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Article

Expression Profiling of Salt-Responsive Genes and Transcription Factors in Leaf Transcriptome of Arabidopsis thaliana

by
Nahaa M. Alotaibi
1 and
Aala A. Abulfaraj
2,*
1
Department of Biology, College of Sciences, Princess Nourah Bint Abdulrahman University, P.O. Box 84428, Riyadh 11617, Saudi Arabia
2
Biological Sciences Department, College of Science & Arts, King Abdulaziz University, Rabigh 21911, Saudi Arabia
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(11), 1119; https://doi.org/10.3390/d15111119
Submission received: 26 September 2023 / Revised: 16 October 2023 / Accepted: 25 October 2023 / Published: 27 October 2023
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Abstract

:
This investigation discerns the expression profiles of genes within the leaf transcriptome of Arabidopsis thaliana subjected to salt stress (200 mM NaCl). Notably, the pivotal role of indole acetic acid emerged as a keystone orchestrating a multifaceted cascade of regulatory events aimed at enhancing the plant’s adaptability under salt-induced stress. Cluster analysis elucidated upregulation of gene families with pivotal roles in supporting the availability of carbon dioxide, ameliorating photosynthetic processes and mitigating the deleterious effects of reactive oxygen species under salt stress. Analysis also unveiled the participation of several transcription factor families in the orchestration of a multitude of genes under salt stress. The investigation singled out a solitary TF, denominated as BH100, which was validated through RNA-Seq and qPCR, utilizing a VIGS line featuring the knockdown of the BH100 gene. This transcription factor was implicated in the upregulation of the FRO gene, thereby establishing a link between the synchronized expression of these two genes and their role in promoting iron acquisition under salt stress. In summation, our study unveiled the regulatory frameworks and salt-responsive genes underpinning the response of Arabidopsis to salt stress. We present compelling arguments for the potential applicability of this information in the realm of molecular breeding programs.

1. Introduction

Abiotic perturbations, encompassing a spectrum of deleterious environmental contingencies, manifest as significant obstacles affecting plants’ vigour and fertility [1]. These stressors exert a transformative influence upon the expansive array of genes resident within a plant’s transcriptome, engendering novel and spatiotemporally dynamic expression patterns that possess significant implications upon the plant’s developmental determinations during distinct ontogenic phases [2]. In the quest to unravel the intricate molecular orchestration underlying stress-induced gene expression alterations, the use of RNA-Sequencing (RNA-Seq) technology emerges as an indispensable approach [3]. This high-throughput method endows us with the precision to quantify differentially expressed (DE) genes at a granular resolution, while also affording the capacity to identify alternately spliced isoforms within the intricate web of complex transcripts, all in response to their environmental milieu [4]. The advent of publicly accessible plant gene databases has vastly expedited the exploration of plant transcriptomes within the crucible of stress conditions. These repositories are invaluable for ascertaining the expression patterns of stress-responsive genes, exemplified by the Arabidopsis Stress-Responsive Gene Database (ASRGDB) [5], the identification of transcription factors (TFs) carrying out the reins of stress-responsive genes, as evidenced by the Stress-Responsive Genes and Transcription Factor Database (STIFDB2) [6], and the revelation of alternatively spliced isoforms in the progress of stress, as exemplified by the Plant-Specific Alternative Splicing Database (PlaASDB) [7].
Transcription factors (TFs), characterized by their cis-acting attributes and DNA-binding domains (DBDs), emerge as genuine conductors of gene expression symphonies, orchestrating intricate and novel gene expression patterns under the influence of many environmental stresses [8]. These master regulators surpass gene expression orchestration; they craft entire genetic circuits governing specific pathways, modulate signal-mediated gene expression cascades culminating in the synthesis of pivotal enzymes or metabolites, and have the capacity to influence sustainable developmental decisions [9,10]. Upon engaging specific DNA sequences proximate to target genes, TFs may either catalyze or impede the recruitment of RNA polymerase to these downstream loci [11]. In the era of post-genomics, these stress-responsive TFs translate into indispensable tools for genetic manipulation and metabolic engineering, furnishing a channel to confer enhanced abiotic stress tolerance upon plants [12,13].
In the realm of genetic inquiry, Virus-Induced Gene Silencing (VIGS), an RNA interference (RNAi)-mediated antiviral defense process, emerges as an invaluable modality for the targeted suppression of specific gene expression, an effective avenue for unraveling gene function [14]. This innovative approach extends its utility to the subtle interrogation of the effects ensuing from the knockdown of TFs within a transcriptomic context. VIGS harnesses the potent virulence of Agrobacterium tumefaciens Ti plasmids to fashion recombinant viruses, such as the Tobacco Rattle Virus (TRV), housing fragments of antisense sequences specific to the genes earmarked for silencing. Introduction of these antisense moieties into plant cells through genetic transmutation activates the endogenous RNAi machinery, thereby provoking dicer-mediated downregulation of target gene expression within the confines of the cell nucleus [15].
A precedent in this avenue of exploration reveals the coexistence of TFs and heat stress-responsive genes in conferring augmented stress tolerance, notably in the contexts of heat and water deficit stresses [16]. Further strides were undertaken in the investigation of concordant TF and stress-responsive gene expression via RNA-Seq modalities, focusing on botanical exemplars such as the love apple (Paris polyphylla) and wheat (Triticum aestivum), unveiling salient associations through the prism of knockout methodologies [17,18]. Against this backdrop, the motivation behind this investigation, aiming to harness RNA-Seq datasets of Arabidopsis thaliana under salt stress to unearth TFs characterized by their concordant expression alongside salt stress-responsive genes. Within this context, we undertake an exploratory endeavor to delineate the putative regulatory mantle of these TFs in shaping the expression milieu of their gene counterparts.

2. Materials and Methods

2.1. Salt Stress Induction in A. thaliana

Cultivation of A. thaliana of the Col-0 ecotype and tobacco (Nicotiana benthamiana) plants was executed within the controlled confines of the greenhouse facility at King Abdulaziz University, Jeddah, Saudi Arabia, whilst rigorously adhering to institutional protocols and prevailing environmental parameters. Notably, no ethical considerations necessitated formal approval for the conduct of the research. The commencement of this investigation witnessed the initiation of surface-sterilized A. thaliana seeds, which were carefully germinated on sterile MS medium in Petri dishes and thereafter maintained in a light-deprived environment at a temperature of 4 °C for a duration spanning four days. Subsequently, these rooted seedlings were transplanted into pots (measuring 9 cm in diameter) replete with a blend of sterilized soil and vermiculate, configured at a ratio of 1:1. The growth chamber was maintained at a steady diurnal temperature of 21 ± 2 °C, punctuated by a luminous flux density approximating ~175 umoles m−2 s−1, with a 16 h photoperiod. Concomitant with this controlled growth regimen, diligent irrigation with deionized double-distilled water was administered on a daily basis, nurturing the seedlings to maturity over a span of three weeks. Thereafter, the crucible of salt stress imposition was initiated, commencing with a salinity exposure of 50 mM NaCl, sustained over a diurnal interval. This preliminary salinity challenge served as the harbinger for subsequent escalations in salinity stress, each increment marked by an addition of 75 mM NaCl, as per established protocols [19]. The pivotal leaf tissues of 4-week-old plants, subjected to an ultimate salt concentration of 200 mM NaCl, were scrupulously harvested in triplicates at 2 h and 12 h intervals following the second incremental salinity escalation. In parallel, leaves originating from 4-week-old unstressed plants, herein termed “0 h”, were concomitantly procured and employed as a control cohort for comparative analysis. Furthermore, the comparative investigation encompassed an analysis of the growth performance of Arabidopsis T-DNA SALK line CS927067 (Col-0 ecotype), genetically manipulated to render it deficient in the STOP1 gene, encoding sensitive to proton rhizotoxicity 1. This genetically modified line was contrasted against its unaltered wild-type (WT) counterpart.

2.2. RNA-Seq and Bioinformatics

Isolation of total RNA was executed, involving the extraction of approximately 50 mg of leaf tissue derived from 4-week-old A. thaliana plants cultivated under diverse environmental conditions. The RNA extraction process was undertaken employing Trizol (Invitrogen, Waltham, MA, USA), supplemented with RNase-free DNase (Promega, Madison, WI, USA) and RNasin® Plus RNase Inhibitor (Promega), the latter of which was administered for a duration of 2 h at a temperature of 37 °C to obviate DNA contamination. The pristine RNA samples thus obtained were expedited to BGI, Beijing, China, for subsequent next-generation sequencing endeavors. Recovered raw data were deposited in the National Center for Biotechnology Information (NCBI) and obtained Bioproject no. PRJNA1018458. The generated sequencing data underwent a series of rigorous processing steps, commencing with the excision of adapter sequences, followed by the alignment of high-quality sequences (possessing ≤ 2 mismatches) to the A. thaliana genome (http://www.arabidopsis.org/, accessed 20 January 2023) facilitated through the utilization of RSEM v1.1.6 and the Bowtie aligner (Bowtie v0.12.1). The determination of differential expression (DE) and concomitant gene clustering were conducted employing EdgeR (R version 2.1.5). A pivotal Blastx analysis was further executed, bearing an E-value threshold of 1 × 10−5, while the centered log2 (fpkm+1) values were determined for samples manifesting differential clustering. Comparative analysis of fpkm read counts among the distinct temporal stages of salt stress induction was accomplished via the likelihood ratio test [20]. Subsequent to extensive permutation analysis, noteworthy Pearson correlations were discerned, and principle component analysis (PCA) was effectuated using Trinity (v2.3.2), conforming to the default parameters.

2.3. Generation of VIGS Lines in Tobacco

The task of constructing VIGS vectors and their subsequent transformation was accomplished through the cultivation of Agrobacterium tumefaciens and Escherichia coli strains within LB medium, sustained at temperatures of 30 °C and 37 °C, respectively. The antibiotic agents Kanamycin (50 μg/mL) and rifampicin (100 μg/mL) were employed in the selective cultivation of transformed A. tumefaciens strains, whereas ampicillin was administered at a concentration of 100 μg/mL for E. coli strains. The design of primers, instrumental in the construction of gateway-compatible pTRV2 vectors for tobacco TFs, analogously aligned with those employed for Arabidopsis, was orchestrated with the aid of Netprimer software (http://www.premierbiosoft.com/netprimer/index.html, accessed 20 January 2023), abiding by conventional protocol. As a discerning touchstone for the success of VIGS, A. tumefaciens transformed strains featuring pTRV2 alone [21] and pTRV2::PDS (phytoene desaturase) genes [22], which were harnessed as negative and positive controls, respectively; the latter eliciting characteristic photo-bleaching of plant leaves. The rigors of the salt stress experiment and the concurrent procurement of total RNA were replicated in 4-week-old tobacco VIGSTF lines, featuring six selected TFs, in strict accordance with the previously described protocol, and adherence to the stipulated criteria.

2.4. Validation of Concordant Expression of TFs and Stress-Responsive Genes in Tobacco VIGS Lines

To validate the concordant expression of selected TFs and salt-responsive genes under salt stress conditions in tobacco VIGS lines, the expression levels of 4-week-old tobacco VIGS lines were quantified through quantitative Polymerase Chain Reaction (qPCR), as outlined in the extant literature [17]. A comprehensive roster of the six concordantly expressed TFs and salt-responsive genes, germane to the validation experiment, is presented in Table S1, with actin and ubiquitin genes serving as dual housekeeping genes throughout the qPCR validation process.

3. Results

3.1. Validating RNA-Seq Datasets of A. thaliana Leaves under Salt Stress

The validation of differentially expressed transcriptomes in A. thaliana leaves across the three time points during salt stress was diligently undertaken through two comprehensive analytical approaches: hierarchical cluster analysis (Figure 1) and PCA (Figure 2). These complementary methods unequivocally elucidated the dynamics of transcriptomic variations across the experimental conditions. The heatmap, as depicted in Figure 1, exhibited significant genetic distance between transcriptomes at the initial time point (0 h) and those at either 2 h or 12 h time points, underscoring the profound impact of salt stress on the plant’s gene expression. Meanwhile, the results derived from PCA, illustrated in Figure 2, underscored the stark divergence between transcriptomes at 0 h and 12 h, with the former existing in the negative quadrant of both PC1 and PC2, while the latter were prominently positioned in the positive quadrants of PC1 and PC2. Intriguingly, a partial congruence between transcriptomes at 2 h and 12 h time points was discerned, with these transcriptomes being positioned on the right side of PC1 and diverging along the orthogonal axis of PC2 (Figure 2).

3.2. Cluster Analysis

Within the context of this study, the term “TF genes” pertains to genes encoding transcription factors, whereas “salt-responsive genes” encompasses those genes not encoding TFs. Across the three distinct time points of salt stress (200 mM NaCl), approximately 1500 differentially expressed (DE) transcripts, showing a ≥4-fold change, were identified within A. thaliana leaves. These transcripts were subsequently categorized into 76 clusters, each exhibiting distinct patterns of expression (Figure S1 and Table S2). A subset of these transcripts, totaling 139, encoded TFs and were distributed among 54 clusters. However, this study focused exclusively on clusters harboring upregulated genes at one or more time points (i.e., 2 h, 12 h, or 2 h/12 h) during salt stress, encompassing a total of 34 clusters out of the 76. Clusters with downregulated genes or those lacking coherence in expression patterns within each time point were excluded from further analysis. Specifically, there were 9 clusters demonstrating upregulated genes at the 2 h time point, 15 clusters exhibiting upregulation at the 12 h time point, and 10 clusters displaying upregulated genes at the 2 h/12 h time points (Figure S1 and Table 1 and Table S2). Remarkably, among these clusters, 12 were devoid of TF genes, including clusters 5 and 76 at the 2 h time point, clusters 19, 27, 39 and 46 at the 12 h time point, and clusters 12, 29, 34, 44, 59 and 72 at the 2 h/12 h time points (Table 1).

3.3. Upregulated Auxin-Related Gene Families under Salt Stress

The comprehensive gene annotation results unveiled a notable abundance of auxin-related genes and gene families within cluster 4, characterized by upregulation at the 12 h time point (Figure S2 and Table S3). This cluster notably featured four auxin-related enzymes: anthranilate synthase (TRPE, EC 4.1.3.27), anthranilate phosphoribosyltransferase (TRP1, EC 2.4.2.18), indole-3-glycerol phosphate synthase (IGPS, EC 4.1.1.48), and tryptophan synthase (TSA, EC 4.2.1.20), all of which participate in the “Phenylalanine, tyrosine, and tryptophan biosynthesis” pathway (Figures S2 and S9). Additionally, two other enzymes, namely tryptophan-pyruvate aminotransferase 1 (TAA1, EC 2.6.1.99) and indole-3-pyruvate monooxygenase (YUC2 or flavin, EC 1.14.13.168), play pivotal roles in “Tryptophan metabolism”, contributing to the biosynthesis of indole acetic acid (IAA), an essential auxin (Figures S2 and S10). This intricate network of auxin-related gene families also encompassed auxin response factors (ARF family), indole-3-acetic acid-amido synthetase (GH3 family), Auxin-responsive protein (IAA family), indole-3-glycerol phosphate synthase (IGPS family), auxin transporter (LAX family), auxin efflux carrier component (PIN family), and auxin-responsive SAUR (SAU family). Specifically, there were seven, six, eleven, one, three, one, and eighteen upregulated auxin-related genes (or gene isoforms) spanning diverse families at the 12 h salt stress time point (Figure S2 and Table S3). These auxin-related gene families collectively participate in the “Plant hormone signal transduction” pathway (Figures S2 and S11).

3.4. Other Upregulated Gene Families under Salt Stress

In addition to the auxin-related gene families, several other salt-responsive gene families experienced upregulation at one or more time points during salt stress in A. thaliana. These gene families encompassed chlorophyll a-b binding (CA and CB), ribulose bisphosphate carboxylase (RBS and RCA), and peroxidase (PER) enzymes (Figure S3 and Tables S2 and S4–S6). Remarkably, all thirteen genes in the CA/CB families (Table S4), all four in the ribulose bisphosphate carboxylase families (Table S5), and two out of five in the peroxidase families (Table S6) were upregulated across various time points of salt stress (Figure S3). Furthermore, certain transcription factor (TF) gene families renowned for their influence under abiotic stress were also upregulated at one or more time points during salt stress in A. thaliana. These included TF families encoding zinc finger CONSTANS (COL), basic leucine zipper (bZIP), zinc finger/RING zinc finger (C3H, GIS, ZAT and ZHD), and NAC domain-containing (NAC) TFs (Tables S2 and S7–S10). Specifically, three out of eight, two out of seven, six out of eleven, and three out of eight TF genes (or gene isoforms) within these families, respectively, were upregulated at various time points of salt stress (Figure S4).

3.5. Concordant Expression of TF and Stress-Responsive Genes

The investigation into TF genes exhibiting concordant expression patterns with salt-responsive genes revealed six distinct instances distributed across clusters 9 (two instances), 13, 55, 64 and 67 (Figure 3). Cluster 9, characterized by upregulation at the 2 h time point of salt stress, encompassed two instances of concordant expression: one involving the TF gene AI5L6 encoding abscisic acid-insensitive 5 protein and the salt-responsive PA gene encoding phosphatidic acid, and the other featuring the TF gene NFYC2 encoding nuclear transcription factor Y subunit C-2 and the salt-responsive PLA gene encoding phospholipase A. Cluster 13, exhibiting upregulated genes under salt stress at both 2 h and 12 h time points, featured the concordantly expressed TF gene STOP1 encoding sensitive to proton rhizotoxicity 1 and the salt-responsive gene AAE1 encoding an acyl-activating enzyme. Clusters 55 and 67, characterized by upregulation at the 12 h time point of salt stress, likewise demonstrated instances of concordant expression. Cluster 55 included the concordantly expressed TF gene BH100 encoding transcription factor bHLH100 and the stress-responsive FRO gene encoding a ferric reduction oxidase enzyme.
While cluster 67 featured the concordantly expressed TF gene ERF55 encoding an ethylene-responsive factor and the stress-responsive gene SSY1 encoding a starch synthase enzyme (Figure 3). Cluster 64 included the TF gene NFYC3 encoding nuclear transcription factor Y C-3 subunit and the salt-responsive gene RFS6 encoding galactinol-sucrose galactosyltransferase 6. In light of our prior research on the COL1 gene encoding zinc finger CONSTANS transcription factors under drought stress in bread wheat (Triticum aestivum), which demonstrated a relationship via qPCR between the COL1 gene and the stress-responsive gene TSA encoding tryptophan synthase [17], the present study has corroborated this finding (Figure 4). In the current investigation, two versions of the COL gene, namely COL2 in cluster 30 and COL8 in cluster 11, exhibited analogous expression patterns, characterized by upregulation at the 12 h time point of salt (200 mM NaCl) stress, akin to the salt-responsive gene TSA within cluster 4 in A. thaliana leaves (Figure 4 and Tables S2, S3 and S7).
The ensuing results (Figure S5 and Tables S11–S14) delineate the salt-responsive gene families in A. thaliana leaves that co-expressed with TF genes. These salt-responsive gene families encompassed the AAE1 gene family encoding acyl-activating enzyme (Table S11), the FRO gene family encoding ferric reduction oxidase (Table S12), the RFS6 gene family encoding galactinol-sucrose galactosyltransferase 6 (Table S13), and the SSY1 gene family encoding starch synthase (Table S14). Notably, three out of five, two out of three, four out of five, four out of four, and three out of three upregulated salt-responsive genes (or gene isoforms) within these families, respectively, displayed coordinated expression across different salt stress time points (Figure S5). The ensuing results (Figure S6 and Tables S15–S18) delineate the TF gene families in A. thaliana leaves that co-expressed with salt-responsive genes. These TF gene families encompassed the AAI gene encoding abscisic acid-insensitive 5 (ABI5, AI5L5 and AI5L6) (Table S15), the NFY gene family encoding nuclear transcription factor Y subunit (NFYA1, NFYB2, NFYB8, NFYC1, NFYC2, NFYC3 and NFYC4) (Table S16), the BH gene family encoding basic helix-loop-helix (bHLH) (Table S17), and the TF gene family ERF encoding ethylene-responsive factor (Table S18). Notably, these TF gene families collectively encompassed four out of four, six out of eight, three out of eight, two out of two, nine out of sixteen, and one out of nine upregulated genes (or gene isoforms), respectively, across different salt stress time points (Figure S6). However, it is important to note that only a single member of the TF gene family STOP1 encoding sensitive to proton rhizotoxicity 1 was identified, and as such, it was not displayed in Figure S6.

3.6. Validating Gene Knockdown of Tobacco VIGSTF Lines

To validate the gene knockdown in tobacco VIGSTF lines, VIGSPDS of the PDS gene encoding the phytoene desaturase enzyme was utilized. The results (Figure 5) confirmed the occurrence of PDS gene silencing induced by RNA interference, as evidenced by photo-bleaching of newly developed leaves, indicative of partial inhibition of carotenoid biosynthesis in tobacco VIGSPDS leaves.

3.7. Validating Concordant Expression of TFs and Salt-Responsive Genes

The generated tobacco VIGSTF lines of the six TFs were employed to validate instances of concordant expression with stress-responsive genes through qPCR. These tobacco TFs and their concordantly expressed stress-responsive genes mirror their counterparts in Arabidopsis, as displayed in Figure 3. qPCR analysis encompassed the six tobacco (N. benthamiana) VIGSTF lines along with the wild-type control for 4-week-old plants subjected to salt stress (200 mM) at 0 h, 2 h and 12 h time points. The overall results obtained for the wild-type control in all six instances of concordant TF/stress-responsive gene expression demonstrated consistent outcomes with the RNA-Seq datasets (Figure S7). Conversely, the results for VIGSTF lines indicated low expression levels in the genes encoding the six TFs at the expected time point(s) of gene upregulation. However, in the case of the corresponding stress-responsive genes, concordant expression with these TFs was detected via qPCR only in two instances: clusters 13 and 55 in VIGSSTOP1 and VIGSBH100 lines, respectively (Figure S7). These two instances comprised the genes encoding the TFs STOP1 and BH100 and their respective stress-responsive genes AAE1 and FRO3. In contrast, the results for the other four instances of TFs and their corresponding stress-responsive genes revealed no concordant expression in tobacco VIGSAI5L6, VIGSNFYC2 and VIGSERF55 lines in qPCR at the expected time point(s) of gene upregulation (Figure S7). This suggests that TFs and corresponding stress-responsive genes in these four instances were coincidentally co-expressed. As a follow-up to the two instances in cluster 9 characterized by upregulation at the 2 h time point, an investigation into the possible reciprocal relationship between the TF gene AI5L6 and the stress-responsive gene PLA in the VIGSAI5L6 line was conducted. Likewise, a parallel analysis of the TF gene NFYC2 and the stress-responsive gene PA in the VIGSNFYC2 line was performed. The results of qPCR for these new combinations similarly revealed negative relationships (Figure S8).

4. Discussion

Terrestrial plants, exemplified by the model organism Arabidopsis, predominantly manifest glycophytic characteristics that confer a tolerance to low to moderately saline soil conditions. The elucidation of intricate mechanisms governing salt tolerance at the molecular level stands as a paramount pursuit within the realm of plant biology research [23]. Such comprehension of these mechanisms holds the promise of devising strategies to mitigate the deleterious effects of salt-induced stress on plant life. Salt stress, in general, exerts a detrimental influence on diverse facets of plant growth and development. This deleterious impact is observable at multiple levels, including morphological, physiological, and biochemical traits. At the morphological level, salt stress is characterized by reduced seed germination rates and the imposition of stunted plant growth. Physiologically, salt stress perturbs hormonal balance and stifles photosynthesis, thereby impeding nutrient uptake. Biochemically, the salient indicators of salt stress encompass oxidative stress, the accumulation of reactive oxygen species (ROS), and the perturbation of membrane fluidity and permeability [24]. Notably, sodium (Na+) and chloride (Cl−) ions, as major contributors to soil salinity, have the intension to disturb ion transport and exchange across cellular membranes, thereby engendering intracellular ion homeostasis disturbances [25]. This, in turn, results in a diminished capacity of plant cells to imbibe water [26].

4.1. Auxin-Based Coordination of Response to Salt Stress in Arabidopsis

Phytohormones constitute pivotal endogenous regulators that intricately orchestrate tissue architecture and all facets of plant growth and development. Of particular note are auxins, which were the initial phytohormones identified for their involvement in stress tolerance mechanisms within the plant kingdom [27]. Nonetheless, the perturbation of auxin homeostasis under salt stress exacts a profound toll on plant growth and development, culminating in the aberrant progression of morphogenesis [28]. The biosynthetic pathways of auxins can be classified into two categories: tryptophan-independent and tryptophan-dependent. Among the latter, the “tryptophan metabolism” pathway features prominently in Arabidopsis, where tryptophan-dependent routes, specifically tryptamine (TAM) and indole-3-pyruvic acid (IPA), are harnessed for the biosynthesis of the auxin indole-3-acetic acid (IAA) [29]. The latter seems to be the pathway of choice in Arabidopsis to be utilized in biosynthesizing the auxin IAA (Figure S10).
The present investigation reveals a multitude of upregulated auxin-related genes in Arabidopsis subjected to salt stress, with a predominance of these alterations manifesting at the 12 h time point (Figure S2 and Tables S2 and S3). These genes traverse the spectrum from those acting upstream of IAA biosynthesis to those downstream of it. This coordinated response to salt stress is underpinned by three distinct crosstalking pathways: the “phenylalanine, tyrosine and tryptophan biosynthesis” pathway, which culminates in the biosynthesis of tryptophan; the “tryptophan metabolism” pathway, which encompasses the conversion of tryptophan into IAA; and the “plant hormone signal transduction” pathway, which comprises an array of auxin-responsive gene families in Arabidopsis as summarized in Figure 6. The “phenylalanine, tyrosine and tryptophan biosynthesis” pathway results in the biosynthesis of tryptophan through a route with chorismate as the precursor and tryptophan as the end product (Figure S9). This route involves the upregulated genes in Arabidopsis that encode anthranilate synthase (TRPE, EC 4.1.3.27), anthranilate phosphoribosyltransferase (TRP1, EC 2.4.2.18), indole-3-glycerol phosphate synthase (IGPS, EC 4.1.1.48) and tryptophan synthase (TSA, EC 4.2.1.20) (Figure S2). The “tryptophan metabolism” pathway contains another route, namely IPA, with tryptophan as the precursor and IAA as the end product (Figure S10). This route involves the upregulated genes in Arabidopsis that encode tryptophan-pyruvate aminotransferase 1 (TAA1, EC 2.6.1.99) and indole-3-pyruvate monooxygenase (YUC2 or flavin, EC 1.14.13.168) (Figure S2). YUC2 was reported to catalyze a rate-limiting step in the pathway [30].
The third pathway, namely “plant hormone signal transduction”, contained a battery of auxin-responsive gene families in Arabidopsis promoted by IAA signal to wire several intracellular biological processes to support plant growth under various environmental conditions [31] (Figure S11). These gene families encompass the likes of auxin influx carrier proteins (AUX/LAX), auxin efflux carrier proteins PIN-FORMED (PIN), auxin-responsive proteins IAA, auxin repressor protein SCFTIR1-E3 ligase, auxin response factors (ARF), Gretchen Hagen 3 (GH3), and small auxin-up RNA (SAUR) [31]. The purview of biological processes governed by auxin encompasses embryogenesis, lateral root initiation, vascular elongation, differentiation, and fruit development [31].
A plethora of reports attests to the context-dependent nature of indole acetic acid (IAA) signaling, which is initiated at the transcriptional level and subsequently cascades through downstream expression levels, culminating in post-translational modifications [32]. The recruitment of the auxin repressor protein SCFTIR1-E3 ligase, encoded by the TIR1 gene, serves as a regulatory check on auxin activity and the downstream auxin-responsive genes [27]. Of particular significance, in the context of our study, is the upregulation of the TIR1 gene family in Arabidopsis during the 12 h time point of salt stress (Figure S2). Consequently, the presence and degradation dynamics of the encoded TIR1 protein emerge as key arbiters of auxin homeostasis. Facilitating bidirectional auxin movement, are the influx carrier (AUX/LAX) and the efflux carrier PIN [31]. AUX/LAX orchestrates tissue-specific developmental processes, whereas PIN governs the direction of auxin flow, thereby establishing local auxin maxima across various stages of plant ontogenesis [33]. Notably, a specific member of the PIN gene family, namely PIN4, plays a pivotal role in channeling auxin into columella cells, thereby promoting auxin gradient formation and homeostasis, which, in turn, sustains root growth, patterning, and architecture [34]. This action promotes auxin gradient and homeostasis towards the maintenance of root growth, pattern and architecture [35]. Intriguingly, under salt stress conditions at the 12 h time point, we observe an upregulation of this particular PIN gene family member in Arabidopsis (Figure S2). AUX/LAX further exerts its regulatory influence on root gravitropism, hair development, vascular development, lateral root development and leaf phyllotactic patterning [36].
The downstream transduction cascades of auxin signals ensue with the interaction between auxin response factors (ARFs) and the repressors known as AUX/IAA proteins. Initially, ARFs bind to the promoters of auxin-responsive genes, while AUX/IAA proteins engage in binding ARFs, thereby obstructing their action and negatively modulating the transcription of auxin-induced genes [37]. Consequently, the interplay between AUX/IAA and ARF proteins plays a pivotal role in modulating intracellular auxin levels [38]. This interaction culminates in intricate dynamics that ultimately regulate the expression of auxin-responsive genes across various developmental stages [39]. Notably, ARF19, a specific member of the ARF gene family, has the unique ability to amplify the auxin signal and expedite a positive feedback signaling loop [40]. Remarkably, ARF19 emerges as one of the regulated genes within the ARF family in Arabidopsis (Table S3). Concurrently, members of GH3, AUX/IAA and SAUR gene families respond to auxin signaling in the early stages of the auxin signal transduction cascade [31]. The GH3 gene family assumes the role of degrading AUX/IAA proteins, thereby contributing to auxin homeostasis, especially under unfavorable environmental conditions. In parallel, the SAUR gene family acts as a stimulator of cell elongation and shoot elongation under both normal and adverse conditions [31,38].

4.2. Other Upregulated Salt-Responsive and Transcription Factor Gene Families

Salt stress eventually culminates in osmotic stress, accompanied by the accumulation of reactive oxygen species (ROS), and disrupts ionic equilibrium within plant cells. In stomatal guard cells, these perturbations trigger abscisic acid (ABA) biosynthesis, leading to stomatal closure and a subsequent reduction in carbon dioxide assimilation, thereby influencing crop yield [41]. This impairs the plant’s ability to photosynthesize adequately for its growth in adverse conditions. Central to this process, the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) governs the availability of carbon dioxide (CO2) within chloroplasts, thereby influencing the efficiency of the photosystem II (PSII) process [42]. Notably, four genes encoding RUBISCO exhibit upregulation in Arabidopsis at the 2 h and 12 h time points under salt stress (Figure S3). These genes serve to secure a continuous supply of CO2 requisite for photosynthesis. Furthermore, a myriad of 13 genes or isoforms encoding light-harvesting chlorophyll a-b binding (LHCB) proteins witness upregulation in response to salt stress at the 2 h and 12 h time points, thereby complementing the role of RUBISCO by facilitating PSII function [42]. In addition to their role in photosynthesis, LHCB proteins have been implicated in the modulation of plant growth and development [43] and the modulation of reactive oxygen species (ROS) homeostasis under abiotic stress in Arabidopsis [44]. Mitigating the deleterious effects of ROS are three critical antioxidant enzymes: superoxide dismutase (SOD), peroxidase (PER), and catalase (CAT) [45]. Within Arabidopsis, two upregulated genes or isoforms encoding PER are detected under salt stress conditions at the 12 h time point (Figure 3). This enzyme serves as a detoxifying agent for hydrogen peroxide (H2O2), an ROS species capable of compromising plant cells, thereby preserving redox homeostasis in the face of salt stress [46]. In summary, the interplay among gene families encoding RUBISCO, LHCB and PER assumes a critical role in ensuring the availability of CO2, the proper functioning of photosynthesis, and the detoxification of ROS under conditions of stress.
Plant transcription factors (TFs) represent a realm of remarkable complexity and diversity, particularly when compared to their counterparts in other eukaryotic organisms, as evidenced by the zinc-finger TFs [47]. In the domain of conserved TFs, basic region/leucine zipper (bZIP), basic helix-loop-helix (bHLH), and MYB families are eukaryotic, while plant-specific TFs encompass AP2, ERF, NAC, WRKY, TCP, ABI3-VP1 (B3) and EIL [47]. Among these, the B-Box zinc finger (BBX) proteins occupy a unique niche, distinguished by their possession of an N-terminal B-box domain and, in some instances, a CONSTANS, CO-like, or TOC1 (CCT) domain in the C terminus [48]. Individual TFs have the capacity to serve as master switches, orchestrating entire genetic circuits comprising cohorts of genes, spanning one or more pathways, to elicit specific expression patterns and engender desired end products [49]. Conversely, individual genes can yield multiple expression windows under the regulatory aegis of various TFs, resulting in divergent tissue-specific patterns, gene expression profiles, rates of transcription, and responses to the surrounding environmental milieu.
Emerging insights from recent investigations underscore the paramount role of transcription factors (TFs) in facilitating plant adaptation to abiotic stresses. Noteworthy examples include TF families such as the basic leucine zipper domain (bZIP) [50], zinc finger (ZF) [51], nuclear factor-Y (NF-Y) [52], basic helix-loop-helix (bHLH) [53], and ethylene response factor (ERF) [54]. A multitude of members from these TF families exhibit upregulation in Arabidopsis under diverse time points of salt stress (Tables S2, S7–S9 and S16–S18). Within the bZIP TF family, structural architecture is characterized by two pivotal domains: the basic region and the Leu zipper [50]. Furthermore, members of the bZIP TF family are known to act as transcriptional activators [55], and their first domain engages in major groove DNA binding. In contrast, the second domain mediates dimerization, thus creating a zipper-like structure [55]. Several members of the bZIP TF family have been shown to confer tolerance to salt and drought stresses by regulating stress-responsive genes, and they play an indispensable role in signaling pathways, eliciting responses to stimuli such as abscisic acid (ABA) and ethylene, among others, under both biotic and abiotic stresses [56].
Zinc finger (ZF) proteins, with their characteristic finger-like structure [57], are ubiquitously distributed in eukaryotes and encompass several RING zinc finger proteins within Arabidopsis thaliana [58]. These ZF proteins play an instrumental role in signaling cascades and plant adaptation to adverse environmental conditions, including drought, temperature, and salinity [59]. Moreover, they have influence over plant growth and development [57]. The nuclear factor-Y (NF-Y) TF, a ubiquitous presence among plants [60], assumes a central role in promoting plant growth and development in response to abiotic stress [52]. The NF-Y complex, a heterotrimeric entity composed of three conserved subunits—NF-YA, NF-YB and NF-YC—collaborates in the regulation of target gene expression through direct binding to the CCAAT cis-acting promoter box [61].
Within the bHLH TF family, the highly conserved alkaline/helix-loop-helix domain is a hallmark [62]. This domain, comprising a basic region binding to the E-box or G-box motif upstream of target genes and a helix-loop-helix region (HLH region) housing two alpha helices that facilitate dimerization, underpins the capacity of bHLH proteins to modulate the expression of target genes across various signaling pathways. Classifications within the bHLH family encompass six primary groups (A, B, C, D, E and F) [62] with group B being the most prevalent in Arabidopsis [63,64], favoring binding to the G-box motif [65]. Members of the bHLH family wield substantial influence over plant growth, development and response to abiotic stresses [53,66]. Moreover, they orchestrate the crosstalk among hormones such as ABA, jasmonic acid (JA) and ethylene (ET), amplifying and transducing signals through a cascade of events to activate downstream target genes [53,62,67].
The APETALA2/ethylene responsive factor (AP2/ERF) family exerts far-reaching influence over plant growth, development, hormone regulation and responses to abiotic stresses [68]. This family contains a DNA-binding domain—the AP2 domain, spanning 60–70 amino acids [69]. The AP2 domain features YRG elements for DNA binding and an α-helix element for interaction with other proteins or DNA [70]. Moreover, ERF includes two subfamilies: ethylene-related ERF and ethylene-independent DREB (Dehydration Responsive Element Binding Protein) [71]. Like their bHLH counterparts, members of the ERF family play a pivotal role in plant growth and development, hormonal sensitivity and ROS homeostasis, particularly under abiotic stresses [72].

4.3. Validation of VIGS TF Lines and Concordant Expression with Salt-Responsive Genes

In the realm of functional genomics, the utilization of VIGS lines in Nicotiana benthamiana emerges as a preferred strategy for investigating gene function, particularly in instances where target genes encode transcription factors (TFs) of anticipated multifunctionality. These TFs play pivotal roles in orchestrating genetic circuits pivotal for developmental processes and stress responses. A discrepancy arises between the perturbed growth dynamics of TF knockout lines in Arabidopsis thaliana, achieved through T-DNA insertion, and the relatively milder impacts observed in VIGS-induced knockdown lines in N. benthamiana, underlining the potential for misinterpreting stress-induced growth constraints. The current investigation interrogates this discrepancy, focusing on the TF STOP1, subjected to knockout in Arabidopsis and knockdown in N. benthamiana, revealing divergent growth responses. These findings align with prior research delineating stunted root growth in STOP1-knockout Arabidopsis as compared with their respective wild type plants, a feature that was complemented by a STOP1-regulated gene, namely CIPK23 [73]. This recent research substantiated our motivation to harness VIGS for TF functional dissection (Figure S12).
This study undertook an extensive literature review to contextualize the biological roles of the six concordantly expressed gene pairs. These gene pairs exhibited diverse functionalities, dependent on the time course of salt stress. VIGSTF lines were generated for the six genes encoding TFs that were concordantly expressed with salt-responsive genes. Validation of TF knockdown was executed through VIGS of the phytoene desaturase (PDS) gene, inducing conspicuous leaf photo-bleaching (Figure 5). Subsequent analyses examined the consequence of TF knockdowns on the expression profiles of both TFs and their respective salt-responsive gene counterparts (Figure S7). Remarkably, quantitative PCR (qPCR) unveiled potential relationships between two TFs, STOP1 and BH100, and their associated stress-responsive genes, AAE1 and FRO3, respectively. Functional validation studies were initiated to elucidate the mechanistic underpinnings of these TF–gene associations (Figure 3).
The first instance of cluster 9 featured the salt-responsive phosphatidic acid (PA) gene, pivotal for ABA signaling [74] and for binding osmotic stress-activated protein kinase (MAPK) families, in order to maintain root system architecture and promote salt stress tolerance [75]. The abscisic acid-insensitive 5 (AI5L6) TF also associates with ABA signaling [76] but regulates seed-specific genes HVA1 and HVA22 implicated in seed embryo survival during desiccation. This duality underscores AI5L6’s role in both drought stress responses and seed development. In the second instance of cluster 9, the salt-responsive phospholipase A (PLA) gene and the nuclear transcription factor Y subunit C-2 (NFYC2) TF gene were co-expressed. The PLA enzyme is multifaceted, contributing to membrane lipid homeostasis, stress responses and various developmental processes [77]. Additionally, PLA modulates hormone signaling pathways such as jasmonic acid (JA) and ABA biosynthesis [78]. NFYC2, part of the NF-Y complex [79], is implicated in diverse cellular processes, including DNA damage repair, cell cycle regulation and ABA signaling, emphasizing its pivotal role in coordinating stress responses [80].
Cluster 13 featured the salt-responsive acyl-activating (AAE1) gene and the sensitive to proton rhizotoxicity 1 (STOP1) TF gene. AAE1 gene, a versatile player, contributes to growth, metabolic pathways [81], jasmonic acid biosynthesis [82] and metal resistance [83]. STOP1, on the other hand, is involved in abscisic acid-regulated stomatal closure and ion transporter regulation [73]. While qPCR confirmed a relationship between STOP1 and AAE1 genes, the precise functional link remains elusive, warranting further investigations. Cluster 55 comprised the salt-responsive ferric reduction oxidase (FRO) gene and the bHLH100 TF gene, BH100. The FRO gene participates in iron homeostasis and phytohormone regulation under abiotic stresses [84]. The gene also has a vital role in iron uptake from the soil [85]. The BH100 gene emerges as a key regulator of iron-deficiency responses and iron distribution within the plant [86]. Integrated RNA-Seq (Figure 3) and qPCR (Figure S7) results suggest a regulatory relationship between BH100 and FRO genes, aligning with their roles in iron metabolism. Cluster 64 entailed the salt-responsive galactinol-sucrose galactosyltransferase 6 (RFS6) gene and the nuclear transcription factor Y subunit C-3 (NFYC3) TF gene. Although limited information exists regarding RFS6’s role in abiotic stress, it catalyzes reactions in the “galactose metabolism” pathway [87] to biosynthesize myo-inositol that impacts stress-responsive gene expression and enhances stress tolerance by the production of antioxidants [88]. NFYC3’s gene function, encompassing DNA damage repair, cell cycle regulation and abscisic acid signaling [80], does not readily correlate with RFS6, suggesting an incidental concordance. Lastly, cluster 67 featured the salt-responsive starch synthase (SSY1) gene acting on starch biosynthesis of starch granules in seeds with an uncertain role during drought stress [89] and the ethylene-responsive transcription factor ERF55 (ERF55) TF gene. While SSY1’s regulation under salt stress in Arabidopsis lacks precedent, ERF55’s role in this context remains elusive. The concordant expression between SSY1 and ERF55 is likely coincidental, with no prior evidence supporting their functional association. With the exception of the instance of the FRO/BH100 gene pair, which demonstrated an association based on RNA-Seq, qPCR and function, the latter conclusion applies to all the other instances of concordant expression in this study.
In conclusion, this study elucidates Arabidopsis gene expression dynamics at distinct salt stress time points, emphasizing the pivotal role of auxin-related genes across three crosstalking KEGG pathways in conferring salt stress tolerance in A. thaliana. Additionally, the present study underscores the involvement of gene families, including bZIP, bHLH and ERF, in orchestrating stress-responsive gene networks, which sheds light on the regulatory architecture governing salt stress responses in plants. Concordant expression was proven between the BH100 and FRO genes at the experimental and functional levels. The insights gleaned here hold promise for advancing metabolic engineering and genetic transformation strategies to enhance salt stress resilience in economically significant plant crop species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d15111119/s1, Figure S1. Cluster analysis of assembled leaf transcriptome datasets of Arabidopsis ecotype Columbia (Col) of three replicates under salt (200 mM NaCl) stress at different time points (0 h, 2 h and 12 h). Figure S2. Expression pattern of assembled auxin-related genes in cluster 4 that were upregulated at 12 h time point and in clusters 3 and 5 that were upregulated at 2 h time point under salt (200 mM NaCl) in leaves of Arabidopsis ecotype Columbia (Col). Figure S3. Expression profiles of salt-responsive gene families in Arabidopsis plant leaves treated with NaCl (200 mM) for 0 h, 2 h and 12 h. These genes are known for their influence under different abiotic stresses. Figure S4. Expression profiles of genes encoding members of TF families in Arabidopsis plant leaves treated with NaCl (200 mM) for 0 h, 2 h and 12 h. These gene families are known for their influence under different abiotic stresses. Figure S5. Expression profiles of salt-responsive genes that are concordantly expressed with TF genes in Arabidopsis plant leaves treated with NaCl (200 mM) for 0 h, 2 h and 12 h. Note that salt-responsive genes PA encoding phosphatidic acid and PLA encoding phospholipase enzyme and DIRL1 encoding lipid-transfer protein are the only regulated genes of their families. Figure S6. Expression profiles of genes encoding members of TF families that are concordantly expressed with salt-responsive genes in Arabidopsis plant leaves treated with NaCl (200 mM) for 0 h, 2 h and 12 h. Note that TFs gene STOP1 encoding sensitive to proton rhizotoxicity 1 is the only regulated TF of the STOP family and ERF55 is the only upregulated ethylene-responsive TF of the ERF family. Figure S7. qPCR profiles to validate concordant expression of selected transcription factors and salt-responsive genes in leaves of tobacco (N. benthamiana) VIGS lines and WT plants treated with salt (200 mM NaCl) at 0 h, 2 h and 12 h time points. Cases in cluster 9 (upregulation at 2 h time point) refer to co-expression of TF gene AI5L6 and salt-responsive gene PA in VIGSAI5L6 line and co-expression of TF gene NFYC2 and PLA gene in VIGSNFYC2 line. Case of cluster 13 (upregulation at 2 h and 12 h time points) refers to TF gene STOP1 and salt-responsive gene AAE1 in VIGSSTOP1 line. Case of cluster 55 (upregulation at 12 h time point) refers to co-expression of TF gene BH100 and salt-responsive gene FRO in VIGSBH100 line. Case of cluster 64 (upregulation at 12 h time point) refers to TF gene NFYC3 and salt-responsive gene RFS6 in VIGSNFYC3 line. Case of cluster 67 refers to co-expression of TF gene ERF55 and salt-responsive gene SSY1 in VIGSERF55 line. Figure S8. qPCR profiles to possible relationships between concordantly expressed transcription factors and salt-responsive genes of cluster 9 (upregulation at 2 h time point) in leaves of tobacco (N. benthamiana) VIGS lines and WT plants treated with salt (200 mM NaCl) at 0 h, 2 h and 12 h time points. Validated cases involved TF gene AI5L6 and salt-responsive gene PLA of VIGSAI5L6 line and TF gene NFYC2 and PA gene of VIGSNFYC2 line. Figure S9. Enriched enzymes in the “Phenylalanine, tyrosine and tryptophan biosynthesis” in Arabidopsis at 12 h time point of salt stress (200 mM NaCl). Red boxes refer to the four enriched enzymes. EC 4.1.3.27 = anthranilate synthase. EC 2.4.2.18 = anthranilate phosphoribosyltransferase. EC 4.1.1.48 = indole-3-glycerol phosphate synthase. EC 4.2.1.20 = tryptophan synthase. Figure S10. Enriched enzymes in the “Tryptophan metabolism” that participate in the indole-3-pyruvic acid (IPA) route in Arabidopsis at 12 h time point of salt stress (200 mM NaCl). Red boxes refer to the two enriched enzymes of this route. EC 2.6.1.99 = tryptophan-pyruvate aminotransferase 1. EC 1.14.13.168 = indole-3-pyruvate monooxygenase. Figure S11. Enriched enzymes in the “Plant hormone signal transduction” that participate in the indole-3-pyruvic acid (IPA) route in Arabidopsis at 12 h time point of salt stress (200 mM NaCl). Red boxes refer to the two enriched AUX/LAX = auxin influx carrier proteins. ARF = auxin response factors. GH3 = Gretchen Hagen 3. SAUR = small auxin-up RNA. Figure S12. Growth performance of Arabidopsis (Arabidopsis thaliana) (a) and tobacco (Nicotiana benthamiana) (b) wild type (WT) plants and those modified for the STOP1 gene encoding sensitive to proton rhizotoxicity 1. The gene was knocked out (KO) in Arabidopsis in the form of T-DNA SALK line (ABRC T-DNA Salk line CS927067), while knocked down in tobacco in the form of virus induced gene silencing (VIGS) RNA interference line. Table S1. Primer sequences along with the annealing temperature and expected amplicon size (bp) utilized in qPCR in scoring expression of salt-responsive genes and concordantly expressed transcription factors (TFs) knocked-down utilied in VIGS in leaves of A. thaliana. Table S2. Cluster analysis referring to fpkm values of assembled leaf transcripts of Arabidopsis plants treated with NaCl (200 mM) for 0, 2 and 12 h. Raws in rose color refer to regulated transcription factors (TFs). Blue raws refer to salt-responsive genes concordently expressed with TFs. Table S3. The fpkm values of assembled leaf auxin-related genes of cluster 4 that were upregulated at 12 h time point in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S4. Expression levels in terms of fpkm values for members of the chlorophyll a-b binding (CA/B) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S5. Expression levels in terms of fpkm values for members of the RUBISCO (RBS/RCA) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S6. Expression levels in terms of fpkm values for members of the peroxidase (PER) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S7. Expression levels in terms of fpkm values for members of the TF COL gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S8. Expression levels in terms of fpkm values for members of the TF BZIP gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S9. Expression levels in terms of fpkm values for members of the TF ZINC FINGER (ZF) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S10. Expression levels in terms of fpkm values for members of the TF NAC gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S11. Expression levels in terms of fpkm values for members of the acyl-activating enzyme (AAE) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S12. Expression levels in terms of fpkm values for members of the Ferric reduction oxidase (FRO) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S13. Expression levels in terms of fpkm values for members of the galactinol-sucrose galactosyltransferase (RFS) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S14. Expression levels in terms of fpkm values for members of theStarch synthase (SSY) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S15. Expression levels in terms of fpkm values for members of the TF ABSCISIC ACID-INSENSITIVE (AAI) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S16. Expression levels in terms of fpkm values for members of the TF Nuclear transcription factor Y (NFY) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S17. Expression levels in terms of fpkm values for members of the TF basic helix-loop-helix (bHLH) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h. Table S18. Expression levels in terms of fpkm values for members of the TF ethylene-responsive (ERF) gene family in Arabidopsis plant leaves treated with NaCl (200 mM) for 0, 2 and 12 h.

Author Contributions

Conceptualization, N.M.A. and A.A.A.; methodology, N.M.A. and A.A.A.; software, N.M.A. and A.A.A.; validation, N.M.A. and A.A.A.; formal analysis, N.M.A. and A.A.A.; investigation, N.M.A. and A.A.A.; resources, N.M.A. and A.A.A.; data curation, N.M.A. and A.A.A.; writing—original draft preparation, N.M.A. and A.A.A.; writing—review and editing, N.M.A. and A.A.A.; visualization, N.M.A. and A.A.A.; supervision, N.M.A. and A.A.A.; project administration, N.M.A. and A.A.A.; funding acquisition, N.M.A. and A.A.A. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Princess Nourah bint Abdulrahman University Researchers Supporting Project No. (PNURSP2023R356), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This work was supported by the Princess Nourah bint Abdulrahman University Researchers Supporting Project No. (PNURSP2023R356), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Heatmap referring to hierarchical cluster analysis of differentially expressed (DEs) genes to describe interrelation among leaf transcriptomes of A. thaliana plants treated with salt (200 mM NaCl) for 0 h, 2 h and 12 h in a replicated experiment. A detailed list of genes and their clusters is shown in Table S2.
Figure 1. Heatmap referring to hierarchical cluster analysis of differentially expressed (DEs) genes to describe interrelation among leaf transcriptomes of A. thaliana plants treated with salt (200 mM NaCl) for 0 h, 2 h and 12 h in a replicated experiment. A detailed list of genes and their clusters is shown in Table S2.
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Figure 2. Principle component analysis (PCA) for leaf transcriptomes of A. thaliana plants treated with salt (200 mM NaCl) for 0 h, 2 h and 12 h in a replicated experiment.
Figure 2. Principle component analysis (PCA) for leaf transcriptomes of A. thaliana plants treated with salt (200 mM NaCl) for 0 h, 2 h and 12 h in a replicated experiment.
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Figure 3. Clusters with transcription factors co-expressed with salt-responsive genes in leaves of A. thaliana plants treated with salt (200 mM NaCl) for 0 h, 2 h and 12 h in a replicated experiment. PA gene of cluster 9 encodes phosphatidic acid while TF gene AI5L6 encodes abscisic acid-insensitive 5 protein. PLA gene of cluster 9 encodes phospholipase enzyme, while TF gene NFYC2 encodes nuclear transcription factor Y subunit C-2. AAE1 gene of cluster 13 encodes probable acyl-activating enzyme, while TF gene STOP1 encodes sensitive to proton rhizotoxicity 1. FRO gene of cluster 55 encodes ferric reduction oxidase enzyme, while TF gene BH100 encodes transcription factor bHLH100. RFS6 gene of cluster 64 encodes galactinol-sucrose galactosyltransferase 6, while TF gene NFYC3 encodes nuclear transcription factor Y subunit C-3. SSY1 gene of cluster 67 encodes starch synthase enzyme, while TF gene ERF55 encodes ethylene-responsive transcription factor ERF55. Descriptions of all genes and TFs along with their clusters are shown in Table S2.
Figure 3. Clusters with transcription factors co-expressed with salt-responsive genes in leaves of A. thaliana plants treated with salt (200 mM NaCl) for 0 h, 2 h and 12 h in a replicated experiment. PA gene of cluster 9 encodes phosphatidic acid while TF gene AI5L6 encodes abscisic acid-insensitive 5 protein. PLA gene of cluster 9 encodes phospholipase enzyme, while TF gene NFYC2 encodes nuclear transcription factor Y subunit C-2. AAE1 gene of cluster 13 encodes probable acyl-activating enzyme, while TF gene STOP1 encodes sensitive to proton rhizotoxicity 1. FRO gene of cluster 55 encodes ferric reduction oxidase enzyme, while TF gene BH100 encodes transcription factor bHLH100. RFS6 gene of cluster 64 encodes galactinol-sucrose galactosyltransferase 6, while TF gene NFYC3 encodes nuclear transcription factor Y subunit C-3. SSY1 gene of cluster 67 encodes starch synthase enzyme, while TF gene ERF55 encodes ethylene-responsive transcription factor ERF55. Descriptions of all genes and TFs along with their clusters are shown in Table S2.
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Figure 4. Concordant expression of two genes encoding members of zinc finger CONSTANS transcription factors, e.g., COL2 in cluster 30 and COL8 in cluster 11, and the salt-responsive gene TSA in cluster 4 encoding tryptophan synthase enzyme in leaves of A. thaliana plants treated with salt (200 mM NaCl). The three genes were upregulated at the 12 h time point. More information is available in Tables S2, S3 and S7.
Figure 4. Concordant expression of two genes encoding members of zinc finger CONSTANS transcription factors, e.g., COL2 in cluster 30 and COL8 in cluster 11, and the salt-responsive gene TSA in cluster 4 encoding tryptophan synthase enzyme in leaves of A. thaliana plants treated with salt (200 mM NaCl). The three genes were upregulated at the 12 h time point. More information is available in Tables S2, S3 and S7.
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Figure 5. Performance of 8-week-old tobacco (N. benthamiana) VIGSPDS line knocked down for the phytoene desaturase (PDS) gene that resulted in the occurrence of photo-bleaching in tobacco plant leaves. The gene represents a model to morphologically confirm the occurrence of RNA interference and gene silencing.
Figure 5. Performance of 8-week-old tobacco (N. benthamiana) VIGSPDS line knocked down for the phytoene desaturase (PDS) gene that resulted in the occurrence of photo-bleaching in tobacco plant leaves. The gene represents a model to morphologically confirm the occurrence of RNA interference and gene silencing.
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Figure 6. Crosstalking KEGG pathways emphasizing the role of auxin-related genes in conferring salt stress tolerance in A. Thaliana. EC 4.1.3.27 = anthranilate synthase. EC 2.4.2.18 = anthranilate phosphoribosyltransferase. EC 4.1.1.48 = indole-3-glycerol phosphate synthase. EC 4.2.1.20 = tryptophan synthase. EC 2.6.1.99 = tryptophan-pyruvate aminotransferase 1. EC 1.14.13.168 = indole-3-pyruvate monooxygenase. IAA = indole acetic acid. AUX/LAX = auxin influx carrier protein. ARF = auxin response factors. GH3 = Gretchen Hagen 3. SAUR = small auxin-up RNA.
Figure 6. Crosstalking KEGG pathways emphasizing the role of auxin-related genes in conferring salt stress tolerance in A. Thaliana. EC 4.1.3.27 = anthranilate synthase. EC 2.4.2.18 = anthranilate phosphoribosyltransferase. EC 4.1.1.48 = indole-3-glycerol phosphate synthase. EC 4.2.1.20 = tryptophan synthase. EC 2.6.1.99 = tryptophan-pyruvate aminotransferase 1. EC 1.14.13.168 = indole-3-pyruvate monooxygenase. IAA = indole acetic acid. AUX/LAX = auxin influx carrier protein. ARF = auxin response factors. GH3 = Gretchen Hagen 3. SAUR = small auxin-up RNA.
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Table 1. List of clusters with upregulated genes under salt (200 mM NaCl) stress at different time points (e.g., 2 h, 12 h and 2 h/12 h) in leaves of A. thaliana. Colored boxes refer to upregulated clusters that were analyzed further. Red box color refers to clusters with regulated transcription factors (TFs), while blue box color refers to clusters with no regulated TFs. * and ** refer to codes of stress-related and TF gene families, respectively.
Table 1. List of clusters with upregulated genes under salt (200 mM NaCl) stress at different time points (e.g., 2 h, 12 h and 2 h/12 h) in leaves of A. thaliana. Colored boxes refer to upregulated clusters that were analyzed further. Red box color refers to clusters with regulated transcription factors (TFs), while blue box color refers to clusters with no regulated TFs. * and ** refer to codes of stress-related and TF gene families, respectively.
ClusterTime Point (h)Gene FamilyClusterTime Point (h)Gene Family
2122/12Stress Related *TF **2122/12Stress RelatedTF
1 g39
2 40
3 41
4 42
5 43
6 44
7 45
8 46 6
9 e, f, g47 d
10 48
11 1,2a49
12 1,5 50 e
13 4 51
14 e52
15 53
16 54
17 c, f55 5g
18 56
19 3 57 1a
20 5g58
21 59 1,2
22 60
23 61 c, d, e, g
24 62
25 f63
26 b, c64 6f
27 65
28 66
29 1,2 67 7c, h
30 1a68 c
31 69
32 70
33 4,5c, d, f71
34 72
35 73
36 74
37 75 3, 6
38 76
1 = Chlorophyl/a-b, 2 = RUBISCO, 3 = PER, 4 = AAE, 5 = FRO, 6 = RFS, 7 = SSY, a = COL, b = BZIP, c = ZINC FINGER, d = NAC, e = AAI, f = NFY, g = bHLH, h = ERF.
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Alotaibi, N.M.; Abulfaraj, A.A. Expression Profiling of Salt-Responsive Genes and Transcription Factors in Leaf Transcriptome of Arabidopsis thaliana. Diversity 2023, 15, 1119. https://doi.org/10.3390/d15111119

AMA Style

Alotaibi NM, Abulfaraj AA. Expression Profiling of Salt-Responsive Genes and Transcription Factors in Leaf Transcriptome of Arabidopsis thaliana. Diversity. 2023; 15(11):1119. https://doi.org/10.3390/d15111119

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Alotaibi, Nahaa M., and Aala A. Abulfaraj. 2023. "Expression Profiling of Salt-Responsive Genes and Transcription Factors in Leaf Transcriptome of Arabidopsis thaliana" Diversity 15, no. 11: 1119. https://doi.org/10.3390/d15111119

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