1. Introduction
Plant transcription factors of the bZIP family play a significant role in biological advancements inclusive of morphogenesis, seed maturation, flower development, and environmental factors [1] [2] . However, bZIP transcription factors are involved in multiple abiotic stress tolerances. Several reports have indicated that members of the bZIP transcription factor family function in plant hormone signaling and environmental stresses, which include drought and salt stress [3] [4] [5] [6] .
Among all the plant transcription factors, bZIP genes have been identified in monocotyledonous and dicotyledonous plants. Nowadays, it is reported that 75 bZIP genes have been classified in Arabidopsis thaliana [7] , 89 in Oryza sativa [8] , 125 in Zea mays [9] , 160 in Glycine max [10] , and 55 in Vitis vinifera [11] . AREB1, AREB2, and ABF3 up-regulate ABA, drought tolerance, and water stresses in Arabidopsis, and they also function as master transcription factors [12] [13] . Furthermore, AtABF3 increased tolerance to various abiotic stresses in Alfalfa [14] . Overexpression of AtbZIP3 is involved in leaf development and response to the sugar signaling pathway in Arabidopsis [15] . However, AtbZIP19 and AtbZIP23 have been related to the regulation of zinc deficiency in Arabidopsis [16] .
In plants, some researchers have confirmed that transcription factors of the bZIP family are induced in seed maturation [1] , light response [17] , sucrose signaling [18] [19] , pathogen resistance [3] , flower development and fertility [20] , phytohormone response, development of organs and response to various stresses [2] . AtbZIP63 may participate in the interaction of ABA and glucose in Arabidopsis [19] . Kang et al. [21] , state that overexpression of ABF3 and ABF4 functions in ABA hypersensitivity, whereas it reduces transpiration and enhances tolerance in Arabidopsis. The function of bZIP16 was developed in seed germination and seedling development of Arabidopsis [22] . Besides, overexpression of GmbZIP1 affected the response of ABA in seed germination stages and abiotic stress tolerance in soybeans [23] . AtbZIP17 might play roles as an ABA signaling pathway and is involved in seed germination and seed filling in Arabidopsis. Furthermore, it was genetically demonstrated that AtbZIP17 plays a crucial role in osmotic stress and is affected as a negative regulator of storage and seed germination in Arabidopsis [24] .
SlbZIP38, SlbZIP1 and LebZIP2 functions were found to be involved in tomato responses to various environmental stresses [5] [6] [25] . Furthermore, SlAREB1 was characterized by ABA, salt, drought, and cold resistance from tomato [26] . In Vitis vinifera, VIbZIP36 improved tolerance to drought treatment in seed germination [27] . Overexpression of VIbZIP30 not only improved seed germination under dehydration stress but was also involved in drought stress in grapevine [28] .
Tomato (Solanum lycopersicum L.) is a dicotyledonous plant that is one of 69 SlbZIP gene family members classified into 14 groups. In this study, we performed further experimental design by overexpressing the SlbZIP39 gene in a tomato to investigate its expression profiles in the different tissues, abiotic stresses, hormonal treatments, and physiological characteristics in tomato.
2. Materials and Methods
2.1. Plant Materials and Growth Conditions
The WT tomato (Solanum lycopersicum L., cv. Micro-Tom) and transgenic seeds were sterilized before germinating, and the seedlings were then grown in 1/2 MS medium. The plants were cultivated in a greenhouse under standard conditions with 18 h light (25˚C)/6h dark (18˚C) cycles and 80% relative humidity. The different tissue samples were gathered to carry out the gene expression analysis, which included roots, stems, leaves, shoots, petioles, flowers, buds, sepals, petals, stamens, pistils, pedicles, immature, mature green, breaker, and 5-day breaker. All the tissue samples were immediately frozen in liquid nitrogen and stored at −80˚C.
2.2. Vector Construction and Transformation
The full length of SlbZIP39 CDS without the stop codon was amplified with a primer (Table 1). The PCR fragments were cloned, then digested by NotI and SbfI and cloned into the K303 expression vector (Gateway technology) under the CaMV 35S promoter. This construction was transformed into Solanum lycopersicum L., cv. Micro-Tom by Agrobacterium tumefaciens strain GV3101 [29] .
2.3. RNA Isolation and Quantitative Real-Time PCR Analysis
Total RNA from different tissues was extracted by using the E.Z.N.A.® Plant RNA Kit (Omega Bio-Tek, USA, R6827-01), according to the manufacturer’s instructions. cDNAs were synthesized with the Prime-ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TAKARA, Japan). Quantitative real-time PCR was performed according to the instructions provided for the Bio-Rad-CFX system (Bio-Rad, USA), using SYBR Green PCR Master Mix (CWBIO, China). The tomato UBI gene (Solyc07g064130) was used as an internal control for normalization. The relative quantification of gene expression levels was calculated by the comparative 2−∆∆CT method. All the primers used for the qRT-PCR were listed in Table 1. The experiments are repeated at least three times.
Table 1. List of the primers used in this study.
2.4. Abiotic Stress and Hormone Treatments
Tomato seedlings were grown in a greenhouse under the same conditions, and all the treatments were conducted using one-month-old plants. For abiotic stress treatments, the plants were sprayed with 100 mM D-mannitol and 200 mM NaCl. For the hormone treatments, the plants were sprayed with 100 µM ABA, GA3, and IAA solutions. For the dehydration assay, leaves and fruits were left on a piece of dry filter paper at 25˚C ± 1˚C, and then all samples were collected at the designated time. All the samples were harvested at 0 h, 3 h, 6 h, 12 h, and 24 h after each treatment. The harvested samples at 0 h were used as control and stored at −80˚C. For each treatment, samples were collected from 6 plants, mixed and all the experiments were performed at least three times.
2.5. Statistical Analysis
Statistical analysis was conducted using Prism-6 (GraphPad, San Diego, CA, USA). All the experiments were repeated three times, and all the data was calculated as the expression of mean ± standard error. Comparisons between the two groups were performed using Student’s t-tests. The significance values of (*P < 0.05; **P < 0.01) were considered to be compared between wild-type and transgenic plants.
3. Results
3.1. Expression Patterns of SlbZIP39 Induced by Abiotic and Hormonal Stresses
Previous reports have indicated that SlbZIP transcription factors are involved in abiotic and biotic stresses [5] [6] [25] . We therefore analyzed the transcript levels of SlbZIP39 in leaves under abiotic and hormonal stress. The transcripts of SlbZIP39 are not only induced by NaCl and D-mannitol treatments but also affected by IAA, GA3, and ABA treatments (Figure 1 and Figure 2). In NaCl and D-mannitol treatments, however, the level of SlbZIP39 transcription was relatively down-regulated in NaCl but adequately up-regulated in D-mannitol treatment (Figure 1(a) and Figure 1(b)). During the dehydration treatments, we compared tomato leaves and fruits. The transcript of SlbZIP39 was expressed in tomato leaves and fruits but was slightly down-regulated at 3 h and 6 h and moderately up-regulated at 12 h and 24 h in leaves (Figure 1(c) and Figure 1(d)). The level of SlbZIP39 transcription was significantly reduced during fruit dehydration treatments (Figure 1(d)). In the hormone treatments, the transcript level of SlbZIP39 was affected within 3 h to 24 h of IAA treatments, whereas it was up-regulated at 12 h and down-regulated at 3 h in tomato leaves (Figure 2(a)). The expression level of SlbZIP39 was up-regulated at 24 h and moderately induced at 3 h to 12 h in GA3 treatments (Figure 2(b)). However, the level of SlbZIP39 transcription was relatively responsive to ABA treatment and up-regulated at 3 h in leaves but declined at later intervals (Figure 2(c)). Here, we found that SlbZIP39 responded at the same time interval after treatments
Figure 1. Expression profiles analysis of the SlbZIP39 gene under various abiotic stresses. For control plants, the expression data were set to 0 h. (a) 100 mM D-mannitol; (b) 200 mM NaCl; (c). Dehydration of leaf; (d) Dehydration of fruit. Error bars indicate SD values from three biological replicates (n = 3). Asterisks indicate significant differences using Student’s t-test (*P < 0.05; **P < 0.01).
Figure 2. Expression profiles analysis of the SlbZIP39 gene under hormonal treatments. For control plants, the expression data were set to 0 h. (a) 100 µM IAA; (b) 100 µM GA3; (c) 100 µM ABA. Error bars indicate SD values from three biological replicates (n = 3). Asterisks indicate significant differences using Student’s t-test (*P < 0.05; **P < 0.01).
but SlbZIP39 was more affected by NaCl, dehydration of fruits, GA3, and ABA than others. We inferred that SlbZIP39 in tomato may respond to abiotic stresses and hormonal treatments.
3.2. Expression Profiles of SlbZIP39 Gene in Different Tissues of Tomato
We transgenically generated overexpression of SlbZIP39 under the 35S promoter in tomato plants. Transgenic tomato plants were obtained from three independent lines (L-1, L-4, L-9) (Figure 3(c)). Plant heights of one-month-old WT and OE were measured for physiological processes. The SlbZIP39 overexpressing plants showed decreased plant height compared to the wild-type (Figure 3(a), Figure 3(b)). The expression profiles of SlbZIP39 in wild-type tomato were analyzed by qRT-PCR in various plant tissues, including roots, stems, leaves, shoots, flowers, buds, sepals, petals, stamens, and pistils, respectively (Figure 3(d)). The transcripts of SlbZIP39 are documented as moderately high in stems, leaves, shoots, petioles, flowers, buds, and pistils; the highest transcripts of SlbZIP39 accumulate in stamens, followed by petals and sepals. However, the lower expression level of SlbZIP39 was expressed in roots and pedicles (Figure 3(d)). Moreover, we analyzed fruit development stages by using qRT-PCR (Figure 3(e)). The expression profiles of SlbZIP39 in fruit developmental stages, consisting of immature green, mature green, and breaker stages, were explored. However, the transcripts of SlbZIP39 were highly expressed at the immature green stage (IM), followed by the mature green stage (MG) and the breaker stage (Br, Br5) (Figure 3(e)). These results showed that SlbZIP39 may affect plant architecture and fruit development processes.
Figure 3. Phenotypical characteristics of wild-type and transgenic plants. (a) Plant architecture after 30 days of wild-type and SlbZIP39-OE lines; (b) Plant height; (c) SlbZIP39 expression levels in wild-type and SlbZIP39-OE tomato plants; (d) and (e) SlbZIP39 expression analysis in various tissues such as Rt, roots; St, stems; L, leaves; Sh, shoots; Pet, petioles; Fl, flowers; Bu, buds; Se, sepals; Pe, petals; Sta, stamens; Pi, pistils; Ped, pedicles; IM, immature; MG, mature green; Br, breaker; Br-5, 5 days breaker. Error bars indicate SD values from three biological replicates (n = 3). Asterisks indicate significant differences using Student’s t-test (*P < 0.05; **P < 0.01).
3.3. Phenotypical Characters of SlbZIP39 Overexpressing in Tomato Plants
The objectives of this study were to document and compare WT and OE plants under normal conditions. Numerous physiological parameters were measured, including plant height, flowering times, fruit weight, fruit length, and seed number, respectively. The SlbZIP39 overexpression plant showed earlier flowering times than the WT (Figure 4(c)). In addition, the fruit size of SlbZIP39-OE was smaller than the WT fruits (Figure 4(d)). In tomato, SlbZIP39 overexpression of fruit was decreased in fruit weight, fruit diameter, and the number of seeds compared to WT (Figures 4(e)-(g)). In our results, overexpression of SlbZIP39 significantly differed in fruit weight, seed numbers, and inflorescence architecture (Figure 4). These data suggest that overexpression of SlbZIP39 may regulate seed number and fruit development in tomato.
3.4. Tissue Specificity of SlbZIP Genes Expression Patterns during Fruits Development
Fruit development can be identified in several stages according to the number of days after anthesis and color. So, we investigated the expression levels of four SlbZIP genes in SlbZIP39-OE and WT at different fruit developmental stages. To verify the specific expression of SlbZIP39, the expression levels of SlbZIP04 (Solyc01g079480.2), SlbZIP07 (Solyc01g100460.2), SlbZIP10 (Solyc01g109880.2)
Figure 4. Phenotypical characteristics and growth parameters of SlbZIP39 transgenic tomato plants. (a) Inflorescence architecture of the transgenic tomato plants; (b) Number of inflorescence; (c) Day to flowers; (d) Fruits characters; (e) Fruits weight; (f) Fruits diameter; (g) Number of seeds. Error bars indicate SD values from three biological replicates (n = 3) with n = 10 or 10 plants for each repeat. Asterisks indicate significant differences using Student’s t-test (*P < 0.05; **P < 0.01).
and SlbZIP34 (Solyc04g080740.1) were analyzed but they belong to group IV and contain sequences with close homology to SlbZIP39, as indicated in the phylogenetic tree database [30] . In our results, SlbZIP04 and SlbZIP07 mRNA levels were significantly up-regulated at IM and MG fruit stages, whereas they were moderately up-regulated at Br and Br-5 fruit stages than in WT (Figure 5(a) and Figure 5(b)). In addition, SlbZIP10 transcript levels were abundantly up-regulated at MG and Br compared to WT fruit (Figure 5(c)). SlbZIP10 expression was slightly up-regulated at IM and Br-5 than in WT fruit (Figure 5(c)). Furthermore, SlbZIP34 transcripts were up-regulated at Br and had similar expression patterns in IM, MG, and Br-5 than in WT fruit developmental stages (Figure 5(d)). Significantly, SlbZIP04 and SlbZIP10 mRNA levels were predominantly up-regulated by SlbZIP39-OE and WT in fruit developmental stages (Figure 5(a) and Figure 5(c)). The transcript levels of the SlbZIP transcription factor
Figure 5. Expression of four bZIP transcription factors in WT and OE-fruits. (a) SlbZIP04; (b) SlbZIP07; (c) SlbZIP34 and; (d) SlbZIP10. The tissues of fruit from different stages were used to analyze the experiment. IM, immature; MG, mature green; Br, breaker; Br-5, 5 days breaker. Error bars indicate SD values from three biological replicates (n = 3). Asterisks indicate significant differences using Student’s t-test (*P < 0.05; **P < 0.01).
were predominantly different fruit developmental stages between SlbZIP39-OE and wild-type. These results assume that SlbZIP39 may be concerned with the expression patterns of closely related bZIP genes.
4. Discussion
A number of bZIP transcription factors in plant growth and development are key processes that may participate in responses to environmental stresses. Abscisic acid (ABA) is a universal plant hormone because of its significant effects on the regulation of plant growth and development [31] . Nevertheless, the role of bZIP transcription factors has been involved in oxidative stress, light-dependent, and unfolded protein responses in all eukaryotes [32] .
In tomato, SlAREB1 and SlAREB2 function in response to abiotic and biotic stresses, and may act as a link between ABA signal responses and other plant hormones; SlAREB1 improves resistance to deficit and salt stress [3] [4] . Overexpression of LebZIP2 might have been involved in tolerance to herbicide and oxidative stress and cell development of Nicotiana benthamiana [33] . However, expression of LebZIP2 was increased by NaCl and mannitol treatments, whereas, it responded to ABA treatment [25] . The transcripts of SlbZIP38 and SlbZIP1 were expressed by NaCl, ABA, and GA treatments in tomato [5] [6] . Furthermore, overexpression of MusabZIP53 was strongly increased by drought stress and ABA treatment in bananas [34] . Similarly, the expression level of SlbZIP39 responded to NaCl treatment in tomato (Figure 1(b)). Besides, SlbZIP39 was slightly induced by D-mannitol treatment in tomato leaves (Figure 1(a)). The present work revealed that the expression of SlbZIP39 was also up-regulated as GA3 treatment, whereas it responded to IAA and ABA treatment (Figures 2(a)-(c)). Additionally, overexpression of ABF2 has been shown to induce ABA sensitivity and dehydration tolerance in Arabidopsis [35] . In our research, we performed experiments to document the dehydration tolerance of fruits compared with leaves but they were affected by dehydration treatments (Figure 1(c) and Figure 1(d)). The transcript level of SlbZIP39 was down-regulated by the dehydration of fruits (Figure 1(d)). This indicated that transcription factors of SlbZIP39 may respond to abiotic stress and hormone treatments in tomato.
In expression analysis, SlbZIP39 was detected in the different tissues of tomato (Figure 3). We observed that expression patterns of SlbZIP39 were abundantly induced by sepals, petals, and stamens tissues (Figure 3(d)). The transcript of SlbZIP39 is expressed in immature green fruits (Figure 3(e)). Previous reports documented that the expression patterns of SlbZIP38, SlbZIP1 and LebZIP2 were expressed in all tomato tissues [5] [6] [25] . Moreover, overexpression of CabZIP1 is expressed in flowers and is involved in the plant development of Arabidopsis [36] . In Arabidopsis, the overexpression of AtbZIP3 and AtbZIP24 stimulated plant development [15] [37] . Furthermore, overexpression of MubZIP53 has been linked to banana growth retardation [34] . In the present study, overexpression of SlbZIP39 decreased plant growth in tomato (Figure 3(a) and Figure 3(b)). At the mRNA level, SlbZIP39 was expressed in all tomato tissues (Figure 3(d) and Figure 3(e)). Therefore, we suggest that it might be involved in the regulation of plant growth and development in tomato.
In our study, overexpression of SlbZIP39 developed in the inflorescence of tomato (Figure 4(a)). In addition to the significant decrease in fruit weight, fruit diameter, and seed number, the number of inflorescence in overexpressing SlbZIP39 was increased, whereas the flowering time was earlier, compared to the wild-type (Figures 4(b)-(g)). SlAGO7 overexpression is involved in tomato inflorescence architecture [38] . In tomato, SlGRAS24 and SlGRAS40 responded to fruit sizes in the overexpression line [39] [40] . Moreover, the silencing of SlGRAS2 is affected by smaller fruit sizes in tomato [41] . Overexpression of SlAGL11 was dramatically influenced by tomato fruit size [42] . The transcription factor SlAREB1 is expressed in fruit tissues and is involved in the fruit development of tomato [43] . In this study, we found that overexpression of SlbZIP39 may be involved in fruit and seed development (Figure 4(d) and Figure 4(g)). For qPCR analysis, we performed the expression levels of SlbZIP04, SlbZIP07, SlbZIP10, and SlbZIP34 (Group IV) during fruit development (Figure 5). The transcripts of SlbZIP04 and SlbZIP07 were up-regulated at IM and MG fruit stages (Figure 5(a) and Figure 5(b)). Furthermore, SlbZIP10 and SlbZIP34 were predominantly up-regulated at the Br fruit stage (Figure 5(c) and Figure 5(d)). These data suggest that SlbZIP39 may be involved in the expression patterns of closely related bZIP genes.
5. Conclusion
In this study, we cloned and performed abiotic and hormonal treatments on tomato plants expressing SlbZIP39 under greenhouse conditions. Our results have showed that evaluating the effects of overexpressing plants and wild-type fruits at developmental stages is related to bZIP transcription factors. We analyzed Group-IV bZIP transcription factors in tomato and expressed them in fruit developmental stages. However, overexpression of SlbZIP39 was necessary to further confirm the transcription levels of the fruit size gene in tomato. Our experimental results revealed that overexpression of SlbZIP39 might be regulated by fruit size and plant architecture.
Acknowledgements
We would like to thank my supervisor, Professor Zhengguo Li from the School of Life Sciences, Chongqing University. I am greatly thankful to the Government of Myanmar, and China Scholarship Council (CSC) for their permission for my Ph.D. degree.
Authors’ Contributions
Conceptualization, Y Y L, M K T and M M K; formal analysis, Y Y L and K H Y; investigation, Y Y L and M M K; data curation, Y Y L and M K T; writing-original draft preparation, Y Y L and K H Y; writing-review and editing, M K T and M M K; funding acquisition, Y Y L. All the authors have read and approved the final manuscript.
Funding
This research was supported by the National Key Research and Development Program of China (No.2016YFD0400101), and the National Natural Science Foundation of China (Nos.3177270 and 31572175).
Abbreviations
ABA: Abscisic acid
bZIP: Basic leucine zipper
GA: Gibberellic acid
IAA: Indole-3-acetic acid
NaOCl: Sodium hypochlorite
MS: Murashige and Skoog
TF: Transcription factor
WT: Wild-type
OE: Overexpression
qRT-PCR: Quantitative Real-time PCR