The Overexpression of The VvWRKY2 Transcription Factor in Potato Improved the Agricultural Performance and Tubers’ Physio-Chemical and Industrial Properties Even Under Non ‐ Stress Conditions

The tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors play important roles in plant growth and response to biotic and abiotic stresses. Several studies described WRKY factors overexpression in different plant species. In a number of cases, modication in morphology and seed size were described in transgenic plants overexpressing transcription factors from this family. In this work, we aimed to study the effect of a grapevine VvWRKY2 transcription factor on transgenic potato plants growth, yields and chemical properties of tubers produced out of any stress condition in the greenhouse and in the eld. The results were compared with the wild type commercial variety "Belle de Fontenay" (WT). Data showed that the overexpression of the VvWRKY2 transcription factor increased signicantly the leaf area and stem diameter of transgenic plants in comparison to WT. A modication of leaf shape was also noticed in VvWRKY2 overexpressing potato plants. The tuber yield, caliber, dry matter, starch and mineral content of transgenic tubers was higher than WT, while soluble sugars level decreased. All these results suggest that the VvWRKY2 overexpression in potato plants seems to have positive effect on plant vigor, yield and tuber quality under normal conditions in addition to its positive effect on plant response to stresses.


Introduction
Potato (Solanum tuberosum L.) is the world's leading non-cereal food (FAO 2019). It is considered as an important crop for food security since it is a valuable source of nutrients for the human diet (Beals 2019).
However, the global potato yield is negatively affected by several biotic and abiotic constraints limiting the plant growth and production (Dahal et   In the present study, we overexpressed the VvWRKY2 TF in potato plants and evaluated the agnomical, physiological and morphological performances of transgenic potato plants out of any stress conditions. Both WT and VvWRKY2 overexpressing plants were grown in greenhouse and eld in the absence of any constraints in order to study the effect of the overexpression of the VvWRKY2 TF on the agricultural performance and plant growth and physiology.

Production and selection of transgenic VvWRKY2 potato lines
A French potato cultivar (Belle de Fontenay, BF15) was used to produce transgenic potato plants. The VvWRKY2 cDNA was inserted into the MDC32 vector anked between the Pr35S promoter and terminator ). The Agrobacterium tumefaciens LBGV3010 strain harboring the MDC32::VvWRKY2 plasmid were used for potato transformation as described by Gargouri-Bouzid et al. (2005). Stems and leaves were used for transformation and plant regeneration was performed in a regeneration medium corresponding to MS basal medium (Murashige and Skoog 1962) supplemented with Morel vitamins (Morel and Wetmore 1951) and containing sucrose 20 g l -1 , phytagel 4 g l -1 and indole-3-acetic acid (IAA) 0.05 mg l -1 , Zeatin 3 mg l -1 and GA3 3 mg l -1 and antibiotics (Hygromycin 3 µg ml -1 and cefotaxime 250 µg ml -1 ). The culture was performed at 24°C in a culture room with a 16/8 hours day/night alternation.
The explants were transplanted every two weeks until production of adventive buds. The transgenic plants were multiplied in MS medium that contains 3 µg ml -1 Hygromycin. Plants rooted in this medium were selected for further analysis.
The transcription of the VvWRKY2 transgene was veri ed by RT-PCR analysis on total RNA extracted as described by Verwoerd et al. (1989) and modi ed by Saïdi et al. (2009). The extracted RNA was used as template for cDNA synthesis by the reverse transcriptase MoMLV (Bio Basic Inc) using an Oligo-dT as primer. The obtained cDNA, was used for the PCR ampli cation reaction using the VvWRKY2 gene speci c primers. The PCR and RT-PCR products were separated by electrophoresis on a 1% agarose gel.

Greenhouse and eld culture conditions
Vitro-plants from one transgenic potato line overexpressing the VvWRKY2 TF and WT plants were used in this experiment. These plants were micro-propagated by node culture according to Nozeran and Rossignol-Bancilhon (1977) in liquid MS medium enriched with vitamins (Morel and Wetmore 1951). The culture was carried out in a culture chamber at 24°C and a photoperiod of 13/11h light/dark cycle with light intensity of 250 µmol m -2 S -1 . Two-weeks-old in vitro, plants that have developed roots in liquid MS medium were transferred into plastic pots of 10 cm diameter. These pots contained a mix (v/v) of soil and fertilizer (Potgrond H: Klasmann Deilmann, Geeste, Germany) and were put in a greenhouse (Temperature between 14 to 25°C). The plants were irrigated with tap water every 2 days. After 75 days, the minitubers were harvested and stored at 4°C in the dark for further analysis and they were used as seeds for subsequent eld culture. At the end of dormancy period, minitubers were transferred into a dark chamber at 25°C. One month later, minitubers that have developed sprouts were planted in the eld (from December 2018 to February 2019) in Sfax, Tunisia. The eld preparation involved soil fertilization using a local fertilizer (Biocompost Elbosten), row preparation, seed minitubers spacing, and weed control were applied. The average temperature and humidity of eld culture were around 13°C and 59% respectively (timeanddate.com). During the culture period, the plants were irrigated with tap water once a week. Tuber harvesting was performed after 75 days of eld culture.

Sampling and analysis of growth parameters
During the culture periods in both greenhouse and eld, measurements of growth parameters were conducted in triplicates every 10 days. Plant height was measured using a steel ruler, the diameter of the stem was assessed 2 cm above the ground, and the number of compound leaves was noted for both WT and the VvWRKY2 overexpressing plants. Leaf area and stomatal conductance of three upper leaves from the WT and the transgenic plants were measured after 30 and 60 days of greenhouse and eld cultures using the methods of Pandey and Singh (2011) and a promoter from Decagon Devices Model SC-1 respectively.

Determination of chlorophyll content
The leaf chlorophyll content was determined according to the method of Arnon (1949). Fresh leaves (0.01g) were ground in a mortar with 500 μl of pure acetone and 1 ml of 80% acetone. The supernatant containing the chlorophyll pigments was obtained after centrifugation at 12000 rpm for 15 min and was adjusted to 2 ml with 80% acetone. The optical density was then measured at 645nm and 663nm. The chlorophyll (a) and (b) contents were calculated using the following formulae: The total chlorophyll= Chl (a) + Chl (b) Where V: volume of sample and Fw: Fresh weight of leaves 2.6 Determination of tuber's yield, caliber, eyes number and color After 75 days of greenhouse and eld culture, the WT and transgenic potato tubers were harvested, counted and weighed to determine their average number and weight in the different culture conditions. Calibers and number of eyes per tuber were also determined. The skin color of tubers was measured using a Chroma Meter CR-400/410 colorimeter (Konica Minolta). By scanning the potato skin at ve locations, three orthogonal coordinates de ne these parameters: the clarity index L*, the chromatic coordinates a* (red) and b* (yellow).

Determination of the tuber's chemical composition
The percentage of dry matter was determined after drying three fresh samples at 105°C for 48 hours until constant weight (AFNOR 1982) and calculated using the following formulae: Dry matter = (DW/FW) *100, where FW is the fresh weight and DW is the dry weight. The ash content (%) was determined by incineration of the dry matter in a mu e furnace for 4 hours at 550°C (AFNOR, 1982). The determination of the reducing sugar content was carried out according to the method of Miller (1959). The reducing sugars of the sample are recovered in the supernatant and then assayed by dinitro-salicylic acid (DNS).
The optical density is then determined at 550 nm and the content of reducing sugars is determined based on a standard curve of glucose. The lipid content of potato tubers was analyzed according to Rogan et al. (2000). The starch content was determined using the enzymatic colorimetric method described by

Statistical Analysis
All the experiments were performed using three independent biological replicates and the data were presented on average ± the standard deviation (SD). Results were subjected to the ANOVA test and compared by the Newman-Keuls test using the statistical software GraphPad Prism 8 (2019 version). Comparisons with P <0.05 were considered signi cantly different.

Results
Transgenic potato plants overexpressing the VvWRKY2 TF were produced. To investigate whether the VvWRKY2 overexpression could have an effect on the agricultural performance of transgenic plants we performed a greenhouse and eld culture of potato plants overexpressing the VvWRKY2 TF.

Production of transgenic potato line overexpressing the VvWRKY2 TF
A recombinant A. tumefaciens strains harboring the VvWRKY2 cDNA inserted in the MDC32 vector ), was used for potato leaf and stem transformation. The VvWRKY2 cDNA is anked by the Cauli ower Mosaic virus (CaMV) 35S RNA promoter and terminator. Transgenic potato plant regeneration was performed in hygromycin supplemented medium, and rooted plantlets were multiplied by in vitro node culture. The presence of the VvWRKY2 transgene was veri ed by PCR analysis on total plant DNA using the CaMV35S promoter speci c primers leading to the ampli cation of a 300 bp fragment (Fig. 1a). It was further con rmed by the ampli cation of a 1.6 kbp DNA fragment of the VvWRKY2 cDNA (Fig. 1b). VvWRKY2 expression was assessed by RT-PCR using the total RNA extracted from the VvWRKY2 plant leaves (Fig. 1c).

VvWRKY2 overexpression in potato promotes plant growth parameters
One transgenic potato line overexpressing the VvWRKY2 TF was used in this study. The effect of the overexpression of VvWRKY2 on potato plant growth was assessed both in the greenhouse and eld culture conditions. The plant height and compound leaves number measured during the culture period didn't show any signi cant difference between the VvWRKY2 transgenic plants and WT in both culture conditions (Fig. 2). However, the VvWRKY2 transgenic plants exhibited higher stem diameter and leaf area in comparison to WT plants (Fig. 3). The VvWRKY2 overexpressing plants exhibited a distinct foliar shape and displayed longer leaf petioles compared to the WT leaves both in greenhouse and eld culture conditions (Fig. 4). Moreover, the VvWRKY2 transgenic potato plants displayed signi cantly higher stomatal conductance than the WT in both culture conditions (Fig. 5a).

The VvWRKY2 overexpression in potato did not change the leaves chlorophyll content
The evaluation of the chlorophyll content of the WT and VvWRKY2 overexpressing potato plants did not show any signi cant difference (Fig. 5b).

The VvWRKY2 overexpression in potato increased tubers yield and caliber and modi ed tubers' color
The potato plant yields were determined to evaluate the tuber number and weight per plant (Table1). Accordingly, we found that the VvWRKY2 transgenic plants cultivated in the greenhouse produced a higher number of tubers than WT. In contrast, under eld conditions WT plants produced more tubers than VvWRKY2 transgenic plants. The tuber weight per plant was signi cantly higher for VvWRKY2 transgenic plants than WT in both culture conditions. Similarly, the tubers of VvWRKY2 transgenic plants from greenhouse and eld cultures showed larger caliber (based on tuber length) and higher number of eyes than the WT (Table1). Interestingly, a clear difference of the skin color was noticed between the VvWRKY2 and WT tubers (Fig. 6). The tubers from the VvWRKY2 transgenic plants showed lighter yellow color index and higher clarity than WT.

The VvWRKY2 overexpression in potato improved the nutritional qualities of tubers
Our results showed that the tubers of the VvWRKY2 transgenic plants showed higher dry matter, ash, starch and mineral content such as potassium, magnesium, and calcium in comparison to WT. In contrast, higher levels of lipids and reducing sugars were measured in the WT in comparison to the VvWRKY2 overexpressing tubers (Table2).

Discussion
Potato is an important food crop in the world, occupying the fourth rank after wheat, rice, and corn (Zhu et al. 2020). However, because of its high sensitivity, biotic and abiotic constraints limit the productivity of this plant ( (Fig. 2b). The VvWRKY2 transgenic plants exhibited higher stem diameter and leaf area in comparison to WT. These results corroborate those of Freitas-Lima et al. (2017) who reported that transgenic plants overexpressing WRKY TF exhibited increased biomass. These data suggest that VvWRKY2 TFs are involved in the plant growth and biomass production. These data con rm the fact that VvWRKY2 overexpressing plants seem to be more vigorous than the WT in greenhouse (Fig.  4a) and eld cultures (Fig. 4b). The VvWRKY2 overexpressing plants showed a distinct foliar shape in comparison to the WT leaves. Indeed, the transgenic plants displayed longer leaf petioles in regards to the WT leaves either in the greenhouse or in the eld. Similarly, Tian et al. (2017) showed that the overexpression of OsWRKY53 led to an enlarged angle and modi ed architecture of rice leaves. These authors suggest that this leaf enlargement seems to be linked to the involvement of OsWRKY53 TF in the brassinosteroid signaling. Accordingly, we can hypothesize that the VvWRKY2 TF may be involved in the brassinosteroid signaling pathways in potato plants. The leaf area is one of the leading contenders when plant robustness is considered. This parameter is often positively correlated with leaf net photosynthetic rate (Shipley and Lechowicz 2000). Moreover, the increase in the leaf size is bound to plant relative growth rate (Poorter and Remkes 1990) and productivity (Koester et al. 2014). The VvWRKY2 transgenic plants showed signi cantly higher stomatal conductance than the WT plants, in both greenhouse and in eld culture. This result suggests that VvWRKY2 TF may be involved in the promotion of gas exchange parameters and in the stomatal opening and closure. In the same context, Finatto et al. (2018) suggested that WRKY TFs may be involved in stomata closing in response to biotic and abiotic stresses via the ABA regulation pathway.
Similar chlorophyll contents were measured in the leaves of VvWRKY2 transgenic and WT potato plants. These ndings suggest that the VvWRKY2 TF is not involved in chlorophyll biosynthesis pathway. Our results are in agreement with those of Guo et al. (2019) who reported that no signi cant differences were noticed in chlorophyll content between WRKY transgenic plants and WT under normal conditions. These results suggest that the VvWRKY2 TF are not involved in the regulation of chlorophyll biosynthesis or metabolism under non-stressed conditions.
The key objective of the present work was to evaluate the impact of the overexpression of VvWRKY2 TF on the productivity of potato plants under non-stressed conditions. Our data showed that in the greenhouse, the VvWRKY2 transgenic plants produced a higher number of tubers than the WT, while in the eld, this parameter was higher in WT in regards to transgenic plants. Interestingly, the tuber weight per plant was signi cantly higher in VvWRKY2 transgenic plants than WT both in greenhouse and eld cultures. These data suggest that the VvWRKY2 TF overexpression had a positive effect on tuber development. Similarly, the tuber caliber (based on tuber length) and the number of tuber eyes of the VvWRKY2 transgenic plants was higher in comparison to WT. These data corroborated those of Tian et al. (2017) who reported that transgenic rice overexpressing the OsWRKY53 TF displayed higher grain length and width than WT. Raineri et al.(2015) showed also that the HaWRKY76 overexpression in sun ower increased plant biomass and seed yield. Moreover, our results showed a signi cant impairment of the yellow color of the tuber color between the VvWRKY2 and WT. These data con rm the important role of WRKY TFs in the regulation of secondary metabolites production pathways (Schluttenhofer and Yuan 2015). A number of studies revealed the involvement of WRKY TFs in the regulation of the production of several secondary metabolites such as phenolic compounds, lignin, avanols and tannins (review in Phukan et al. 2016).
In potato processing industries, the most important parameter is tuber quality which includes biological, sensorial, and industrial traits (Kumari et al. 2018a). Hence, the composition of potatoes is a very important parameter de ning their potential use in food ( Gravoueille, 1996;Pedreschi et al. 2012). An analysis of the chemical composition of tubers indicated that the transgenic plants produced tubers with higher dry matter, starch, ash, and minerals but markedly lower lipid and reducing sugar content in comparison to the WT tubers. In fact, low reducing sugar concentration reduces the browning of French fries (Gravoueille, 1996). Tuber dry matter and starch contents are crucial factors for the production of The increase of the dry matter and starch content, the decrease of the reducing sugar content in VvWRKY2 transgenic plants constitute key parameters that in uence the nutritional and industrial properties of potatoes (Kumari et al. 2018b). In fact, the non-enzymatic browning related to high levels of reducing sugars is a serious problem in potato French fries, akes, and potato chips since it leads to the dark color of the end products (Marwaha et al. 2008). These data suggest that VvWRKY2 TF overexpressed in potato plants positively regulated the tuber industrial and nutritional qualities, by increasing dry matter, starch and mineral uptake and lowering the reducing sugar contents.

Conclusions
Potato is a sensitive plant and its productivity is altered by biotic and abiotic stresses. The overexpression of VvWRKY2 TF in potato plants improved the plant vigor, tuber yields, size and the nutritional quality in comparison to the WT. Taken together, our data suggest that VvWRKY2 would be a good candidate not only for the improvement of potato culture under biotic and abiotic stresses, but also a candidate to promote potato's agricultural performance under non-stress conditions.

Declarations
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the reported results of this paper.  Zn 0.0001 a ± 0 0.0001 a ± 0 0 a ± 0 0.004 a ± 0 P 0.013 a ± 0.004 0.013 a ± 0.002 0.008 a ± 0 0.008 a ± 0

Tables
Data represent means ± standard deviation (n=3), all values with superscripts (a-b) are signi cantly different (at P ≤ 0.05, ANOVA test). Statistical differences were evaluated between the WT and VvWRKY2 separately in greenhouse and in eld.     Measurement of the elds' tubers skin color. L* represents: the clarity index, a*: chromatic coordinates of red color, and b*: chromatic coordinates of yellow color. All Data are represented by means of three independent repeats ± standard deviation. Values with (*) and (**) indicate signi cant differences at P < 0.05 and P < 0.01 (ANOVA test), respectively.