*Article* **Genome-Wide Identification and Characterization of the Polyamine Uptake Transporter (Put) Gene Family in Tomatoes and the Role of Put2 in Response to Salt Stress**

**Min Zhong 1,†, Lingqi Yue 1,†, Wei Liu 1, Hongyi Qin 1, Bingfu Lei 2, Riming Huang 3, Xian Yang 1,\* and Yunyan Kang 1,\***

<sup>1</sup> College of Horticulture, South China Agricultural University, Guangzhou 510642, China

<sup>2</sup> Key Laboratory for Biobased Materials and Energy of Ministry of Education, Guangdong Provincial Engineering Technology Research Center for Optical Agriculture, College of Materials and Energy, South China Agricultural University, Guangzhou 510642, China

<sup>3</sup> College of Food Science, South China Agricultural University, Guangzhou 510642, China

**\*** Correspondence: yangxian@scau.edu.cn (X.Y.); kangyunyan@scau.edu.cn (Y.K.)

† These authors contributed equally to this work.

**Abstract:** The polyamine uptake transporter (Put), an important polyamines-related protein, is involved in plant cell growth, developmental processes, and abiotic stimuli, but no research on the Put family has been carried out in the tomato. Herein, eight tomato Put were identified and scattered across four chromosomes, which were classified into three primary groups by phylogenetic analysis. Protein domains and gene structural organization also showed a significant degree of similarity, and the *Put* genes were significantly induced by various hormones and polyamines. Tissuespecific expression analysis indicated that *Put* genes were expressed in all tissues of the tomato. The majority of *Put* genes were induced by different abiotic stresses. Furthermore, *Put2* transcription was found to be responsive to salt stress, and overexpression of *Put2* in yeast conferred salinity tolerance and polyamine uptake. Moreover, overexpression of *Put2* in tomatoes promoted salinity tolerance accompanied by a decrease in the Na+/K+ ratio, restricting the generation of reactive oxygen and increasing polyamine metabolism and catabolism, antioxidant enzyme activity (SOD, CAT, APX, and POD), and nonenzymatic antioxidant activity (GSH/GSSG and ASA/DHA ratios, GABA, and flavonoid content); loss of function of *put2* produced opposite effects. These findings highlight that Put2 plays a pivotal role in mediating polyamine synthesis and catabolism, and the antioxidant capacity in tomatoes, providing a valuable gene for salinity tolerance in plants.

**Keywords:** polyamine uptake protein; Put2; antioxidants; reactive oxygen species; salt stress; tomato

Academic Editor: Nafees A. Khan

Received: 17 December 2022 Revised: 16 January 2023 Accepted: 17 January 2023 Published: 18 January 2023

**Citation:** Zhong, M.; Yue, L.; Liu, W.; Qin, H.; Lei, B.; Huang, R.; Yang, X.; Kang, Y. Genome-Wide Identification and Characterization of the Polyamine Uptake Transporter (Put) Gene Family in Tomatoes and the Role of Put2 in Response to Salt Stress. *Antioxidants* **2023**, *12*, 228. https://doi.org/10.3390/ antiox12020228

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**1. Introduction**

Polyamines, one of the organic polycations, are abundant in various plant organisms and are involved in various cellular processes such as cell growth, nucleic acid stability, and protein synthesis [1,2]. The most abundant polyamines in plant cells, diamine putrescine, triamine spermidine, and tetraamine spermine, are strongly associated with plant responses to biotic and abiotic cues [3,4]. Manipulation of putrescine, spermidine, and spermine by chemical or genetic means is essential for many developmental processes [5,6]. The crucial roles of polyamine synthesis and metabolism in response to numerous stresses have been demonstrated by genetic manipulation [7]. Intracellular polyamine pools are critical for the intermediary part of nitrogen metabolism, and also crosstalk with other metabolic pathways, such as hormones, small molecule signals, and stress-response complexes [8,9].

Besides, increasing evidence indicates that the uptake of polyamine plays essential functions in coordinating the response of plants to a variety of environmental stresses. A salt-sensitive cultivar of rice supplied with putrescine in roots exhibited an increased grain

yield [10]. The *arginine decarboxylase* (*adc*) mutant in *Arabidopsis* showed hypersensitivity to low-temperature stress, but the tolerance was enhanced after being fed putrescine in *adc* mutants [11]. These studies show that polyamine transport could be an important component of diverse environmental protection. In *Arabidopsis*, methyl viologen 1 (At5g05630, *AtRMV1*), a L-type amino acid transporter, was recently found to be a protein important for paraquat (PQ) and uptake of polyamine [12]. The *Arabidopsis* mutant (AT1G31830, *pqr2*/*AtPut2*) also encodes a polyamine transporter and negatively responds to ABA signaling [13,14]. The protective influences of polyamine in opposition to PQ toxicity are partly attributed to transport interactions between polyamines and PQ because both have similar uptake characteristics. In addition, the *paraquat resistant1 (pr1)* mutant exhibited inefficient absorption of PQ [13]. The *put5* plants produced fewer flowers, flowered earlier, and had smaller leaves than wild-type (WT) plants, while the *OsPut1* or *OsPut3* over-expression plants showed a buildup of spermidine and conjugated-spermidine in leaves, larger leaves, more flowers, and a delay in the flowering time, which is implicated in polyamine transport [15]. The rice *OsPut1/2/3* mutant was created by CRISPR/Cas9 gene editing, and the *OsPut1/2/3* mutant increases PQ tolerance without significant yield loss [16]. Moreover, *OsPut1*-*OsPut3* was shown to have a high affinity for spermidine uptake through the substrate assay with a yeast polyamine uptake mutant (*agp2*Δ), and *AtPut1*-*AtPut3* has similar properties [17]. However, much less attention has been paid to transport proteins in plants. To our knowledge, very little has been unraveled regarding the tomato polyamine uptake protein (Put) family and their functions in abiotic stress.

The tomato is one of the world's most significant cash crops and is sensitive to biotic and abiotic pressures such as salt stress, low and high temperatures, and so on. These unfavorable environmental factors seriously compromise tomato growth and yield. Under stressful conditions, polyamine anabolism and catabolism have been found to have important roles involving a multitude of mechanisms [18]. For example, polyamine oxidase, arginine decarboxylase, *S*-adenosylmethione synthetase, and spermine synthase act as critical mediators in multiple stress conditions [19–23]. To date, it has been widely accepted that polyamine acts as a crucial antioxidant in plants [24]. The increase of endogenous polyamines levels saves cells by eliminating reactive oxygen species (ROS) and boosting the antioxidant capacity in response to oxidative stress [25]. Nevertheless, the implications of Put in response to environmental stresses remains elusive. In particular, the role of Put in polyamine anabolism and catabolism, as well as in antioxidant activity, remains largely unknown. In this study, by identifying and characterizing members of the Put gene family in the tomato, a family known for polyamines uptake, we unraveled that Put2, a candidate of the Put family, had a favorable function in salt tolerance via modulating polyamine metabolism and antioxidants. This study sheds fresh light on the important role of Put-mediated polyamine homeostasis in tomatoes, as well as its significance for plant fitness.

#### **2. Material and Methods**

#### *2.1. Indentation and Sequence Analysis of the Tomato Put Family*

The protein sequence of tomato Put and *Arabidopsis* AtPut were downloaded from the NCBI (http://www.ncbi.nlm.nih.gov/, accessed on 10 January 2022). The SGN database (https://solgenomics.net/, accessed on 10 January 2022) was used to get the tomato (SL4.0) reference genome sequence and annotations. We identified eight putative Put proteins encoded in the tomato genome based on investigations of the Arabidopsis Put protein. The eight Put genes in tomato were called by their chromosomal locations. Expasy, an online software, was used to gather basic information for all Put proteins, including their molecular weight (Mw) and isoelectric point (pI).

#### *2.2. Alignment of the Protein Sequence and Phylogenetic Tree Construction*

Using MEGA X, the protein sequences of Put in tomato, rice, and *Arabidopsis* were aligned [26]. Poor alignment areas from all protein sequences were removed using the trimAl tool, and a phylogenetic tree was created using the maximum-likelihood (ML) technique with the Poisson correction and 1000 bootstrap repetitions in IQ-TREE [27]. The depiction of the phylogenetic tree was constructed by Evolview (www.evolgenius.info/, accessed on 22 January 2022). A phylogenetic tree of the tomato Put protein was also built independently. The Put protein sequences are provided in Supplemental data S1.

#### *2.3. Analyses of Conserved Motifs, Conserved Domains, Cis-Acting Elements in Promoters, and miRNA Prediction*

The conserved motifs of the tomato Put proteins were performed by the MEME tool (5.05) (http://meme.nbcr.net/meme/, accessed on 22 January 2022), and Pfam (http: //pfam.xfam.org/, accessed on 22 January 2022) was used to predict the conserved domains of the tomato Puts proteins. The promoter regions of Put were created using the 2.0 kb genomic DNA sequence upstream of the translation start codon (ATG). PlantCare took the cis elements from the Put promoter regions. In Supplemental data S2, the cis elements are listed. The relevant data visualization was conducted using TBtools. According to the targeted candidate, as described previously, the Put coding sequences were submitted to the psRNATarget serve to predict the miRNAs (https://www.zhaolab.org/psRNATarget/, accessed on 22 January 2022) [28]. The Puts protein's transmembrane domains were predicted using the TMHMM program [29].

#### *2.4. Plant Material and Treatments*

Three-week-old tomato (*Solanum lycopersicum* L. cv. Ailsa Craig) seedlings were treated with different exogenous polyamines, hormones, and oxidative stress, or abiotic stresses. Briefly, 2.0 mM Put, 1.0 mM Spd, or 2.0 mM Spm were sprayed over the seedlings; for hormone treatments and oxidative stress, 100 μM ABA, 2 mM SA, 100 μM GA3, 40% ethylene (ETH), 100 μM paraquat, and water were also sprayed onto the tomato plants, respectively. For RNA extraction, at 0, 30 min, 1, 3, 6, and 12 h, samples of leaves were taken, accordingly. The control was the water treatment at 0 h.

We then investigated Put genes response to different abiotic stresses. Salt and drought stress were initiated by irrigating the plants with 200 mM NaCl or 20% PEG6000 solution, respectively. Tomato seedlings were subjected to 42 ◦C (high temperature), and 4 ◦C (low temperature) for heat and cold stress, respectively. After, the treated samples were respectively collected after 0, 30 min, 1, 3, 6, and 12 h; the samples at 0 h were used as the control. In addition, tissues (root, stem, leaf, bud, flower, and fruit) were harvested for investigation of tissue-specific expression. After each treatment, leaves from different plants (three biological replicates) were quickly frozen in liquid nitrogen and kept at −80 ◦C for further analysis.
