Potato (Solanum tuberosum L.) is an important food crop that is grown and consumed worldwide. The growth and productivity of this crop are severely affected by various abiotic stresses. Basic leucine zipper (bZIP) transcription factors (TFs) in plants are well known for their function during growth and development. However, systematic and in-depth identification and functional characterization of the bZIP gene family of potato is lacking. In the current study, we identified a total of 90 bZIPs (StbZIP) distributed on 12 linkage groups of potato. Based on the previous functional annotation and classification of bZIPs in Arabidopsis, wheat, and rice, a phylogenetic tree of potato bZIPs was constructed and genes were categorized into various functional groups (A to I, S, and U) as previously annotated in Arabidopsis thaliana. Analyses of the transcript sequence (RNA-seq) data led to identifying a total of 18 candidate StbZIPs [four in roots, eight in the tuber, six in mesocarp and endocarp] that were expressed in a tissue-specific manner. Differential expression analysis under the various abiotic conditions (salt, mannitol, water, and heat stress) and treatment with phytohormones (ABA, GA, IAA, and BAP) led to the identification of forty-two [thirteen under salt stress, two under mannitol stress, ten under water stress, and eighteen under heat stress], and eleven [eight and three StbZIPs upon treatment with ABA, and IAA, respectively] candidate StbZIPs, respectively. Using sequence information of candidate StbZIPs, a total of 22 SSR markers were also identified in this study. In conclusion, the genome-wide identification analysis coupled with RNA-Seq expression data led to identifying candidate StbZIPs, which are dysregulated, and may play a pivotal role under various abiotic stress conditions. This study will pave the way for future functional studies using forward and reverse genetics to improve abiotic stress tolerance in potato.
Citation: Kumar P, Kumar P, Sharma D, Verma SK, Halterman D, Kumar A (2021) Genome-wide identification and expression profiling of basic leucine zipper transcription factors following abiotic stresses in potato (Solanum tuberosum L.). PLoS ONE 16(3): e0247864. https://doi.org/10.1371/journal.pone.0247864
Editor: Farrukh Azeem, Government College University Faisalabad, PAKISTAN
Received: November 30, 2020; Accepted: February 15, 2021; Published: March 12, 2021
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
One of the biggest challenges of current times is to feed the ever-growing population amidst a changing climate with limited available resources. Abiotic stresses such as salinity, water (drought and flooding), and heat are a few of the many factors that directly impact the agronomical traits and yield of crops . Potato (Solanum tuberosum L.) is one of the most important food crops, third-largest after rice and wheat in terms of human consumption . This crop can also be used commercially as a health food because it is rich in antioxidants, minerals, and dietary fibers . However, as a consequence of global warming, a continuous increase in temperature leads to multiple stresses, including heat and salinity. Consequently, excess reactive oxygen species (ROS) are generated inside the cells, which, if not scavenged, can lead to severe oxidative stress to plants . ROS can be scavenged using an array of enzymatic [such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase]  and non-enzymatic mechanisms (glutathione, α-tocopherol, carotenoids, flavonoids) [3–5]. Impaired function or decreased activity of any of these ROS scavenging activities can also have a devastating impact on the plant’s growth and yield . Stress also affects the quality of potato tubers by altering sugar and solid content . A limited understanding of heat and salt tolerance regulation mechanisms through TFs (transcription factors) hinders elite cultivars’ development with higher stress tolerance [7, 8]. The complete genome sequence data of potato (haploid genome content of 840 Mb) offers an excellent opportunity to explore gene families, their downstream pathways, and regulation mechanisms under stress conditions.
In the current study, we mined and functionally annotated the basic leucine zipper family (bZIP) of TFs in potato using its genome information and previous annotation [9–11]. Generally, the bZIP proteins contain an essential region that consists of a DNA-binding part and a leucine zipper dimerization motif [9–12]. Structurally, the bZIP family is highly conserved among the species with signature sequences of the domains (60–80 amino acids long). These bZIPs can be subdivided further into two essential parts depending on their structure: the basic region and the leucine zipper region. The basic region has an invariant motif N-X7-R / K-X9, which links nuclear localization to the target DNA [10, 12]. The leucine zipper region consists of Leu heptad repeats, and often with Ile, Val, Phe, or Met [9–12]. Recent findings of bZIP TFs in specific plant families have shown that they are involved in diverse biological roles in plant physiology [13–16]. The functions of bZIP TFs are well characterized in various plants during stress conditions, such as salt, heat, and hormone signaling [16–20]. To date, genome-wide analysis has led to the identification of the bZIP gene family in several plants, including 75 in Arabidopsis thaliana (AtbZIPs), 89 in Oryza sativa (OsbZIPs), 160 in Glycine max (GmbZIPs), 166 in Sorghum bicolor (SbbZIPs), 125 in Zea mays (ZmbZIPs), 136 in Brassica napus (BrbZIPs), and 370 in Triticum aestivum (TabZIPs), and 41 in Ipomoea trifida [10, 11, 21–23, 25, 26]. Recently, 65 bZIPs were identified in the potato genome using a single database source viz. Spud DB . In our current study, we used the previous genome sequence databases available in SpudDB  as well as the latest genome assembly in the Ensembl plants database (http://plants.ensembl.org/Solanum_tuberosum) of potato for a comprehensive investigation of the StbZIP family that includes transcripts and genes. The present work led to the identification of 25 more bZIPs at the genome-wide scale.
A previous study has shown the role of bZIP StABF1 in inducing tolerance to salt, drought, and abscisic acid (ABA) stress in potatoes . However, a detailed identification and characterization of the StbZIP gene family in response to abiotic and hormonal stress in potato is lacking. A thorough study of the gene expression pattern of StbZIPs under various stress conditions in the potato will help us to understand stress regulatory mechanisms. Here, we identified members of the StbZIP gene family using computational approaches, and the subfamily classification was performed based on prior annotations in Arabidopsis . The distribution and conserved domain analysis of the StbZIP family were studied and validated using proteome sequence data and next-generation sequencing (NGS) data available in the public domain. Based on this information, a few candidate StbZIPs that are expressed under various stress conditions were identified. This research will be valuable in further functional validation of the StbZIPs and developing stress-tolerant elite varieties either using trans/cis-genic route or via molecular breeding programs.
Material and methods
Identification of bZIP transcription factors in the potato genome
A two-step identification process was performed to categorize bZIPs of potato. In the first step, the curated Hidden Markov Model profiles of the bZIP domain family, viz. PF00170 downloaded from Pfam , was used as a query to search the bZIP proteins in the potato proteome Ensembl database (http://plants.ensembl.org/index.html) using HMMER3.0 . In the second step, a local BLASTp search was performed to identify the predicted potato bZIPs by HMMER3.0 from already known bZIPs in Arabidopsis , maize , rice , and barley  with the E-value of 0.00001. Such potential potato bZIPs were further explored with NCBI-CDD and InterproScan  for the existence and integrity of the bZIP domain. CDD is the conserved domain database at NCBI for the annotation of protein sequences along with the location of conserved domain footprints within the sequence. InterproScan enables extensive scanning of protein sequences against the Interpro database by predicting domains and important sites and classify them into families .
Sequence alignment and phylogenetic analysis
The bZIP protein sequences from potato, rice, maize, barley, and Arabidopsis were aligned with gap opening and gap extension penalties of 10 and 0.1, respectively, using Clustal W. A Neighbor-Joining (NJ) method was used to develop a cladogram of all bZIP protein sequences. The associated taxa clustered together in the bootstrap test of 1000 replicas. The phylogenetic tree was constructed using MEGA 7 software, version 7 , and visualized through EvolView v2 .
Protein properties and sequence analysis of potato bZIPs for detection of conserved motifs
The standalone Multiple Em (Expectation Maximization) for the Motif Elicitation (MEME version 4.11.2)  was used for the prediction of conserved motifs in StbZIPs. The limits specified for minimum and maximum width, and the maximum numbers of motifs were 1, 7, and 9, respectively, and motifs were numbered according to their order displayed by MEME .
Chromosomal distribution and gene duplication study
The chromosomal distribution of StbZIPs was identified using the MCScanX (http:/chibba.pgml.uga.edu/mcscan2/) program to map the positions of the StbZIP genes within the Solanum tuberosum genome (plant ensemble and Spud DB (http:/solanaceae.plantbiology.msu.edu/pgsc%20download.shtml). Based on the condition of BLAST aligned query sequence (> 80 percent) of the StbZIPs, tandem and collinear gene duplications were recognized (Fig 1).
RNA-Seq gene expression analysis
The reference genome sequence information of Solanum tuberosum Group Phureja DM1-3 and the FPKM values of all the representative transcripts were retrieved from Spud DB. The FPKM values were calculated by Cufflinks (v1.3.0) using v3.4 in the Spud DB database representative models for complete annotation (http://solanaceae.plantbiology.msu.edu/data/Potato_dm_v404). The relative intensity levels (heat map) and linkage hierarchical cluster analysis were performed using multiple experiments viewer (MeV 4.9.0) with differential gene expression of StbZIPs under various stress conditions [36–38]. Expression levels are shown as FPKM’s log 2 ratios between control and treatment samples using Pearson uncentered distance as a statistical method. The color represents the logarithmic intensity of the expressed StbZIPs, in color with high and low expression.
Mining of simple sequence repeats (SSRs) from the candidate StbZIPs
The coding sequences of candidate StbZIPs were retrieved from the Ensembl plant reference database of potato, and the SSRs were predicted using MISA (http://pgrc.ipk103%20gatersleben.de/misa/) with criteria of a minimum length of six for dinucleotide repeats and five for trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats, and hexanucleotide repeats. Mix type SSRs were considered disrupted by a non-repetitive repeats length of 100 base pair sequence.
Results and discussion
Identification of bZIP transcription factor family in potato
The genome-wide analysis resulted in identifying a total of 90 potato StbZIPs using a combination of Hidden Markov Model (HMM) profile PF00170, a search against the whole potato proteome using Ensembl database by HMMER3.0, and BLASTp search using Arabidopsis, maize, rice, and barley bZIP sequences. Each potato bZIP protein was assigned a unique identifier from StbZIP1 to StbZIP90, and the information on StbZIPs is depicted in Table 1 and Fig 2. Since the cultivated potato (Solanum tuberosum L.) is highly heterozygous and presents challenges in genome analyses and breeding, the information derived from diploid potatoes genomic resources will aid in the identification of gene variants such as copy number variation, single nucleotide polymorphism, and genomic-enabled development of inbred diploid potatoes and improvement of cultivated tetraploid potatoes.
The conserved motifs of StbZIPs were identified using the MEME database. Based on the clustering relationship, the StbZIPs were classified into ten groups shown as A to I, and S.
Phylogenetic analysis of potato bZIPs
The ninety bZIP sequences from potato and representative bZIP sequences from each group were analyzed from rice (Oryza sativa), barley (Hordeum vulgare), maize (Zea mays), and Arabidopsis (Arabidopsis thaliana). Multiple sequence alignment of StbZIPs with rice, barley, maize, and Arabidopsis bZIPs was performed with ClustalW (with gap open penalty 10 and gap extension penalty 0.05) and is represented in S1 Fig. The Neighbor-Joining (NJ) phylogenetic tree was constructed using MEGA7.0 and visualized by EvolView (Fig 3). The phylogenetic tree analysis grouped StbZIPs proteins into 11 clades, designated as groups A to I, S, and U, following those in Arabidopsis . Among these, groups A and D constituted the largest clades with 11 and 22 members, respectively. StbZIP members were placed within category C and B (3 StbZIPs each), group I (8 StbZIPs), group G (9 StbZIPs), group S (16 StbZIPs), group E (7 StbZIPs), group F (1 StbZIP), and group H (3 StbZIPs) (Figs 2 and 3). Group A is one of the major groups comprising 11 StbZIPs, (StbZIPs 3, 14, 15, 16, 17, 18, 19, 20, 21, 22). It is evident from prior studies that group A StbZIPs have an important role in abscisic acid (ABA) signaling and tolerance to abiotic stresses as evident by transcriptional and post-translational regulation of its group members [10, 39]. Recently, in rice, OsbZIP23 was reported to have a significant role in tolerance to salinity and drought (ABA-dependent) . Group D is composed of 22 StbZIPs, one of the largest potato groups. This group’s genes are primarily involved in plant development and defense-related pathways [10, 14]. Earlier findings on the bZIP domain containing TGA family (TGACG sequence binding protein) in Arabidopsis and tobacco reported a pivotal role in stress defense mechanisms [41, 42]. The TGA family generally interacts with the Non-Expressor of PR1 (NPR1) proteins, so this protein is a central component in the defense signaling related to salicylic acid [43, 44]. The Arabidopsis TGA2 protein interacts with the xenobiotic responsive promoters that trigger the reaction process linked to salicylic acid pathways. A similar form of the process has been found in tobacco, as pathways are activated by interaction with TGA1a [14, 41, 44]. Group B and C are among the smallest groups in the potato bZIP family that consists of three StbZIPs. The function-related information of group B is limited. The earlier published reports showed that Group B bZIPs have a transmembrane domain with specific stretches of amino acids at the C-terminus and play significant roles in salt stress response via endoplasmic reticulum stress signaling . The members of Group C bZIPs have an essential role in modulating seed-specific gene expression [10, 14]. The rice OsbZIP52 from group C showed a significant increase in the susceptivity to drought and cold stresses during the seed germination process .
The phylogenetic tree with representative bZIPs of each group from Arabidopsis, rice, maize, barley, and potato (denoted using red, blue, purple, brown, and green stars, respectively) was constructed using the Neighbor-joining (NJ) method by MEGA 7 with 1000 bootstrap values. The StbZIPs were categorized into 11 groups and are shown by colored branches A to I, S, and U.
Group E and F are constituted by seven and one bZIP members, respectively. The members of these groups, e.g. bZIP 34 and 61, are reported to have a role in the pollen germination and tube growth in Arabidopsis . Group F is primarily responsible for regulating genes involved in zinc transport, thus promoting plant adaptation through binding to zinc-deficiency-response elements (ZDREs; RTGTCGACAY) under zinc-starving conditions . Group G consists of nine StbZIPs. The name assigned by the letter ‘G’ refers to G-box, which also function as binding factors for bZIPs . The G-box binding factor bZIPs in Arabidopsis are attributed to the regulation of light-responsive elements and perform a function in cell protection during both biotic and abiotic stress [49, 50]. Group H is comprised of three StbZIPs. For several plant species possessing a complete conserved pattern of the bZIP domain, this group’s name is often known as the HY5 gene. In Arabidopsis, the gene HY5 attaches to the G-box and regulates root and hypocotyl development . Potato Group I consists of eight bZIPs, and all members of the group I have a lysine (K) instead of arginine (R) at the 10th position [10, 14]. Members of group I bZIPs are reported to be functionally involved in vascular development [10, 11, 12]. Group S bZIPs in potato is the second largest group, with sixteen bZIPs, although it is the main group in Arabidopsis. The small size of proteins in this group specifies their requirement to interact with certain other transcriptional activation factors [10, 52]. The bZIP proteins from Arabidopsis (AtbZIP1), Medicago (MtbZIP2, and MtbZIP26), and rice (OsbZIP16) showed higher expression during salt treatment, resulting in an improvement in salt stress tolerance in several crops . Group U in potato consisted of five StbZIP members which share high sequence similarity to the same members from other crops, including maize and rice. Conserved domains for members of this group have a set of signature sequences in the hinge and basic regions, and a conserved amino acid residue isoleucine is replaced with arginine. This substitution affects the DNA binding specificity as reported in earlier studies, and such a substitution has shown to inhibit the affinity of bZIP for AP1 protein in yeast  and rice for promoters containing G-boxes .
StbZIP properties and conserved motif analysis
Analysis of structure and divergence using the MEME tool led to the identification of a total of 10 motifs in all the 90 StbZIPs (S1 Table, Fig 2). The basic leucine zipper domain of motif 1 is commonly present in all the StbZIP proteins. Besides group D, motif 1 to 6 is present in all the groups. Groups A, B, C, F, G, and I share motifs 1, 7, 8, and 9. Present results show that StbZIPs share similar sequences and are clustered in the same group. Prior studies reported that monocots usually have more bZIP and motifs than the others . In wheat, a total of 370 bZIPs were reported and categorized into 11 groups (A to I, S, and U) . Similarly, in potato, these bZIPs were grouped into ten groups (lacking less members in group U) based on functional annotation (Fig 3). Even the same conserved pattern was observed in the MEME motif pattern in all the groups (Fig 2). The StbZIP proteins from a similar group had the same motifs arranged in the same order (S1 Table).
Genome-wide expression atlas of StbZIPs
Using FPKM data, tissue-specific expression dynamics of StbZIPs were studied. The effect of various abiotic stresses and hormones on the expression of StbZIPs was also analyzed.
Tissue-specific expression dynamics (leaf, root, tuber, sepal, petal, mesocarp & endocarp).
The leaf-stage expression study showed an expression of a total of forty-seven StbZIPs. Out of these, thirty-four exhibited an expression value of >2 FPKM. In roots, fifty-five StbZIPs were expressed, and forty of them had the FPKM value of >2. Out of these, thirty-six StbZIPs had expression >5 FPKM, and StbZIP 25, 34, 50, and 89 had a maximum expression. Forty-eight StbZIPs were expressed in tubers, and StbZIP 4, 22, 34, 42, 50, 63, 68, and 89 had the highest expression. In the sepal and petal stage, fifty-one and fifty-two StbZIP were expressed, and out of these six StbZIPs, each showed maximum expression in sepal (StbZIP 20, 25, 34, 50, 63, and 89) and petal (StbZIP 20, 25, 34, 75, 87 and 89). During the mesocarp & endocarp development (fruiting stage), expression analysis revealed fifty-four and forty-four StbZIPs with expression > 2 FPKM. However, only thirty-eight StbZIPs showed > 5 FPKM with the highest expression in StbZIP 20, 34, 42, 50, 87, and 89. Expression analysis based on FPKM suggested that most bZIPs are expressed in all tissues. StbZIP 25 had a higher expression in root, sepal, and petals, and StbZIP 89 and 34 had the highest expression in all tissues, i.e., root, tuber, sepal, petal, and fruit. Similarly, StbZIP 50 showed the highest expression in root, tuber, sepal, and fruit. StbZIP 42 showed the common highest expression in tuber and fruit (S2 Table). The expression analysis suggested the tissue-specific expression of a few, and the ubiquitous expression of most, StbZIPs. This expression atlas can further be used to identify candidate StbZIPs for crop improvement by developing cis/trans-genic lines. Using a similar approach, CabZIP25 was identified from Capsicum annum and its role in imparting salt stress tolerance was shown by heterologous expression in Arabidopsis .
Abiotic stress-based expression dynamics (salinity, osmotic, water, and heat).
Abiotic stress, such as salt, mannitol, water, and heat, can significantly impact the production of the potato crop. Salinity affects plant development and growth through interference with the plant’s physiological system. The FPKM expression values were used to calculate the fold change (FC) and log 2 FC by comparing controls with respective treated samples (Fig 4, S3 Table). The comparative analysis showed fifty-five StbZIPs in leaf tissues after treatment with 150 mM NaCl. Out of these, thirteen had an expression of > 2 FC, and thirty-one of < 2 FC. However, differential log 2 FC expression analysis showed that only StbZIP 62 and 66 showed positive differential log 2 FC in salt stress condition. StbZIPs that showed negatively differential log 2 FC expression during this salt stress were StbZIP 7, 2, 4, 6, 23, 32, 38, 48, 49, 75, and 71. These bZIPs could serve as negative regulators during salt stress.
A heat map represents the log 2 FC-FPKM value of StbZIPs after stress induced by NaCl, mannitol, heat, water, and exogenous application of phytohormones BAP, ABA, IAA, and GA3.
Fifty-five StbZIPs were expressed under mannitol stress (concentration of 260 μM for 24 h), out of which only 17 had an expression value of > 2FC. Among these, StbZIPs 21and 66 showed expression of FC >5. Forty-eight StbZIPs were expressed during water stress, out of which six StbZIPs had an FC >5. Among these, StbZIP1, 14, 25, 53, 54 and 82 had log2 FC values of >2. Fifteen StbZIPs were negatively regulated under water stress, and only StbZIP3, 38, 62, and 71 had negative log2FC >2. Based on this data, StbZIPs can be further selected for functional validation using forward and reverse genetics approaches.
Salinity stress can cause osmotic imbalance and toxicity to ions. The major impact of osmotic stress on plants is on growth reduction due to reduced water potential . The salt stress also increases sodium ion (Na+) concentration that directly hinders the absorption of potassium ions (K+) in plants. However, in higher plants, low Na+ levels, and a normal range of K+/Na+ ratios enhance salt tolerance in the cell cytoplasm [55, 56]. The exact mechanism of tolerance to salt and osmotic stress in potato is not well studied, especially how bZIPs can regulate this response. In the present study, initial evidence of expression of StbZIPs during abiotic stress conditions sets out a new hypothesis to understand the regulatory process as validated in prior reports, which showed that expression of bZIP17 increased salt tolerance in Arabidopsis . Similarly, the AREB1 and ABF2 are bZIP family genes, and their overexpression and silencing in tomato and cotton resulted in enhanced salt- and drought-stress tolerance, respectively [58, 59]. The AREB1 gene also regulates other genes related to abiotic and biotic stress [58–60].
The process of tuberization is largely dependent on photoperiod , phytohormones , and day and night temperature . The exact mechanism of potato tuberization and the effect of temperature on potatoes is not well understood. However, heat stress can lead to reduced yield due to less photosynthate available for plant growth and partitioning into potato tubers . Heat stress causes physiological changes in tubers like early sprouting, altered tuber shape, tuber cracking, changes in texture, taste formation, low dry matter, etc. Also, heat stress can induce necrosis, loss of chlorophyll with reduced stem thickness, and an increase in the quantity of reducing sugars . Henceforth, a detailed understanding of the regulatory network is needed to develop lines that provide resistance/tolerance to heat stress. In the present study, we identified fifty-four StbZIPs expressed during heat stress (heating at 35 °C for 24h), and out of these, a total of twelve StbZIPs had FC >5. The StbZIPs 1, 5, 7, 8, 15, 19, 23, 30, 37, 41, 62, 66 and 82 had positive expression of > 2 FC, whereas StbZIP 3, 24, 27, 32, and 53 were negatively expressed. The overall expression study of StbZIPs during abiotic (heat, water, salt, mannitol) stress conditions led to the identification of a total of 33 StbZIPs stresses (Fig 4, S3 and S4 Tables) that can be targeted to improve resistance/tolerance against abiotic stresses.
Hormone-induced expression profiles of StbZIPs
Phytohormones are the critical players in inducing cell divisions, and partitioning assimilates by source-sink interactions  and are active in the transcriptional regulation of various stress-related genes . Therefore, the expression of StbZIPs was studied after treatment with different hormones (Fig 4, S3 and S5 Tables).
BAP (10 μM 24h).
The exogenous application of BAP phytohormones resulted in the expression of a total of 55 StbZIPs, but none of them revealed the expression of more than 2 FPKM. Differential log2 FC FPKM expression analysis suggested the downregulation of most of the StbZIPs.
ABA (50 μM 24h).
The expression analysis after the exogenous supply of ABA in potato leaf tissue revealed 53 StbZIPs, and out of these, only StbZIP88 had an expression of >2 FC. However, relative differential log2 FC expression analysis suggests that four StbZIPs are positively (1, 7, 25, and 88) and negatively (8, 21, 24, and 37) expressed, respectively. ABA plays a vital role in plant physiological processes and, therefore, regulates plant development. The expression of several ABA/stress-responsive genes in plants is regulated by cis-acting elements [66, 67]. The regulation of ABA is reported to be controlled by PYRABACTIN RESISTANCE (PYR / PYL), PROTEIN PHOSPHATASE 2C, SNF1-RELATED PROTEIN KINASE 2 (SnRK2), ion channels, and NADPH oxidases , and most notably by bZIPs. The bZIP has a binding affinity towards the ABA-responsive element-binding protein / ABRE-binding factor (AREB / ABF) . The identified ABA-responsive StbZIPs will facilitate further functional characterization of these subfamily members.
IAA (10 μM 24h).
Auxin, particularly indole-3-acetic acid (IAA), is a plant growth hormone that affects plants’ growth and development . Following treatment of 10 mM IAA, an analysis of the differential log 2-fold expression showed that only three StbZIPs (5, 32, and 59) exhibit positive regulation, while a set of 18 bZIPs are down-regulated. IAA regulates various biological and physiological processes such as cell elongation, induction of root growth, and flower and fruit development . Such responses are controlled by the cis-acting auxin-responsive elements (AuxREs) , which are directly linked by auxin response factors (ARFs) [70, 71] by protein-protein interaction and repressed by AUX / IAA protein transcription . The promoter analysis in prior studies suggests that other cis-elements are also involved in auxin-mediated transcription in addition to AuxREs. For example, the soybean and tobacco GH3 promoters found a conserved organization of G-box-related elements (GREs) located near AuxREs . An enrichment of modules consisting of GREs and AuxREs in auxin-inducible promoters in Arabidopsis and rice was reported using in silico bioinformatics approaches [72, 73]. Based on our expression data, and available literature, it is evident that bZIPs regulate ABA / IAA stress in potato.
GA3 (50 μM 24h).
Gibberellins are a large family of plant growth hormones. The gibberellic acid (GA3) is mainly involved in cell expansion, cell division, seed germination, and stem elongation. GA3 performs a versatile function in the growth and production of Potatoes, and, at a low concentration, it induces the seed tuber weight and sugar content . A total of five StbZIPs (8, 23, 32, 59, and 88) showed expression greater than two-fold (FC) upon treatment with GA3.
Development of SSR markers
Mining existing diversity and functional validation in a large population are vital for germplasm conservation and crop improvement. bZIPs play crucial roles in plants in stress and developmental processes. Therefore, it is important to develop molecular markers that can be used in breeding programs [75, 76]. Out of 30 candidate StbZIPs, only sixteen had SSR containing sequences from which 22 SSRs were identified (Table 2). The highest number of tri repeats was eleven among these SSRs. Detailed information regarding SSR-like motif frequency and frequency of classified repeat types are available in Table 2 and S6 Table. It is important to note that these SSR markers were derived from StbZIPs which were differentially regulated in a stress-specific manner. For example, StbZIP2, 4, 7, 23, 38, 71, 75 were differentially regulated under salt stress, StbZIP25, 54, and 82 under mannitol stress, StbZIP38 under water stress, StbZIP7 and 25 under ABA stress, StbZIP5 under IAA stress, and StbZIP23 under GA3 stress (S6 Table). These candidate StbZIPs and SSRs add to the list of known genes and markers and after validation can be further used in the marker-trait analysis to breed potato for tolerance to abiotic stresses. A similar approach was adopted to identify candidate bZIPs and develop SSR markers in Medicago truncatula  and wheat .
In summary, a total of 90 StbZIPs on a genome-wide level were identified in potato. The FPKM based expression analysis led to the identification of eighteen candidate StbZIPs, expressed in a tissue-specific manner [4 in roots (StbZIP 25, 34, 50, and 89), 8 in tubers (StbZIP 4, 22, 34, 42, 50, 63, 68, and 89), and four each in mesocarp and endocarp (StbZIP 20, 34, 42, 50, 87, and 89)]. Differential expression analysis (log 2 FC) under salt stress identified 2 positively (StbZIP 62 and 66), and 11 negatively (StbZIPs 7, 2, 4, 6, 23, 32, 38, 48, 49, 71, and 75) dysregulated StbZIPs. In response to water stress, StbZIPs 1, 14, 25, 53, 54, 82 showed positive upregulation (log2FC), while StbZIPs 3, 38, 62, and 71 were regulated negatively. The application of mannitol on potato showed the upregulated of StbZIPs 21 and 66. Heat stress led to positive up-regulation of StbZIPs 1, 5, 7, 8, 15, 17, 23, 30, 37, 41, 62, 66, and 82, while StbZIPs 3, 24, 27, 32, 53 were upregulated but negatively. Likewise, the expression analysis upon treatment with phytohormones led to the identification of 8 StbZIP that were dysregulated upon ABA treatment [StbZIP 1, 7, 25, and 88 were positively upregulated, and StbZIPs 8, 21, 24, and 37 were negative downregulated], and three upon IAA treatment (StbZIPs 5, 32, and 59 upregulated positively). No major changes in the expression of StbZIPs were observed upon treatment with IAA and BAP. The phylogenetic analysis, annotation of function, and expression analysis opens the path for further functional validation studies and the use of StbZIPs for engineering potato for tolerance to various abiotic stresses. Similarly, the identified SSRs from candidate StbZIPs will also be useful in potato crop improvement programs. The function of these genes can be validated by using either overexpression or gene knockout approaches.
S1 Fig. Multiple sequence alignment of basic and hinge regions of StbZIPs.
The StbZIP proteins were classified into 11 categories (A-I, S, and U). Each group’s representative bZIP was taken as reference from Arabidopsis, rice, maize, and barley and is shown in the red box. An asterisk marks conserved amino acid residues.
S1 Table. Motif analysis of StbZIPs with the group name, length, motif type, start, and end position.
S2 Table. FPKM expression of StbZIPs in different tissues (leaves, roots, tuber, sepals, petals, and fruit).
The FPKM values were retrieved from the various transcriptomic datasets available at http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml.
S3 Table. FPKM expression during control and abiotic stress conditions were retrieved from http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml in potato tissues.
S4 Table. Differential log 2 FC- FPKM, expression of StbZIPs in abiotic conditions (salt-150 mM NaCl for 24h, heat-35 °C for 24h, mannitol-260 μM for 24h, and water stress) and the controls in potato plants at different times and concentrations.
S5 Table. Differential log 2 FC-FPKM expression analysis after the exogenous supply of phytohormones BAP, ABA, IAA, and GA for control in potato plant at different concentrations and times.
AK acknowledges the Director, CSIR-IHBT, Palampur for providing necessary facilities. This manuscript represents CSIR-IHBT communication no. 4786.
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