Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress

Potato microtuber productions through in vitro techniques are ideal propagules for producing high quality seed potatoes. Microtuber development is influenced by several factors, i.e., high content sucrose and cytokinins are among them. To understand a molecular mechanism of microtuberization using osmotic stress and cytokinin signaling will help us to elucidate this process. We demonstrate in this work a rapid and efficient protocol for microtuber development and gene expression analysis. Medium with high content of sucrose and gelrite supplemented with 2iP as cytokinin under darkness condition produced the higher quantity and quality of microtubers. Gene expression analysis of genes involved in the two-component signaling system (StHK1), cytokinin signaling, (StHK3, StHP4, StRR1) homeodomains (WUSCHEL, POTH1, BEL5), auxin signaling, ARF5, carbon metabolism (TPI, TIM), protein synthesis, NAC5 and a morphogenetic regulator of tuberization (POTH15) was performed by qPCR real time. Differential gene expression was observed during microtuber development. Gene regulation of two component and cytokinin signaling is taking place during this developmental process, yielding more microtubers. Further analysis of each component is required to elucidate it.


Introduction
Potato (Solanum tuberosum L.) is the fourth most important crop worldwide, with an average production of 388 million tons per year of tubers [1,2]. A potato tuber is a specialized stem that arises from the underground organ known as stolon [3]. Tubers are used for plant survival by vegetative propagation; they are sink organs in which surplus photosynthetic assimilates are stored [4], with starch [5,6], vitamins [7] and proteins [8,9] as the main storage components.
Microtuber development has advantages provided by the handling, production and packaging of healthy seeds. The technique accelerates the multiplication process, producing more seed potato faster and cheaper than other methods. Researchers estimate that growers can earn 40% more from apical cuttings than from minitubers [14].
The induction of microtubers under in vitro conditions was first described by Baker [15]; Mes and Menge [16], using high content of sucrose in the medium, as plant pathology tool. auxin signaling, ARF5, carbon metabolism (TPI, TIM), protein synthesis, NAC5 and a morphogenetic regulator of tuberization (POTH15).

Microtuber Induction
Microtuberization was induced from explants derived from in vitro propagated shoots of potato cv. Alpha in solid osmotic stress medium, MR8-G6-2iP (medium MS [62], sucrose 8%, gelrite 6 g/L, 2iP 10 mg/L, −1.5 Mpa) after one month of incubation in darkness condition ( Figure 1A). Microtubers were 7 mm in diameter, 2.3 per explant and 126.3 mg fresh weight. Explants cultured in non-osmotic medium developed only small swellings in the tip of shoot explants ( Figure 1B).

Microtuber Induction
Microtuberization was induced from explants derived from in vitro propagated shoots of potato cv. Alpha in solid osmotic stress medium, MR8-G6-2iP (medium MS [62], sucrose 8%, gelrite 6 g/L, 2iP 10 mg/L, −1.5 Mpa) after one month of incubation in darkness condition ( Figure 1A). Microtubers were 7 mm in diameter, 2.3 per explant and 126.3 mg fresh weight. Explants cultured in non-osmotic medium developed only small swellings in the tip of shoot explants ( Figure 1B). . Microtuber development of potato S. tuberosum cv. Alpha in medium with 8% sucrose, 6 g/L gelrite and activated charcoal after four weeks of incubation in darkness. (B). Shoot explants of potato in medium with low content of sucrose (1%), gelrite 3 g/L and activated charcoal after four weeks of incubation in darkness. Bar represents 1 cm.

Interaction Analysis of Proteins Directly Involved in Microtuberization
To understand a possible regulatory interaction, network of a STRING-based bioinformatic analysis with confidence (0.500) was made based on potato S. tuberosum genome ( Figure 2). . Microtuber development of potato S. tuberosum cv. Alpha in medium with 8% sucrose, 6 g/L gelrite and activated charcoal after four weeks of incubation in darkness. (B). Shoot explants of potato in medium with low content of sucrose (1%), gelrite 3 g/L and activated charcoal after four weeks of incubation in darkness. Bar represents 1 cm.

Interaction Analysis of Proteins Directly Involved in Microtuberization
To understand a possible regulatory interaction, network of a STRING-based bioinformatic analysis with confidence (0.500) was made based on potato S. tuberosum genome ( Figure 2).
A set of two component and cytokinin signaling-related coding genes were evaluated (StHK1, StHK3, StHP4 and StRR1) and according with the theoretical gene network in S. tuberosum, those proteins interact with WUSCHEL, POTH1 and BEL5 homeodomains and with morphogenic genes, such as POTH15, ARF5 (MP), carbon metabolism (TIM, TPI) and protein synthesis (NAC5) whose expression underwent significant changes during the tuberization of S. tuberosum.
Interestingly, StHK1 and POTH15 were pivotal in this network interaction, suggesting a greater importance for those proteins in the theoretical regulatory network in the tuberization of S. tuberosum.
This interaction network represents the first report of theoretical and preliminary evidence in the microtuberization process involving the two component and cytokinin signaling coupled with STBEL5, POTH1. A set of two component and cytokinin signaling-related coding genes were evaluated (StHK1, StHK3, StHP4 and StRR1) and according with the theoretical gene network in S. tuberosum, those proteins interact with WUSCHEL, POTH1 and BEL5 homeodomains and with morphogenic genes, such as POTH15, ARF5 (MP), carbon metabolism (TIM, TPI) and protein synthesis (NAC5) whose expression underwent significant changes during the tuberization of S. tuberosum.
Interestingly, StHK1 and POTH15 were pivotal in this network interaction, suggesting a greater importance for those proteins in the theoretical regulatory network in the tuberization of S. tuberosum.
This interaction network represents the first report of theoretical and preliminary evidence in the microtuberization process involving the two component and cytokinin signaling coupled with STBEL5, POTH1.
Levels of expression of the histidine-containing phosphotransferase, AHP4, was downregulated in the 8 and 31 days, expression level to −3.78 and −0.83 respectively in 15 to 23 days, expression level was 0.53 and 0.43 ( Figure 3, Table 1).
ARF5, an auxin response factor 5, was downregulated −0.32 at 8 days, −1.08 at 23 days and upregulated 1.22 at 15 days and 31 days in osmotic stress medium ( Figure 3, Table 1). rimer design of genes that were analyzed during microtuber induction of potato S. tuberosum cv. Alpha.

Discussion
Potato tuber formation is a complex developmental process that requires the interaction of environmental, biochemical and genetic factors. It involves many important biological processes, including carbon partitioning, signal transduction and meristem determination [47][48][49]. Under optimal crop field conditions, tuberization in potato is activated by signals that function in the leaf and move down into stolon tips to induce and activate tuber formation. The major signals that regulate the onset of tuber formation in potato are: CYCLING DOF FACTOR (StCDF1), StBEL5 and SELF-PRUNING6A (StSP6A) as mobile signals originating in the leaf [63][64][65].
In plant tissue culture medium with 2-3% sucrose lacking plant growth regulators, microtubers can be induced after 4-5 months in culture. However, microtubers are significantly accelerated and improved by using plant growth regulators or by changing culture conditions.
Gelrite is a bacterial (Pseudomonas elodea) polysaccharide composed of glucuronic acid, rhamnose and glucose, and has been used routinely to promote somatic embryo maturation of several plant species with concentrations exceeding the standard 3 g/L up to 12 g/L. It has been reported that high gel strength was associated with reduced water availability from the medium to the explants [66]. Gelrite influences cytokinin-sensitivity in the moss Physcomitrella patens inducing a bud protonema differentiation in the mutant cytokininsensitive PC22 [67]. The gelrite effect is attributable to physical and chemical properties of the gelling agent [68]. We found a positive effect on number, size and germination of microtubers induced in gelrite 6 g/L, compared in non-osmotic conditions. Our results demonstrate a synergism between gelrite concentration and cytokinin signaling, in addition to other genes strongly involved in tuber formation. Furthermore, the cytokinin 2iP was the best to induce microtuber development in our protocol. Lomin et al. [52] found that the highest affinity with potato histidine kinases was 2iP and trans-zeatin, compared with cis-zeatin, BAP, kinetin and thidiazuron.
In the present work, we propose a model of the osmotic-protein regulatory network that integrates the previously reported cross-talk between the CK signaling pathways, with transcriptional regulators that have been shown to be important in tuber development ( Figure 4).  TCS is considered as one of the most crucial signal transduction systems in plants. Evidence suggests that TCS pathways are involved in sensing the environmental stimuli, TCS is considered as one of the most crucial signal transduction systems in plants. Evidence suggests that TCS pathways are involved in sensing the environmental stimuli, ethylene signaling, light perception, circadian rhythm and cytokinin-dependent processes which include shoot and root development, vascular differentiation and leaf senescence [54,55,69]. Cytokinin signaling has been associated with the variety of stress response [70]. Histidine kinase of the TCS is known to function as an oxidative stress sensor [71], involved in the primary cell wall [72]. The genome of potato S. tuberosum contains 4 genes encoding histidine kinases (HK), 7 genes involved in phosphotransferase (HPT), 8 B-type response regulators (B-ARR), 8 A-type response regulators (A-ARR) and 4 C-type response regulators (C-ARR) [52].
According to our results derived from gene expression analysis and the network developed in STRING database, the molecular mechanisms can be interpreted as follows: 1.-Histidine-kinase1 (StHK1) is part of the two-component signaling system and was upregulated in MR8-G6-2iP (osmotic stress) treatment during the first two weeks of microtuber induction. This finding correlates with microtuber development in the first two weeks. Downregulation of StHK1 occurred when potato microtubers were already formed, in the third and fourth week of induction. AtHK1 is induced by cytokinins [73], functions as an osmosensor, a positive regulator of drought in Arabidopsis [74][75][76], salt stress [75,76], ABA signaling [77][78][79][80][81], ethylene [82,83] and stomatal control [84], and is related to MAPK cascade signaling [74] as well as conferring drought tolerance, by regulating levels of ABA accumulation [78][79][80][81]. AtHK1 is required in the regulation of desiccation process during seed formation. AHK1 mutation in Arabidopsis causes a decrease in storage proteins [83]. 2.-AHK1 interact and feeds phosphate to phosphorelay-integrating histidine protein AHP4; in our analysis, it was upregulated in osmotic stress (MR8-G6-2iP) treatment in the second and third week of incubation. Singh et al. [85] found that AHP4 was upregulated in root tissue of Arabidopsis under osmotic stress whereas other members of this family (AHP1, AHP2 and AHP3) were downregulated. 3.-Histidine kinase 3 (StHK3) interacts with AHP4. It was found downregulated in three consecutive weeks (1 to 3) in osmotic stress condition MR8-G6-2iP and upregulated in the fourth week of incubation. AHK3 is a negative regulator of the adaptive response to osmotic stress in Arabidopsis [75]. 4.-The response regulator type B, StRR1, was upregulated in the fourth week of incubation. In the two-component signaling system, phosphate is transferred to an aspartate residue within the receptor domain of the response regulator and this functions as a transcription factor. StRR1 is involved in DNA repair, oxidative stress response, drought tolerance and low temperatures [76,77,[86][87][88], and as a transcriptional activator of WUSCHEL [89] through a cytokinin-dependent signaling, which demonstrates its relevance and confirms its role in the tuberization signaling system. Lomin et al. [52] verified the rapid activation of type A response regulators in the presence of cytokinins in potatoes. 5.-Following the network, WUSCHEL interacts with AHP4. In our analysis, WUSCHEL was upregulated in the first and second week of incubation and downregulated from third to fourth week of incubation. WUSCHEL plays a central role during the specialization of stem cells in meristems [60,90,91]. Upregulation of WUS allows us to infer that this is essential for the initiation of microtuberization and its downward regulation can promote microtubers' dormancy. 6.-WUSCHEL interacts with POTH1. It was found upregulated in the first and second week of incubation similar than WUSCHEL in MR8-G6-2iP medium. This indicates that POTH1 is important in the regulation of the initiation and maturation of tubers. In potato, overexpression of POTH1 results in improved tuberization rate in light and dark conditions [92]. POTH1 has been largely analyzed during tuber development. When overexpressed along with BEL5, it increases cytokinin levels at the tips of stolons and promotes tuberization [44]. In addition, their homeodomain KNOX allows them to form dimers for the suppression of other genes [93]. Interaction of POTH1 protein with seven members of the BEL family has been analyzed in the potato plant. Using mutant analysis, multiple BEL proteins that bind POTH1 were identified, which implies that they are involved in a complex development control system in potato [44]. 7.-Next in the network, POTH1 interacts with BEL5, a bell-like homeodomain protein 5, that was upregulated only in the second week of incubation. BEL5 has the POX homeodomain, which allows it to form heterodimers with POTH1 and these dimers are determining factors in the maintenance of the inflorescence meristem in Arabidopsis [94]. BEL5 is a mobile long-range signaling molecule that promotes tuberization in potato [65]. BEL5 is induced by light and has a primary role in tuber maturation [95]. 8.-WUSCHEL also interacts with POTH15, an ortholog of STM in Arabidopsis. In our analysis, POTH15 was upregulated in the second and fourth week of incubation. POTH15 (STM) mutant causes defects in the formation and maintenance of SAM [96]. According to Endrizzi et al. [97], STM regulates the expression of WUS. Scofield et al. [96] showed that STM prevents the meristem organizing center cells by adopting specific lateral destinations. WUSCHEL and STM gene regulation converge in suppression of differentiation, coexpression of both genes produces a synergistic effect [98]. 9.-ARF5, an auxin response factor 5, interacts with AHP4, POTH15, WUSCHEL, CLAVATA1 and PIN1. ARF5 was upregulated in the second and fourth week of incubation in MR8-G6-2iP (osmotic stress) medium. ARF5 mediate organ formation and vascular tissues throughout the Arabidopsis life cycle, and its expression is gradually restricted to the vasculature as the organs mature [99,100]. Upregulation in the second week is coincident with tuber enlargement. 10.-Alfa-nascent chain-associated (NAC5) complex is a highly conserved protein complex and was upregulated in the second and third week of incubation. αNAC plays an important role in conjunction with its heterodimer βNAC as regulator and as a chaperone in protein synthesis during translation, joining newly synthesized polypeptides to protect them from proteolysis and facilitate their folding in ribosome biogenesis and protein assembly; and in transport to other organelles, there is evidence that translation is less intense when NAC is absent from the ribosome [101]. The substantial increase in αNAC expression at the second week is attributed to cell division and growth in tuber formation. In the network, αNAC interacts with S10 ribosomal protein, that interacts with THY-1 and thereby indirectly to the isoforms TIM and TPI. Furthermore, THY-1 interacts with two histidine-kinase proteins, HK1, part of a two-component signaling system and HK4 (WOL/CRE1) which is a primary cytokinin receptor [102]. 11.-In the interaction network, triosephosphate isomerase (TPI and TIM) interacts with the bifunctional dihydrofolate reductase-thymidylate synthase, THY-1, involved in the de novo biosynthesis of dTMP nucleotide, and metabolism of folate [103]. TIM and TPI were found expressed opposed. TIM was upregulated in the first three weeks and TPI downregulated. Both TPI and TIM play crucial roles in glycolysis and glycogenesis [104]. TIM was found to be upregulated in a proteomic analysis of rice roots under hydric stress, due mainly to the supply of additional energy in form of ATP required to maintain homeostasis under these conditions [105]. Similar regulation pattern under hydric stress conditions was reported in maize (cytosolic) [106] and rice leaves (cytosolic) [107]. Upregulation during induction, initiation and enlargement of the tuber in the transcriptional analysis of TIM, and the primary interactions of TIM and TPI in the network, indicate its importance to carry out the primary mechanisms of tuber induction, initiation and enlargement, and the enzymes are essential in carbohydrate metabolism and energy generation.
Further analysis of each component is required to validate this network. Transcriptomic analysis, overexpression and downregulation of these genes are currently in evaluation.

Isolation of RNA and qPCR Analysis
Total RNA derived from four times (8, 15, 23, 31 dayss) was isolated using Trizol (Invitrogen, Carlsbad, CA, USA), RNA concentration was measured by its absorbance at 260 nm, ratio 260 nm/280 nm was assessed, and its integrity confirmed by electrophoresis in agarose 1% (w/v) gels. Samples of cDNA were amplified by PCR using SYBR TM Green (ThermoFisher CAT: 4312704, Waltham, MA, USA) in Real-Time PCR Systems (CFX96 BioRad, Herules, CA, USA). The expression of EF1 and SEC3 was used as reference for calculating the relative amount of target gene expression using the 2−∆∆ CT method [108,109]. qPCR analysis was based on at least five biological replicates for each sample with three technical replicates.

1.
Microtubers were suitable plant material for the analysis of gene expression.
Differential gene expression of genes analyzed in microtubers induced under osmotic stress confirmed the hypothesis that TCS and cytokinin signaling are coupled with genes that have been associated in tuberization.

4.
Improvement of the understanding in molecular mechanisms involved in potato microtuberization was achieved by STRING database bioinformatic tool.