Next Article in Journal
Microalga Broths Synthesize Antibacterial and Non-Cytotoxic Silver Nanoparticles Showing Synergy with Antibiotics and Bacterial ROS Induction and Can Be Reused for Successive AgNP Batches
Previous Article in Journal
Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 22
10.3390/ijms242216184
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress

by
Miaoli Wang
†,
Guanyan Feng
†,
Zhongfu Yang
,
Jiahui Wu
,
Bingyan Liu
,
Xiaoheng Xu
,
Gang Nie
,
Linkai Huang
* and
Xinquan Zhang
*
College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(22), 16184; https://doi.org/10.3390/ijms242216184
Submission received: 14 September 2023 / Revised: 6 November 2023 / Accepted: 9 November 2023 / Published: 10 November 2023
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Review Reports Versions Notes

Abstract

:
Drought stress is an important factor that reduces plant biomass production and quality. As one of the most important economic forage grasses, orchardgrass (Dactylis glomerata) has high drought tolerance. Auxin/indole-3-acetic acid (Aux/IAA) is one of the early responsive gene families of auxin and plays a key role in the response to drought stress. However, the characteristics of the Aux/IAA gene family in orchardgrass and their potential function in responding to drought stress remain unclear. Here, 30 Aux/IAA members were identified in orchardgrass. Segmental duplication may be an important driving force in the evolution of the Aux/IAA gene family in orchardgrass. Some Aux/IAA genes were induced by IAA, drought, salt, and temperature stresses, implying that these genes may play important roles in responding to abiotic stresses. Heterologous expression in yeast revealed that DgIAA21 can reduce drought tolerance. Similarly, the overexpression of DgIAA21 also reduced drought tolerance in transgenic Arabidopsis, which was supported by lower total chlorophyll content and relative water content as well as higher relative electrolyte leakage and malondialdehyde content (MDA) than Col-0 plants under drought conditions. The results of this study provided valuable insight into the function of DgIAAs in response to drought stress, which can be further used to improve forage grass breeding programs.

1. Introduction

Orchardgrass (Dactylis glomerata) is one of the top four economically important forage grasses and is extensively cultivated; it plays a vital role in livestock husbandry and ecological remediation [1,2,3]. Compared with other perennial grass species, orchardgrass has more drought tolerance and shade tolerance. Auxin plays an important role in sensing and signaling related to varied abiotic stress conditions, such as drought, temperature, and salt stress [4]. Aux/IAA is a nuclear protein with a short life cycle that plays a key role in auxin balance in plants [5,6]. The Aux/IAA gene family contains four domains, including the LxLxLx leucine-rich repeat motif domain I, as well as a highly conserved sequence with GWPPV domain II, domain III, and domain IV, which are located at the C-terminus and bind to the auxin response factor [7,8]. Previous studies have identified the features of Aux/IAAs in plant species such as Malus domestica [9], Hordeum vulgare [10], Hedychium coronarium [11], Zea mays [12], Brassica rapa [13], and Brassica napus [14].
Drought is a widespread problem that negatively impacts the economy. At present, approximately 40% of the Earth’s total landmasses are suffering drought stress [15,16]. Drought stress in plants occurs if the evaporation and transpiration rate exceed the rate of water uptake by the roots [17,18]. Drought stress has adverse effects on seed germination, vegetative growth, flowering, and fruit ripening [19,20,21,22]. As sessile organisms, plant populations adapt to stresses in wild and agricultural habitats through changing their genetic and physiological–biochemical characteristics, as well as their morphological traits [23]. Closing the stomatal aperture to retain the leaf water potential is the first step in the response to drought stress [24]. A decrease in the entrance of CO2 into leaves due to stomatal closure can suppress the rate of photosynthesis. In addition, drought stress also creates ionic and osmotic imbalance, reactive oxygen species (ROS), and changes to cellular metabolism that could limit plant growth and development [21,25,26,27].
Increasing evidence has shown that Aux/IAA genes can respond to drought stress. Water loss by stomates was adjusted in IAA12 and IAA17 mutants through strongly clustering to respond water deficit stress [28,29,30]. Plants with mutations of IAA5, IAA6, and IAA19 showed reduced drought resistance through downregulating aliphatic glucosinolate levels [31]. The overexpression of OsIAA20 in rice increased the expression level of the abscisic acid (ABA)-responsive gene OsRab21 in response to drought stress [32]. The overexpression of MdIAA9 of tobacco had lower relative electrolyte leakage and MDA levels, as well as higher total chlorophyll content and proline content to enhance resistance to drought stress [33].
However, limited information is available about the Aux/IAA gene family in orchardgrass and the functional characteristics of Aux/IAA genes in response to drought stress. Therefore, in this study, a genome-wide characterization of the Aux/IAA gene family in orchardgrass was presented. Furthermore, this study analyzed the physiochemical information, cladogram, gene structure, conserved motif, chromosome location, and synteny of the Aux/IAA gene family in orchardgrass. The expression patterns of Aux/IAAs under different stress conditions were determined. Moreover, the overexpression of DgIAA21 in Arabidopsis plants further verified the role of DgIAA21 in drought stress. The results of this study provide insight into the mechanism through which the Aux/IAA gene family responds to abiotic stress conditions.

2. Results

2.1. Identification of the Aux/IAA Gene Family in Orchardgrass

Thirty Aux/IAA proteins were identified in orchardgrass; the protein sequences are shown in Table S1. The lengths of DgAux/IAA proteins varied from 89 to 530 amino acids, with an isoelectric point ranging from 4.62 to 10.16 (Table S2). The lowest molecular weight was 23.45 KDa, and the highest molecular weight was 56.59 KDa. Furthermore, the instability index of DgIAA16b, DgIAA24, and DgIAA26 was low, which showed that these three proteins were relatively stable. The grand average of hydropathicity of DgAux/IAA proteins was a negative value, except for DgIAA27, indicating that nearly all proteins were hydrophilic and unstable in water. The physiochemical analysis showed that the proteins of DgIAAs are acid, alkaline, or acid–base, most of which are unstable and hydrophilic. Subcellular localization indicated that DgAux/IAA proteins were mainly located in the nucleus and chloroplasts, implying that DgAux/IAA proteins may play an important role in the nucleus and chloroplasts.

2.2. Motif Analysis and Phylogenetic Relationships Reveal the Evolution of Aux/IAA Genes

To explore the evolution of the DgAux/IAA family, a phylogenetic tree was constructed, the structure of exons and introns was investigated, and conserved motifs were further analyzed (Figure 1). Based on the phylogenetic tree, DgAux/IAA proteins were separated into two clades: A and B. Clade A had higher proportion of DgAux/IAA protein members (60%). The number of introns varied from zero to seven. Almost all DgAux/IAA genes had an intron, except for DgIAA9. DgIAA31a, DgIAA31b, DgIAA23, DgIAA18a, and DgIAA33 had one intron, while the other genes had two or more introns. Additionally, Aux/IAA genes in cluster A had at most four introns, but many genes in clade B had more than four introns, such as DgIAA27, DgIAA6, DgIAA7, and DgIAA18b. Motif 2 existed in all DgIAA proteins, and motifs 1, 3, and 4 existed in nearly all DgIAA proteins. Motif 5, motif 9, and motif 10 only existed in clade A, while motif 8 was only present in clade B, suggesting DgIAA proteins were highly conserved in the clades.
To investigate the phylogenetic relationships among the Aux/IAA proteins, a phylogenetic tree was constructed using 90 protein sequences, including 30 sequences from Dactylis glomerata, 29 sequences from Arabidopsis thaliana, and 31 sequences from Oryza sativa (Figure 2). All of the 90 Aux/IAA proteins were divided into two classes (A and B) based on the classification of the Arabidopsis thaliana Aux/IAA gene family in previous research [34]. The Aux/IAA proteins of orchardgrass were tightly clustered with rice but distant to Arabidopsis; for example, OsIAA21 and DgIAA21, AtIAA14 and AtIAA17 were found together. These results illustrated that divergence has occurred between dicots and monocots in Aux/IAAs.

2.3. Chromosome Distribution and Synteny Analysis of DgAux/IAAs

Figure 3 shows the results of the chromosome distribution and synteny analyses of DgAux/IAAs. DgAux/IAA genes were randomly located on six chromosomes, but no DgIAAs were found on Chromosome 3 (Figure 3A). Eight predicted genes were present on Chromosome 4, followed by six genes and five genes on Chromosomes 5 and 6, respectively. Four genes were found on Chromosomes 2 and 7, while two genes were distributed on Chromosome 1, which had lower gene density than the other chromosomes. Furthermore, we found that some DgIAA genes were adjacent to each other. For example, DgIAA12 and DgIAA30a; DgIAA15a and DgIAA16b; and DgIAA31a, DgIAA31b, and DgIAA30b were connected in series, indicating that tandem duplication occurred in these DgAux/IAAs.
To uncover the origin and evolution of the DgAux/IAA members, a synteny analysis was performed in orchardgrass as well as among orchardgrass, Arabidopsis, and rice. Seven segmental duplication pairs of DgIAAs were found in orchardgrass, while Chromosomes 5 and 6 had the most duplication regions (Figure 3B). The number of collinear gene pairs between orchardgrass and rice was significantly higher than that between orchardgrass and Arabidopsis, revealing that orchardgrass DgIAAs showed more homology with rice than Arabidopsis, supporting a close relationship with monocotyledons during evolution (Figure 3C). DgIAA4 and DgIAA13 in orchardgrass had collinearity both with Aux/IAA genes in Arabidopsis and rice, suggesting that DgIAA4 and DgIAA13 may have existed before the differentiation of dicots and monocots.

2.4. Expression of DgIAA Genes in Different Tissues and Their Response to Different Stresses

The expression patterns of DgIAA genes were observed in the spike, stem, leaf, flower, and root tissues (Figure 4A). The expression of DgAux/IAA genes varied in different tissues, with the majority of DgAux/IAA genes highly expressed in the spike and root. For example, DgIAA1, 6, 9, 12, 18a, and 24 were specifically highly expressed in the root tissue. These results implied that DgAux/IAAs may be involved in both spike and root development.
Previous research has indicated that the Aux/IAA gene family responds to different stresses; therefore, we investigated the expression of DgAux/IAA gene family members under five stress conditions (Figure 4B). For IAA treatment, the expression level of the DgIAA6 and DgIAA21 genes maintained a significant decreasing trend, while the expression level of the DgIAA16a and DgIAA33 genes maintained a significant increasing trend. The expression level of the DgIAA4, 16a, and 21 genes maintained a significant decreasing trend under drought stress. For salt stress, the expression level of the DgIAA3, 4, 6, 16a, 21, and 33 genes exhibited oscillatory changes. But the expression level of the DgIAA30a gene sustained a significant decrease. For high-temperature stress, the expression level of the DgIAA6 gene sustained a significant reduction, and the expression level of the DgIAA33 gene sustained a significant increase. The expression level of the DgIAA3, 4, 6, 21, 30a, and 33 genes maintained a significant decreasing trend under low-temperature stress.

2.5. Heterologous Expression of DgIAA3, DgIAA4, DgIAA6, and DgIAA14 Genes Enhances Drought Tolerance in Yeast

To further analyze the function of DgIAAs under drought stress, some genes were transformed into the pYES2 vector and then introduced into INVScI. DgIAA3-, DgIAA4-, DgIAA6-, and DgIAA14-pYES2 stains appeared to be highly insensitive due to higher cell recovery in 100- and 1000-fold dilution rates under a high sorbitol level (2.5 M), apart from DgIAA21-pYES2 stains. This indicates that DgIAAs may exhibit different patterns of behavior when subjected to drought stress conditions (Figure 5).

2.6. Overexpression of DgIAA21 Negatively Regulates Root Length under Drought Stress

The fluorescence signal of DgIAA21-GFP fusion vector was obtained primarily in the nucleus, and minority in nucleus and cytoplasm (Figure 6). To verify the gene function under drought stress, we selected 3 transgenic DgIAA21 Arabidopsis lines for further analysis (Figure S1). The germination rate was lower in DgIAA21-overexpressing lines than Col-0 in 300 mM mannitol (Figure 7A). The root lengths of Col-0 plants increased significantly compared to OE plants in 300 mM mannitol concentration (Figure 7B).

2.7. Overexpression of DgIAA21 Negatively Regulated Drought Tolerance in Transgenic Arabidopsis Plants

Three-week-old DgIAA21-overexpressing and Col-0 plants were treated without water for 14 days and re-watered for 4 days, and the result showed that the DgIAA21-overexpressing plants had fewer green leaves than Col-0 plants (Figure 8A). Under control conditions, the photosynthesis parameters showed that transgenic plants had a greater intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr) than Col-0 plants, while the net photosynthetic rate (Pn) and water use efficiency (WUE) were lower than those in Col-0 plants (Figure 8B–F). Moreover, transgenic DgIAA21 lines had higher water loss and lower total chlorophyll content than Col-0 plants (Figure 8G–I). Under drought conditions, the relative electrolyte leak in transgenic lines was higher, whereas the relative water content was lower than in Col-0 plants (Figure 8H,K). In addition, the transgenic plants, except for the OE3 lines, exhibited significantly higher MDA content, while transgenic plants had lower Fv/Fm and total chlorophyll content than those of Col-0 plants under drought stress conditions (Figure 8I,J,L). We measured eight chlorophyll-related genes to explore the impact on total chlorophyll content. Chlorophyll synthesis gene CDR1 was significantly reduced under normal conditions (6.4-fold) and drought conditions (2.3-fold), suggesting that the overexpression of DgIAA21 transgenic plants could decrease the total chlorophyll content through reducing the expression level of the CDR1 gene (Figure 9B,C).
To determine the molecular mechanism of sensitive phenotypes of DgIAA21-overexpressing transgenic plants, 13 stress-related genes were analysis under drought stress (Figure 9A). APX1 and NHX2 gene expression levels were decreased in the transgenic line compared with the Col-0 type. CAT1 and SOD1 gene expression levels were steady within 24 h after treatment. SOS1 (12h, 2.0-fold), RD29B (6 h, 2.2-fold), and ABI1 (6 h, 2.0-fold) gene expression levels in the transgenic line were significantly decreased than those of the Col-0 type. In general, DgIAA21 transgenic plants had lower drought tolerance than Col-0 plants.

3. Discussion

As one of the most outstanding gene families in mediated auxin signal transduction, the Aux/IAA family not only plays a key role in plant growth and development, but it also plays a significant role in responses to abiotic stresses [5,35]. The function of Aux/IAA genes has been deeply studied, mainly in model plants such as Arabidopsis thaliana and Oryza sativa. For Arabidopsis thaliana, AtIAA8 interactions with AtARF6 and AtARF8 lead to abnormal flower organ development through a change in jasmonic acid level [36]. Mutants of iaa16 display smaller rosettes and fewer lateral roots than the wild type [37]. For Oryza sativa, the overexpression of OsIAA4 shows dwarfism and insensitivity to exogenous auxin [38]. Overexpressing OsIAA31 reduces crown root formation and produces short leaf blades [39]. OsIAA20 RNAi transgenic rice exhibits reduced drought and salt tolerance [32].
We identified 30 members of the Aux/IAA gene family in orchardgrass. The gene members of the Aux/IAA family exist with huge variation, from 1 to 119 members in different species, such as 1 in Marchantia polymorpha [40], 16 in Solanum melongena [41], 31 in Oryza sativa [42], 63 in Glycine max [43], and 119 in Brassica napus [14]. Gene duplication events could play a decisive role in functional diversity and evolutionary mechanisms [44]. In this study, four pairs of tandem duplications and seven pairs of segmental duplications were confirmed in orchardgrass. The rate of gene duplication was lower than that in Arabidopsis and rice [8,34,42].
Drought is one of the most serious threats for agriculture worldwide [45]. Increasing evidence has shown that Aux/IAA proteins can enhance drought stress response [5,46]. The overexpression of OsIAA6 in rice enhanced drought stress tolerance through potentially regulating the auxin biosynthesis gene’s expression level [47]. In addition, some genes of the Aux/IAA family, including MdIAA9 in apple and OsIAA18 in rice, have also been shown to increase drought stress tolerance [33,48]. In this study, DgIAA6 was clustered with OsIAA18 and OsIAA6. DgIAA6 transformed into INVScI yeast showed that DgIAA6 can enhance drought resistance. However, Aux/IAAs also play a role as a negative regulator in response to drought stress. For example, the expression level of IAA4 in Vitis vinifera was decreased under drought treatment [46]. Low cell recovery in yeast and a low gene expression level under drought stress showed that DgIAA21 could play a role as a negative regulator in response to drought stress.
Previous studies have shown that photosynthetic machinery is remarkably impacted by drought stress and can especially cause a decrease in chlorophyll content [49,50]. Photosynthesis fuels lots of metabolic processes through triggering the process of conversion of light energy into chemical energy. The Pn and total chlorophyll content of DgIAA21-overexpressing plants was significantly decreased compared to Col-0 plants under control conditions, implying that the overexpressing plants may have less energy for defense when suffering abiotic stress. The chlorophyll fluorescence and total chlorophyll content in overexpressing lines were lower than in Col-0 lines under drought stress conditions, which can cause a reduction in PQ and PSII electronic transport [51]. In addition, the chlorophyll synthesis gene CDR1′s expression level was significantly decreased in DgIAA21-overexpressing plants under normal and drought conditions, suggesting that DgIAA21 may negatively regulate chlorophyll biosynthesis to reduce drought stress.
It is important for plants to maintain a moisture balance to defend against drought stress [52,53]. Longer roots can enhance the ability of a plant obtain water from deeper soil compared with short roots to cope with drought conditions [54,55]. The relative water content and root length of transgenic Arabidopsis plants containing DgIAA21 were lower than those of control plants under drought stress conditions. Moreover, DgIAA21-overexpressing plants exhibited poor WUE and higher water loss. These results indicated that DgIAA21-overexpressing plants were less able to maintain a moisture balance, which may be an important factor that decreases drought tolerance.
Drought can cause lipid peroxidation, membrane damage, oxidation, and even cell death through a rapid increase in ROS [56,57]. The antioxidant enzyme gene APX1 and the Na+/H+ antiporter gene NHX2 in DgIAA21-overexpressing plants showed lower expression levels than Col-0, suggesting DgIAA21 may negatively regulate drought responses via reducing the expression levels of the APX1 and NHX2 genes in Arabidopsis. As an indicator of ROS, MDA concentration reflects the degree of stress [58]. Our results showed that DgIAA21-overexpressing plants were more sensitive than Col-0 plants under drought stress conditions, as indicated by the increased level of MDA.

4. Materials and Methods

4.1. Analyzing Expression Patterns of Aux/IAAs in Orchardgrass

The expression patterns of Aux/IAA genes in root, stem, leaf, spike, and flower tissues were obtained from the orchardgrass reference genome database [59]. To analyze expression patterns under different stresses, the seeds of the ‘2006-1′ cultivar were germinated and grown in pots filled with quartz. Seedlings were irrigated using Hoagland’s solution for four weeks. Then, these plants were treated with exogenous auxin (100 μM IAA), drought stress (20% PEG 6000 (w/v)), salt stress (200 mM NaCl), high-temperature stress (37 °C) and low-temperature stress (4 °C), respectively. Samples of leaves were collected after 0 h, 2 h, 4 h, and 8 h under different stress treatments. Three biological replicates from each plant were harvested at each time point. All samples were placed into liquid nitrogen and stored at −80 °C. Total RNA was extracted using an RNA kit (RNA simply total RNA Kit, Tiangen, Beijing, China). cDNA was synthesized using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Finally, gene expression was determined with qRT-PCR using an SYBR Primix Ex Taq kit (TaKaRa). Primers were designed using NCBI. The reference genes were the β-actin gene and GADPH gene of orchardgrass (Table S3). The relative expression level of selected genes was calculated using the 2−∆∆Ct calculation method [60].

4.2. Yeast Transformation and Verification of Drought Stress

To further analyze the functions in drought of DgIAAs, some genes of clade A (DgIAA3, DgIAA14, DgIAA21) and clade B (DgIAA4, DgIAA6) of orchardgrass were randomly selected to connect the yeast expression system. The open reading frames (ORFs) of these genes were inserted into the pYES2 vector. The specific primers for gene amplification are listed in Table S4. Then, the recombinant plasmid and empty vector were transformed into the yeast strain INVScI (Weidi Biotechnology Co., Ltd., Shanghai, China). The correct single colony was selected and then incubated in SD-Ura liquid medium containing 2% (w/v) galactose at 28 °C and 200 rpm until cell densities reached an OD600 of 2.0. Yeast suspensions were diluted to 100, 10−1, 10−2, 10−3, 10−4, and 10−5 with ddH2O. Finally, diluted yeast cells were spotted on yeast peptone glucose (YPG) plates containing 0, 1.5 mol/L, and 2.5 mol/L sorbitol for phenotypic analysis.

4.3. Germination and Root Length Assays

Sterilized seeds from T3 transgenic and Col-0 Arabidopsis plants were sown on 1/2 MS medium containing 0 mM, 200 mM, or 300 mM mannitol. The germination rate was computed 10 days after sowing. The seeds of T3 transgenic and Col-0 lines were sown on 1/2 MS medium and then transferred to 1/2 MS medium containing 0 mM, 200 mM, or 300 mM mannitol for 7 days to measure the root length of plants.

4.4. Drought Tolerance Analysis of Transgenic A. thaliana Plants

The seeds of Col-0 and homozygous T3 DgIAA21-overexpressing Arabidopsis were sown on soil and kept in a phytotron for 3 weeks to use for further treatments. In the drought treatment, water was withheld from plants for 14 days, and then watered for 4 days before plant recovery was recorded [61]. Normally watered Col-0 and DgIAA21-overexpressing Arabidopsis plants were considered the control. Malondialdehyde content was measured using the MDA Assay Kit (Grace Biotechnology Co., Ltd., Suzhou, China). The water loss, relative water content, relative electrolyte leak, and total chlorophyll content were determined using a previously described method [62]. Chlorophyll fluorescence was measured and recorded using a Pocket PEA Chlorophyll Fluorimeter (Hansatech, King’s Lynn, UK). The photosynthesis parameters, including intercellular CO2 concentration, stomatal conductance, transpiration rate, net photosynthetic rate, and water use efficiency, were measured via CIRAS-3 (Boston, MA, USA).
Three-week-old plants of the Col-0 and DgIAA21-overexpressing varieties were treated with 20% PEG 6000 (w/v), and the leaves were collected 0 h, 2 h, 4 h, 6 h, 12 h, and 24 h after treatment. Then, 13 stress-related genes and 8 chlorophyll-related gene were further analyzed via qRT-PCR [24,61]. The reference genes were the Actin gene and the Actin2 gene of Arabidopsis. The specific primers are listed in Table S3.

4.5. Subcellular Localization of DgIAA21 Protein

In order to explore the location of DgIAA21, the ORF of DgIAA21 was cloned into a pAN580-35S-GFP vector. The specific primers are presented in Table S4. The empty vector of pAN580-35S-GFP was used as the control. The recombined fusion vector and empty control vector were transferred into the rice protoplasts as described in [63]. Subcellular localizations were visualized using a Nikon C2-ER laser confocal microscope system with excitation at 488 nm and emission at 510 nm.

4.6. Plasmid Construction and Transformation of A. thaliana

The ORF of DgIAA21 was inserted into a pCAMBIA1300-35S-GUS vector, and the specific primers are listed in Table S4. The recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101, which was transformed into Arabidopsis via the floral dip method [64]. The transgenic Arabidopsis plants were screened on a Petri dish with 1/2 Morishige and Skoog (MS) medium containing 30 ug/mL hygromycin. Transgenic plants were verified via qRT-PCR amplification with DgIAA21-qPCR-F/R primers (Table S3). Homozygous lines in the T3 generation overexpressing DgIAA21 were used in subsequent phenotypic and stress assays.

4.7. Identification and Protein Properties of the Aux/IAA Gene Family in Orchardgrass

The total protein sequences of orchardgrass were obtained from the latest version of the orchardgrass reference genome data [59]. The Aux/IAA protein sequences of Arabidopsis thaliana and Oryza sativa were downloaded from the NCBI gene database (https://www.ncbi.nlm.nih.gov, accessed on 1 August 2023) according to the Gene ID [34,65]. The Arabidopsis thaliana Aux/IAA protein sequences were used as queries to search the orchardgrass genome for putative Aux/IAA sequences, which were obtained based on a BLASTP search in NCBI. Further support for the putative Aux/IAA protein sequences was gained using hidden Markov model (HMM) profiles with a domain of Aux/IAA (PF02309). The protein length in amino acids, molecular weight, isoelectric point, instability index, aliphatic index, and grand average of hydropathicity of the Aux/IAA gene family were calculated using ProtParam (https://web.expasy.org/protparam/, accessed on 5 August 2023). The subcellular localization of proteins was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 5 August 2023).

4.8. Phylogenetic and Synteny Analysis

The intron–exon structure and chromosome location of orchardgrass Aux/IAAs were analyzed using TBtools. MEME Suite was used to analyze the conserved motifs of orchardgrass Aux/IAA proteins (https://meme-suite.org/meme/tools/meme, accessed on 7 August 2023). A maximum likelihood (ML) phylogenetic tree was built using MEGA 7.0 software with 1000 bootstrap iterations [66]. The tree was further edited using the Interactive Tree of Life (iTOL) v5.7 web tool [67]. The collinear regions in orchardgrass Aux/IAA genes, as well as the collinear regions of Aux/IAAs among orchardgrass, Arabidopsis, and rice, were explored using MCScanX software https://mybiosoftware.com/tag/mcscanx (accessed on 5 November 2023) [68].

4.9. Statistical Analysis

The data of histogram was analyzed through an analysis of variance (ANOVA) using SPSS 22.0 software (IBM, Armonk, NY, USA), followed by a Tukey’s test (p < 0.05). The histogram was further produced using GraphPad Prism 9.2.0 software (GraphPad, Boston, MA, USA).

5. Conclusions

We identified 30 Aux/IAAs in orchardgrass, which were classified into two clades that showed divergence between dicots and monocots. Segmental duplication may have played a leading role during the evolution of the Aux/IAA gene family in orchardgrass. We isolated and identified the DgIAA21 gene in orchardgrass. Heterologous expression of the DgIAA21 gene reduced drought tolerance in yeast. The overexpression of DgIAA21 in Arabidopsis had poor WUE and low total chlorophyll content and Pn in control conditions, which may have caused decreased tolerance when overexpressing plants suffered drought stress. Moreover, compared with Col-0 plants, higher levels of MDA content and relative electrolyte leakage and lower APX1 and NHX2 gene expression levels in transgenic plants were observed under drought stress, which also proved that DgIAA21 transgenic plants were more sensitive than Col-0 plants. This study provides valuable information for the biological function of DgAux/IAA in the regulatory mechanisms of abiotic stress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242216184/s1.

Author Contributions

Conceptualization, M.W. and G.F.; methodology, M.W., G.F., Z.Y., J.W. and B.L.; software, M.W., G.F. and Z.Y.; validation, M.W. and G.F.; formal analysis, M.W., G.F. and Z.Y.; investigation, M.W. and G.F.; resources, G.F. and M.W.; data curation, M.W. and G.F.; writing—original draft preparation, M.W.; writing—review and editing, M.W., G.F., J.W. and X.X.; visualization, M.W., G.F., B.L. and G.N.; supervision, X.Z.; project administration, X.Z. and L.H.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Modern Agro-industry Technology Research System (No. CARS-34), Sichuan Province’s Science Fund for Distinguished Young Scholars under Grant (2021JDJQ001), Sichuan Province’s Science Fund for International Cooperation (2022YFH0058), and National Natural Science Foundation of China (NSFC 32101422).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Huang, L.; Yan, H.; Jiang, X.; Zhang, Y.; Zhang, X.; Ji, Y.; Zeng, B.; Xu, B.; Yin, G.; Lee, S.; et al. Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses. Gene 2014, 553, 158–165. [Google Scholar] [CrossRef]
  2. Casler, M.; Fales, S.; Undersander, D.; McElroy, A. Genetic progress from 40 years of orchardgrass breeding in North America measured under management-intensive rotational grazing. Can. J. Plant Sci. 2001, 81, 713–721. [Google Scholar] [CrossRef]
  3. Yang, Z.; Yan, H.; Wang, J.; Nie, G.; Feng, G.; Xu, X.; Li, D.; Huang, L.; Zhang, X. DNA hypermethylation promotes the flowering of orchardgrass during vernalization. Plant Physiol. 2022, 190, 1490–1505. [Google Scholar] [CrossRef] [PubMed]
  4. Lymperopoulos, P.; Msanne, J.; Rabara, R. Phytochrome and phytohormones: Working in tandem for plant growth and development. Front. Plant Sci. 2018, 9, 1037. [Google Scholar] [CrossRef]
  5. Luo, J.; Zhou, J.; Zhang, J. Aux/IAA gene family in plants: Molecular structure, regulation, and function. Int. J. Mol. Sci. 2018, 19, 259. [Google Scholar] [CrossRef] [PubMed]
  6. Nemhauser, J.L. Back to basics: What is the function of an Aux/IAA in auxin response? New Phytol. 2018, 218, 1295–1297. [Google Scholar] [CrossRef] [PubMed]
  7. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [PubMed]
  8. Wu, W.; Liu, Y.; Wang, Y.; Li, H.; Liu, J.; Tan, J.; He, J.; Bai, J.; Ma, H. Evolution analysis of the Aux/IAA gene family in plants shows dual origins and variable nuclear localization signals. Int. J. Mol. Sci. 2017, 18, 2107. [Google Scholar] [PubMed]
  9. Su, Y.; He, H.; Wang, P.; Ma, Z.; Mao, J.; Chen, B. Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid (Aux/IAA) gene family in apple (Malus domestica). Gene 2021, 768, 145302. [Google Scholar] [CrossRef]
  10. Shi, Q.; Zhang, Y.; To, V.-T.; Shi, J.; Zhang, D.; Cai, W. Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid (Aux/IAA) gene family in barley (Hordeum vulgare L.). Sci. Rep. 2020, 10, 10242. [Google Scholar] [CrossRef] [PubMed]
  11. Ke, Y.; Abbas, F.; Zhou, Y.; Yu, R.; Yue, Y.; Li, X.; Yu, Y.; Fan, Y. Genome-wide analysis and characterization of the Aux/IAA family genes related to floral scent formation in Hedychium coronarium. Int. J. Mol. Sci. 2019, 20, 3235. [Google Scholar] [CrossRef]
  12. Wang, Y.; Deng, D.; Bian, Y.; Lv, Y.; Xie, Q. Genome-wide analysis of primary auxin-responsive Aux/IAA gene family in maize (Zea mays. L.). Mol. Biol. Rep. 2010, 37, 3991–4001. [Google Scholar] [CrossRef] [PubMed]
  13. Paul, P.; Dhandapani, V.; Rameneni, J.J.; Li, X.; Sivanandhan, G.; Choi, S.R.; Pang, W.; Im, S.; Lim, Y.P. Genome-wide analysis and characterization of Aux/IAA family genes in Brassica rapa. PLoS ONE 2016, 11, e0151522. [Google Scholar] [CrossRef]
  14. Li, H.; Wang, B.; Zhang, Q.; Wang, J.; King, G.J.; Liu, K. Genome-wide analysis of the auxin/indoleacetic acid (Aux/IAA) gene family in allotetraploid rapeseed (Brassica napus L.). BMC Plant Biol. 2017, 17, 204. [Google Scholar] [CrossRef]
  15. Zhang, X.; Lu, G.; Long, W.; Zou, X.; Li, F.; Nishio, T. Recent progress in drought and salt tolerance studies in Brassica crops. Breed. Sci. 2014, 64, 60–73. [Google Scholar] [CrossRef]
  16. Urbanavičiūtė, I.; Bonfiglioli, L.; Pagnotta, M.A. One hundred candidate genes and their roles in drought and salt tolerance in wheat. Int. J. Mol. Sci. 2021, 22, 6378. [Google Scholar] [CrossRef] [PubMed]
  17. Chawla, N. Defensive role of nitric oxide (NO) under drought stress in plants: Mini. J. Emerg. Technol. Innov. Res. 2019, 6, 898–901. [Google Scholar] [CrossRef]
  18. Sakcali, M.; Bahadir, H.; Ozturk, M. Ecophysiology of Capparis spinosa L.: A plant suitable for combating desertification. Pak. J. Bot. 2008, 40, 1481–1486. [Google Scholar]
  19. Quan, R.; Lin, H.; Mendoza, I.; Zhang, Y.; Cao, W.; Yang, Y.; Shang, M.; Chen, S.; Pardo, J.M.; Guo, Y. SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress. Plant Cell 2007, 19, 1415–1431. [Google Scholar] [CrossRef]
  20. Munns, R.; Tester, M. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 2008, 59, 651–681. [Google Scholar] [CrossRef]
  21. Kapoor, D.; Bhardwaj, S.; Landi, M.; Sharma, A.; Ramakrishnan, M.; Sharma, A. The impact of drought in plant metabolism: How to exploit tolerance mechanisms to increase crop production. Appl. Sci. 2020, 10, 5692. [Google Scholar] [CrossRef]
  22. Pequeno, D.N.; Hernandez-Ochoa, I.M.; Reynolds, M.; Sonder, K.; MoleroMilan, A.; Robertson, R.D.; Lopes, M.S.; Xiong, W.; Kropff, M.; Asseng, S. Climate impact and adaptation to heat and drought stress of regional and global wheat production. Environ. Res. Lett. 2021, 16, 054070. [Google Scholar] [CrossRef]
  23. Ahmed, S.; Kouser, S.; Asgher, M.; Gandhi, S.G. Plant aquaporins: A frontward to make crop plants drought resistant. Physiol. Plant. 2021, 172, 1089–1105. [Google Scholar] [CrossRef]
  24. Laxa, M.; Liebthal, M.; Telman, W.; Chibani, K.; Dietz, K.-J. The role of the plant antioxidant system in drought tolerance. Antioxidants 2019, 8, 94. [Google Scholar] [CrossRef] [PubMed]
  25. González-Gordo, S.; Rodríguez-Ruiz, M.; Palma, J.M.; Corpas, F.J. Superoxide radical metabolism in sweet pepper (Capsicum annuum L.) fruits is regulated by ripening and by a NO-enriched environment. Front. Plant Sci. 2020, 11, 485. [Google Scholar] [CrossRef] [PubMed]
  26. Sharma, A.; Kumar, V.; Shahzad, B.; Ramakrishnan, M.; Singh Sidhu, G.P.; Bali, A.S.; Handa, N.; Kapoor, D.; Yadav, P.; Khanna, K.; et al. Photosynthetic response of plants under different abiotic stresses: A review. J. Plant Growth Regul. 2020, 39, 509–531. [Google Scholar] [CrossRef]
  27. Corpas, F.J.; González-Gordo, S.; Palma, J.M. Plant peroxisomes: A factory of reactive species. Front. Plant Sci. 2020, 11, 853. [Google Scholar] [CrossRef] [PubMed]
  28. Lohse, G.; Hedrich, R. Characterization of the plasma-membrane H+-ATPase from Vicia faba guard cells. Planta 1992, 188, 206–214. [Google Scholar] [CrossRef]
  29. Balcerowicz, M.; Ranjan, A.; Rupprecht, L.; Fiene, G.; Hoecker, U. Auxin represses stomatal development in dark-grown seedlings via Aux/IAA proteins. Development 2014, 141, 3165–3176. [Google Scholar] [CrossRef]
  30. Zhang, J.-Y.; He, S.-B.; Li, L.; Yang, H.-Q. Auxin inhibits stomatal development through MONOPTEROS repression of a mobile peptide gene STOMAGEN in mesophyll. Proc. Nat. Acad. Sci. USA 2014, 111, E3015–E3023. [Google Scholar] [CrossRef] [PubMed]
  31. Salehin, M.; Li, B.; Tang, M.; Katz, E.; Song, L.; Ecker, J.R.; Kliebenstein, D.J.; Estelle, M. Auxin-sensitive Aux/IAA proteins mediate drought tolerance in Arabidopsis by regulating glucosinolate levels. Nat. Commun. 2019, 10, 4021. [Google Scholar] [CrossRef] [PubMed]
  32. Zhang, A.; Yang, X.; Lu, J.; Song, F.; Sun, J.; Wang, C.; Lian, J.; Zhao, L.; Zhao, B. OsIAA20, an Aux/IAA protein, mediates abiotic stress tolerance in rice through an ABA pathway. Plant Sci. 2021, 308, 110903. [Google Scholar] [CrossRef]
  33. Huang, D.; Wang, Q.; Duan, D.; Dong, Q.; Zhao, S.; Zhang, M.; Jing, G.; Liu, C.; van Nocker, S.; Ma, F.; et al. Overexpression of MdIAA9 confers high tolerance to osmotic stress in transgenic tobacco. PeerJ 2019, 7, e7935. [Google Scholar] [CrossRef] [PubMed]
  34. Liscum, E.; Reed, J. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Mol. Biol. 2002, 49, 387–400. [Google Scholar] [CrossRef]
  35. Shani, E.; Salehin, M.; Zhang, Y.; Sanchez, S.E.; Doherty, C.; Wang, R.; Mangado, C.C.; Song, L.; Tal, I.; Pisanty, O. Plant stress tolerance requires auxin-sensitive Aux/IAA transcriptional repressors. Curr. Biol. 2017, 27, 437–444. [Google Scholar] [CrossRef]
  36. Wang, J.; Yan, D.-W.; Yuan, T.-T.; Gao, X.; Lu, Y.-T. A gain-of-function mutation in IAA8 alters Arabidopsis floral organ development by change of jasmonic acid level. Plant Mol. Biol. 2013, 82, 71–83. [Google Scholar] [CrossRef]
  37. Rinaldi, M.A.; Liu, J.; Enders, T.A.; Bartel, B.; Strader, L.C. A gain-of-function mutation in IAA16 confers reduced responses to auxin and abscisic acid and impedes plant growth and fertility. Plant Mol. Biol. 2012, 79, 359–373. [Google Scholar] [CrossRef] [PubMed]
  38. Song, Y.; Xu, Z.-F. Ectopic overexpression of an auxin/indole-3-acetic acid (Aux/IAA) gene OsIAA4 in rice induces morphological changes and reduces responsiveness to auxin. Int. J. Mol. Sci. 2013, 14, 13645–13656. [Google Scholar] [CrossRef] [PubMed]
  39. Nakamura, A.; Umemura, I.; Gomi, K.; Hasegawa, Y.; Kitano, H.; Sazuka, T.; Matsuoka, M. Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein. Plant J. 2006, 46, 297–306. [Google Scholar] [CrossRef]
  40. Bowman, J.L.; Kohchi, T.; Yamato, K.T.; Jenkins, J.; Shu, S.; Ishizaki, K.; Yamaoka, S.; Nishihama, R.; Nakamura, Y.; Berger, F. Insights into land plant evolution garnered from the Marchantia polymorpha genome. Cell 2017, 171, 287–304.e215. [Google Scholar] [CrossRef]
  41. Kumar, R.; Agarwal, P.; Pareek, A.; Tyagi, A.K.; Sharma, A.K. Genomic survey, gene expression, and interaction analysis suggest diverse roles of ARF and Aux/IAA proteins in Solanaceae. Plant Mol. Biol. Rep. 2015, 33, 1552–1572. [Google Scholar] [CrossRef]
  42. Jain, M.; Kaur, N.; Garg, R.; Thakur, J.K.; Tyagi, A.K.; Khurana, J.P. Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct. Integr. Genom. 2006, 6, 47–59. [Google Scholar] [CrossRef] [PubMed]
  43. Singh, V.K.; Jain, M. Genome-wide survey and comprehensive expression profiling of Aux/IAA gene family in chickpea and soybean. Front. Plant Sci. 2015, 6, 918. [Google Scholar] [CrossRef] [PubMed]
  44. Bowers, J.E.; Chapman, B.A.; Rong, J.; Paterson, A.H. Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature 2003, 422, 433–438. [Google Scholar] [CrossRef] [PubMed]
  45. Lawas, L.M.F.; Zuther, E.; Jagadish, S.K.; Hincha, D.K. Molecular mechanisms of combined heat and drought stress resilience in cereals. Curr. Opin. Plant Biol. 2018, 45, 212–217. [Google Scholar] [CrossRef] [PubMed]
  46. Cakir, B.; Kiliçkaya, O.; Olcay, A.C. Genome-wide analysis of Aux/IAA genes in Vitis vinifera: Cloning and expression profiling of a grape Aux/IAA gene in response to phytohormone and abiotic stresses. Acta Physiol. Plant. 2013, 35, 365–377. [Google Scholar] [CrossRef]
  47. Jung, H.; Lee, D.-K.; Do Choi, Y.; Kim, J.-K. OsIAA6, a member of the rice Aux/IAA gene family, is involved in drought tolerance and tiller outgrowth. Plant Sci. 2015, 236, 304–312. [Google Scholar] [CrossRef]
  48. Li, G.; Ye, Y.; Ren, X.; Qi, M.; Zhao, H.; Zhou, Q.; Chen, X.; Wang, J.; Yuan, C.; Wang, F. The rice Aux/IAA transcription factor gene OsIAA18 enhances salt and osmotic tolerance in Arabidopsis. Biol. Plant. 2020, 64, 454–464. [Google Scholar] [CrossRef]
  49. Sun, J.; Gu, J.; Zeng, J.; Han, S.; Song, A.; Chen, F.; Fang, W.; Jiang, J.; Chen, S. Changes in leaf morphology, antioxidant activity and photosynthesis capacity in two different drought-tolerant cultivars of chrysanthemum during and after water stress. Sci. Hortic. 2013, 161, 249–258. [Google Scholar] [CrossRef]
  50. Bertioli, D.J.; Cannon, S.B.; Froenicke, L.; Huang, G.; Farmer, A.D.; Cannon, E.K.; Liu, X.; Gao, D.; Clevenger, J.; Dash, S. The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nat. Genet. 2016, 48, 438–446. [Google Scholar] [CrossRef]
  51. Batra, N.G.; Sharma, V.; Kumari, N. Drought-induced changes in chlorophyll fluorescence, photosynthetic pigments, and thylakoid membrane proteins of Vigna radiata. J. Plant Interact. 2014, 9, 712–721. [Google Scholar] [CrossRef]
  52. Wang, X.; Gao, F.; Bing, J.; Sun, W.; Feng, X.; Ma, X.; Zhou, Y.; Zhang, G. Overexpression of the Jojoba aquaporin gene, ScPIP1, enhances drought and salt tolerance in transgenic Arabidopsis. Int. J. Mol. Sci. 2019, 20, 153. [Google Scholar] [CrossRef] [PubMed]
  53. Bonfiglioli, L.; Pagnotta, M.A. Diversity in Root Architecture of Durum Wheat at Stem Elongation under Drought Stress. Agronomy 2022, 12, 1329. [Google Scholar] [CrossRef]
  54. Mao, X.; Zhang, H.; Qian, X.; Li, A.; Zhao, G.; Jing, R. TaNAC2, a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis. J. Exp. Bot. 2012, 63, 2933–2946. [Google Scholar] [CrossRef] [PubMed]
  55. Huang, Q.; Wang, Y.; Li, B.; Chang, J.; Chen, M.; Li, K.; Yang, G.; He, G. TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis. BMC Plant Biol. 2015, 15, 268. [Google Scholar] [CrossRef] [PubMed]
  56. Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9, 490–498. [Google Scholar] [CrossRef]
  57. Li, T.; Huang, Y.; Khadr, A.; Wang, Y.-H.; Xu, Z.-S.; Xiong, A.-S. DcDREB1A, a DREB-binding transcription factor from Daucus carota, enhances drought tolerance in transgenic Arabidopsis thaliana and modulates lignin levels by regulating lignin-biosynthesis-related genes. Environ. Exp. Bot. 2020, 169, 103896. [Google Scholar] [CrossRef]
  58. Davey, M.; Stals, E.; Panis, B.; Keulemans, J.; Swennen, R. High-throughput determination of malondialdehyde in plant tissues. Anal. Biochem. 2005, 347, 201–207. [Google Scholar] [CrossRef] [PubMed]
  59. Huang, L.; Feng, G.; Yan, H.; Zhang, Z.; Bushman, B.S.; Wang, J.; Bombarely, A.; Li, M.; Yang, Z.; Nie, G. Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass. Plant Biotechnol. J. 2020, 18, 373–388. [Google Scholar] [CrossRef]
  60. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  61. Wang, M.; Zhuang, J.; Zou, Z.; Li, Q.; Xin, H.; Li, X. Overexpression of a Camellia sinensis DREB Transcription Factor Gene (CsDREB) Increases Salt and Drought Tolerance in Transgenic Arabidopsis thaliana. J. Plant Biol. 2017, 60, 452–461. [Google Scholar] [CrossRef]
  62. Qin, L.; Rao, Y.; Li, L.; Huang, J.; Xu, W.; Li, X. Cotton GalT1 encoding a putative glycosyltransferase is involved in regulation of cell wall pectin biosynthesis during plant development. PLoS ONE 2013, 8, e59115. [Google Scholar] [CrossRef] [PubMed]
  63. Yoo, S.-D.; Cho, Y.-H.; Sheen, J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2007, 2, 1565–1572. [Google Scholar] [CrossRef] [PubMed]
  64. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735–743. [Google Scholar] [CrossRef]
  65. Ulmasov, T.; Murfett, J.; Hagen, G.; Guilfoyle, T.J. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 1997, 9, 1963–1971. [Google Scholar] [CrossRef]
  66. Hall, B.G. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 2013, 30, 1229–1235. [Google Scholar] [CrossRef] [PubMed]
  67. Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Res. 2019, 47, W256–W259. [Google Scholar] [CrossRef] [PubMed]
  68. Wang, Y.; Tang, H.; DeBarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-H.; Jin, H.; Marler, B.; Guo, H. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed]
Ijms 24 16184 g001
Figure 1. Gene cluster, structure, and conserved motifs analysis of DgAux/IAA gene family. The phylogenetic tree, intron–exon structure distribution and conserved motif distribution of DgAux/IAAs were sequenced from left to right. The DgAux/IAAs were divided into two clades: the yellow presents clade (A) and the green presents clade (B) in the phylogenetic tree section. The yellow boxes indicate exons, and the gray lines indicate introns in the intron–exon structure section. Ten putative motifs are shown in different colored boxes in the conserved motif analysis section.
Figure 1. Gene cluster, structure, and conserved motifs analysis of DgAux/IAA gene family. The phylogenetic tree, intron–exon structure distribution and conserved motif distribution of DgAux/IAAs were sequenced from left to right. The DgAux/IAAs were divided into two clades: the yellow presents clade (A) and the green presents clade (B) in the phylogenetic tree section. The yellow boxes indicate exons, and the gray lines indicate introns in the intron–exon structure section. Ten putative motifs are shown in different colored boxes in the conserved motif analysis section.
Ijms 24 16184 g001
Ijms 24 16184 g002
Figure 2. Construction of phylogenetic trees of Aux/IAA gene family among DgIAAs, AtIAAs, and OsIAAs. Yellow regions represent clade A, while the green areas indicate clade B.
Figure 2. Construction of phylogenetic trees of Aux/IAA gene family among DgIAAs, AtIAAs, and OsIAAs. Yellow regions represent clade A, while the green areas indicate clade B.
Ijms 24 16184 g002
Ijms 24 16184 g003
Figure 3. Chromosome location and syntenic analysis of Aux/IAA gene family. (A) Distribution of DgAux/IAAs on orchardgrass chromosomes. (B) Segmental duplication of DgAux/IAA genes. Different gray lines represent different gene pairs on Aux/IAA family of orchardgrass. (C) Syntenic analysis of Dactylis glomerata, Arabidopsis thaliana, and Oryza sativa Aux/IAA genes. Highlighted red lines show the syntenic relationships among Dactylis glomerata, Arabidopsis thaliana, and Oryza sativa Aux/IAA genes.
Figure 3. Chromosome location and syntenic analysis of Aux/IAA gene family. (A) Distribution of DgAux/IAAs on orchardgrass chromosomes. (B) Segmental duplication of DgAux/IAA genes. Different gray lines represent different gene pairs on Aux/IAA family of orchardgrass. (C) Syntenic analysis of Dactylis glomerata, Arabidopsis thaliana, and Oryza sativa Aux/IAA genes. Highlighted red lines show the syntenic relationships among Dactylis glomerata, Arabidopsis thaliana, and Oryza sativa Aux/IAA genes.
Ijms 24 16184 g003
Ijms 24 16184 g004
Figure 4. The expression pattern of DgAux/IAA genes. (A) The expression profiles of Aux/IAA genes in different tissues. (B) The expression analysis of seven genes of Aux/IAA under IAA, drought, salt, heat, and cold stresses. Bars represent mean standard deviation (SD). The different letters above the bars indicate significant differences among different times (p < 0.05, according to Tukey’s multiple range test).
Figure 4. The expression pattern of DgAux/IAA genes. (A) The expression profiles of Aux/IAA genes in different tissues. (B) The expression analysis of seven genes of Aux/IAA under IAA, drought, salt, heat, and cold stresses. Bars represent mean standard deviation (SD). The different letters above the bars indicate significant differences among different times (p < 0.05, according to Tukey’s multiple range test).
Ijms 24 16184 g004
Ijms 24 16184 g005
Figure 5. Phenotypic growth assays of pYES2-DgIAA3, -DgIAA4, -DgIAA6, -DgIAA14, -DgIAA21, and empty vector under drought stress. INVScI yeast cells were dotted on YPG medium in 2 uL aliquots and were diluted 10 times in turn using ddH2O.
Figure 5. Phenotypic growth assays of pYES2-DgIAA3, -DgIAA4, -DgIAA6, -DgIAA14, -DgIAA21, and empty vector under drought stress. INVScI yeast cells were dotted on YPG medium in 2 uL aliquots and were diluted 10 times in turn using ddH2O.
Ijms 24 16184 g005
Ijms 24 16184 g006
Figure 6. Subcellular localization of DgIAA21 in rice protoplasts. The green florescent protein signals obtained via confocal microscopy showed that fusion protein DgIAA21-GFP were localized primarily in the nucleus, and minority in nucleus and cytoplasm. The green florescent protein fields, chlorophyll auto fluorescent fields, bright fields, and merge fields are indicated in images from left to right. Scale bar = 10 um.
Figure 6. Subcellular localization of DgIAA21 in rice protoplasts. The green florescent protein signals obtained via confocal microscopy showed that fusion protein DgIAA21-GFP were localized primarily in the nucleus, and minority in nucleus and cytoplasm. The green florescent protein fields, chlorophyll auto fluorescent fields, bright fields, and merge fields are indicated in images from left to right. Scale bar = 10 um.
Ijms 24 16184 g006
Ijms 24 16184 g007
Figure 7. Effects of drought stress on germination and root length of Col-0 and overexpression DgIAA21 transgenic Arabidopsis lines. (A) Phenotypes and germination rates of Col-0 and DgIAA21 transgenic lines subjected to 1/2 MS, 200 mM Mannitol, and 300 mM Mannitol treatments. (B) Phenotypes and root lengths in DgIAA21 transgenic lines and Col-0 lines under same treatment conditions. Bars represent mean standard deviation (SD). The different letters above the bars indicate significant differences between Col-0 and three transgenic lines (p < 0.05, according to Tukey’s multiple range test).
Figure 7. Effects of drought stress on germination and root length of Col-0 and overexpression DgIAA21 transgenic Arabidopsis lines. (A) Phenotypes and germination rates of Col-0 and DgIAA21 transgenic lines subjected to 1/2 MS, 200 mM Mannitol, and 300 mM Mannitol treatments. (B) Phenotypes and root lengths in DgIAA21 transgenic lines and Col-0 lines under same treatment conditions. Bars represent mean standard deviation (SD). The different letters above the bars indicate significant differences between Col-0 and three transgenic lines (p < 0.05, according to Tukey’s multiple range test).
Ijms 24 16184 g007
Ijms 24 16184 g008
Figure 8. Overexpression of DgIAA21 transgenic Arabidopsis plants were sensitive to drought tolerance. Three-week-old A.thaliana plants with Col-0 and DgIAA21- overexpression were used to drought experiment. (A) Appearance of plants were given drought tolerance through withholding watering for 14 days and recovery watering for 4 days. Comparison of photosynthesis parameters and several physiological parameters in Col-0 and transgenic plants: (B) intercellular CO2 concentration, (C) stomatal conductance, (D) net photosynthetic rate, (E) water use efficiency, (F) transpiration rate, (G) water loss, (H) relative water content, (I) total chlorophyll content, (J) chlorophyll fluorescence, (K) relative electrolyte leakage, and (L) MDA content. (B–G) were measured under normal condition, while (H–L) were detected under normal and drought conditions. Bars represent mean standard deviation (SD). The different letters above the bars indicate significant differences between Col-0 and three transgenic lines (p < 0.05, according to Tukey’s multiple range test).
Figure 8. Overexpression of DgIAA21 transgenic Arabidopsis plants were sensitive to drought tolerance. Three-week-old A.thaliana plants with Col-0 and DgIAA21- overexpression were used to drought experiment. (A) Appearance of plants were given drought tolerance through withholding watering for 14 days and recovery watering for 4 days. Comparison of photosynthesis parameters and several physiological parameters in Col-0 and transgenic plants: (B) intercellular CO2 concentration, (C) stomatal conductance, (D) net photosynthetic rate, (E) water use efficiency, (F) transpiration rate, (G) water loss, (H) relative water content, (I) total chlorophyll content, (J) chlorophyll fluorescence, (K) relative electrolyte leakage, and (L) MDA content. (B–G) were measured under normal condition, while (H–L) were detected under normal and drought conditions. Bars represent mean standard deviation (SD). The different letters above the bars indicate significant differences between Col-0 and three transgenic lines (p < 0.05, according to Tukey’s multiple range test).
Ijms 24 16184 g008
Ijms 24 16184 g009
Figure 9. The expression of stress-related genes and chlorophyll-related genes of Col-0 and DgIAA21 OE1 line under 20% PEG 6000 treatment. (A) The heatmap of expression levels of 13 stress-related genes after 0 h, 2 h, 4 h, 6 h, 12 h, and 24 h under drought stress. (B) The changes in the expression level of chlorophyll degradation pathway genes (C) and chlorophyll synthesis pathway genes (C) after 0 h and 24 h under drought stress. Error bars represent mean standard deviation (SD). Asterisks (*) represent significant differences compared with the Col-0 plants at the same time. *, p < 0.05; **, p < 0.01, ns: non-significant.
Figure 9. The expression of stress-related genes and chlorophyll-related genes of Col-0 and DgIAA21 OE1 line under 20% PEG 6000 treatment. (A) The heatmap of expression levels of 13 stress-related genes after 0 h, 2 h, 4 h, 6 h, 12 h, and 24 h under drought stress. (B) The changes in the expression level of chlorophyll degradation pathway genes (C) and chlorophyll synthesis pathway genes (C) after 0 h and 24 h under drought stress. Error bars represent mean standard deviation (SD). Asterisks (*) represent significant differences compared with the Col-0 plants at the same time. *, p < 0.05; **, p < 0.01, ns: non-significant.
Ijms 24 16184 g009
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, M.; Feng, G.; Yang, Z.; Wu, J.; Liu, B.; Xu, X.; Nie, G.; Huang, L.; Zhang, X. Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress. Int. J. Mol. Sci. 2023, 24, 16184. https://doi.org/10.3390/ijms242216184

AMA Style

Wang M, Feng G, Yang Z, Wu J, Liu B, Xu X, Nie G, Huang L, Zhang X. Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress. International Journal of Molecular Sciences. 2023; 24(22):16184. https://doi.org/10.3390/ijms242216184

Chicago/Turabian Style

Wang, Miaoli, Guanyan Feng, Zhongfu Yang, Jiahui Wu, Bingyan Liu, Xiaoheng Xu, Gang Nie, Linkai Huang, and Xinquan Zhang. 2023. "Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress" International Journal of Molecular Sciences 24, no. 22: 16184. https://doi.org/10.3390/ijms242216184

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop