenesis. In comparison between D − M/N − M, there were two carbohydrate-metabolic-process related genes (Table 3) whose expression level were completely induced in control plants, and it can be associated with normal irrigation condition. Since four induced genes (XM_020321162.1, XM_020321162.1, XM_020329909.1, XM_020310973.1) related to carbohydrate metabolic process, hydrolase activity and xylan biosynthetic process were found in AMF plants (Table 8), it can be deduced that AMF could effectively improve cell behavior by changing its gene expression pattern. Amongst different roles of carbohydrates in plant cells, such as mechanical support imparted by xylan, mannan and xyloglucan in cell wall can be considered as one of their important roles (Houston et al. 2016). It seems that some carbohydrates related to the host plant are allocated to AM development, and accumulation of some carbohydrates, like monosaccharides, can improve the osmotic adjustment and water preservation of the host plant (Wu et al. 2013). It has been shown that carbohydrates play an important role in plant defense response and immunity (Lastdrager et al. 2014). Some participate as elicitors, whilst, others can imply such as phyto-hormones and signaling molecules (Trouvelot et al. 2014), or elicitors which are derived from plant cell wall during plant’s interaction with microorganisms (Boudart et al. 2003). Since water stress can directly affect the cell membrane construction, it is suggested that carbohydrates play a key role in integral redox network and mitigate the destructive effects of drought stress (Keunen et al. 2013). Chen et al. (2014) has previously reported increased the expression of monosaccharide genes like mannose and glucose in symbiotic plants. Hemicellulose, as a complex and important part of the plant cell wall is composed of mannose, glucose or xylose and reinforces the cell wall through interaction with cellulose microfibrils and lignin (Malinovsky et al. 2014). Here we can suggest a mycorrhizal-responsive role for abovementioned monosaccharide related genes which appeared as upregulated DEGs in colonized plants (Tables 5, 7). Also, AMF positively affect the plant immune system by increasing the expression of genes annotated as hemicellulose compartments, such as mannose and xylose, and fortifying the cell wall as a first barrier of plant cell.

The expression data about chitin binding and chitinase activity genes, revealed increasing pattern in presence of AMF, especially under water deficit condition. This result is consistent with Behringer et al. (2015) observation in Picea abies, and other reports demonstrating the role of chitinase enzymes in increasing plant tolerance (Couto et al. 2013; Dana et al. 2006; Hermosa et al. 2012; Lucas et al. 2014).

As fundamental molecule of fungus cell wall, chitin and chitin-derived molecules play a key role as signals to initiate defense responses in plant cells in symbiosis relationship (Shimizu et al. 2010; Hayafune et al. 2014). It could be suggested that chitin-related genes such as chitinase detect chitin molecules of fungus as a signal to trigger a proper defense response, and also increase the plant tolerance.

Cufflink assembly data indicated higher expression of lignin catabolic processes related genes. Gutjahr et al. (2015) have reported plant secondary cell wall genes such as lignin, down-regulated during symbiosis, and it might facilitate mycorrhizal fungus penetration and development through plant cell wall, and this is in agreement with our results.

Conclusion

Our analysis of significantly expressed genes in AMF roots of wheat cv. “Chamran” proposes that a primary defense response related to cell wall and membrane and also some membrane located molecules such as carbohydrates as a very significant group of molecules could be accomplished in mycorrhizal plants. These two important external parts of plant cells could initiate the first step of defense response against water deficiency through signaling processes in which different molecules of cell wall and also membrane transporters and compartments are involved. For the first time, in the present research it has been suggested that modifications and changes in cell wall components and their activities and cell-membrane-located molecules induced by AMF as an invader in the first place, may provide a tolerance-specified system in which structural part, such as cellulose and hemicellulose compartments and lipid molecules, and non-structural molecules, such as many carbohydrates, cooperate toward improving biosynthetic processes and physically strengthening of the outermost parts of the host plant which lead to plant growth improvement as well. Since there are great number of genes addressed to cell wall and membrane, understanding the exact genes and their roles, obviously needs more investigations in the future. Our results represent that symbiotic plants displayed differentially amount of expressed genes, most related to restructuring and strengthening of cell wall and membrane, and also their organization and remodeling to protect the host plant, and this tolerance response can provide more durability and better growth status for the symbiotic plants.

Finally, despite several investigations on cell wall and membrane changes in symbiotic plants and the relationship with plant tolerance to achieve a precise conclusion, this study has tried to focus on some significant genes and introduce some specific genes associated with mycorrhiza under two different irrigation regimes. Notwithstanding the delicate and extended range of water stress condition as one of the most important abiotic stresses, the relationship between cell wall and membrane genes expression pattern and the intensity and duration of water deficit can be very impressive; so the studies domain can be really vast and detailed in this case. Probably, our outcomes can highlight the advantage of transcriptome profiling to identify additional genes and the mycorrhiza-associated genes that have been found, provide a field for further investigations into cell wall-related genes and their relationship with plant tolerance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

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