EFFECT OF DROUGHT ON AQUAPORIN EXPRESSION IN GRAFTED AND UNGRAFTED GRAPEVINE CULTIVARS

Drought stress severely


INTRODUCTION
The Mediterranean region, including Balkans, with a total of 4.2 million hectares meet over 50% of the world's wine grapes and one-third of all table and raisin grapes (Capone et al., 2014). As for other annual or perennial plant species, grapevine cultivars are continuously under pressure of environmental fluctuations. The effects of the everchanging environmental conditions are translated into the impaired physiological and biochemical attributes of the plants, which in turn cause critical reductions in productivity of grape (Gambetta et. al., 2020). The reports anticipate that the incidence of unexpected climatic scenarios such as excessive precipitation or dry seasons might increase in certain regions (IPCC, 2007). Being an iconic plant species such as the olive tree, the vine and related activities are intensely developed in the Mediterranean Basin. Researchers hypothesized that crop yield of grapes will critically decrease due to environmental fluctuations (Iglesias et al., 2007;Brás et al., 2021;Dinis et al., 2022).
Among the stress factors, drought is one of the most devastating abiotic factors on grapevines. In order to cope with the relevant stress, grapevines, as other sessile plants, developed remarkable strategies at physiological, biochemical and molecular levels (Serra et al., 2014). For instance, grapevines effectively use hydraulic conductivity to mitigate the adverse impact of drought stress. Hydraulic conductivity is very critical in early stomatal closure to preserve foliar water, avoiding xylem vessel cavitation and embolism damage, and enhancing water uptake (Lovisolo and Schubert, 2006). Regarding to the regulation of hydraulic conductivity, aquaporins, called water channel proteins, play crucial roles in ensuring the continuous water transport from roots to leaves, controlling the permeability of membranes to water, and altering cellular hydraulic conductivity (Lovisolo and Schubert, 2006;Hayes et al., 2007;Surbanovski and Grand, 2014). Aquaporins belong to the major intrinsic protein (MIP) family, having five sub-groups in grapevines based on their nucleotide sequence similarity and cellular location. Of the aquaporins available, plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) are the most abundant in plant organs. Their locations in the cell suggest that they mainly regulate water transport (Chrispeels and Maurel, 1994;Johanson et al., 2001). PIPs are mostly located in the plasma membrane whereas TIPs occur in the tonoplast (Kapilan et al.,2018). PIPs have higher nucleotide and amino acid similarity than TIPs. The PIP aquaporins are divided into PIP1 and PIP2 subgroups whilst the TIP aquaporins are divided into five different subfamilies (Shelden et al., 2009). Aquaporins control water movement and might be downregulated or upregulated depending on their location and the severity of abiotic stress on the plant (Jang et al., 2013). In this regard, the former reports clearly showed that changes in the aquaporin isoform expression levels differ among grapevine varieties, plant organ types and locations, environmental stress duration and severity, and circadian rhythm (Kaldenhoff et al., 2006;Heinen et al., 2009;Leitão et al., 2012;Pou et al., 2013;Turgay, 2015;Shelden et al., 2017;Abdelhakam et al., 2021). Transpiration removes water from the leaf, resulting high tension. Xylem embolism is avoided by moving water from adjacent living cells to the xylem vessels via aquaporins (Daniela et al., 2021). PIP aquaporins are membrane proteins; hence, they play important roles in cell water exchange, regulating the intracellular traffic by modulating aquaporin gene transcription. PIP1 and PIP2 aquaporins are expressed independently of each other. However, PIP2 aquaporins play active roles in water flow whereas PIP1 aquaporins increase the efficiency of PIP2 aquaporins (Gautam and Pandey, 2021). TIPs regulate cell turgor by moving water to and from the vacuoles across the tonoplasts. TIP aquaporins are abundant along the tonoplast, control water flow between the vacuole and the cytoplasm, regulate the permeability of urea, hydrogen peroxide, and glycerol, and provide osmoregulation (Li et al., 2014). Some TIP aquaporins may be organ-specific. TIP3;1 and TIP3;2 are expressed in seeds whereas TIP5;1 is found in pollen mitochondria and remobilises nitrogen (Mandlik et al., 2022).
The scion × rootstock × environment interaction must be considered to elucidate the mechanisms by which grapevines contend with drought stress. American rootstocks have high tolerance to abiotic stress factors such as drought and biotic stress factors like phylloxera. Hence, the selection of a rootstock suitable for the local soil and climatic conditions and the scion variety is vital in sustainable viticulture development (Serra et al., 2014). Rootstocks might play important roles in drought tolerance by controlling water uptake from the soil and regulating transpiration (Soar et al., 2006;Tramontini et al., 2013). Several studies have been conducted on drought stress in grapevine (Chaves et al., 2010). Nevertheless, few investigations have been performed on rootstock × scion interactions. Hence, under a given terroir condition, it is vital to select a suitable combination of variety and rootstock for a sustainable development of viticulture. For this reason, it was hypothesised that grafting would help to ensure a wide range of tolerance against drought stress. In order to test the hypothesis, a series of aquaporin associated genes (VvPIP2;1, VvPIP2;2, VvTIP1;1, and VvTIP2;1), RWC and proline content of two grapevine cultivars contrasting drought tolerance was estimated.

Plant materials and experimental conditions
The experiment was conducted from February to August 2019 in a semi-controlled greenhouse at Kilis 7 Aralık University (Kilis, Turkey). Droughttolerant ('Horozkarası') and drought-sensitive ('Kabarcık') Vitis vinifera L. cultivars and 'Rupestris du Lot' rootstock were used. The experiment was carried out with four replicates, and each replicate corresponded to five plants. Both ungrafted and grafted plants were cultivated in pots filled with 1:2:1 (w/w/w) peat:soil:perlite and wellwatered at field capacity for six months. Their lateral branches were trained to create and maintain a homogeneous morphology. The plants were then exposed to drought stress by withholding water. The plastic pots containing the soil mixture were monitored with a soil tensiometer and the drought stress phase was terminated by day 7. The control plants were irrigated at field capacity throughout the experiment. After a 7-day period, fully expanded leaves were collected. The experimental design (cultivar*rootstock*drought stress, 2 3 =8) is presented in Table I.

Relative water content (RWC)
To ensure homogeneous sample collection, the leaves were cut 1,5 cm d ameter with a hole punch, and the fresh weight (FW) of the leaf discs was immediately determined. The leaf discs were then stored in double-distilled water for 4 h. Then excess surface water was blotted with paper towels, the turgid weights (TW) of the leaf discs were measured, the leaf discs were then dried at 60 °C for 24 h, and their dry weights (DW) were measured (Dhanda and Sethi, 1998). RWC was calculated according to Equation 1.
ANOVA was carried out to identify significant differences, and Tukey HSD was used as post hoc test among treatments at each sampling time.  RWC decreased in both cultivars in response to drought stress. However, the reduction in RWC was more severe in the sensitive cultivar 'Kabarcık' than the tolerant cultivar 'Horozkarası'. The lower RWC reduction was observed in KGS, whereas the higher RWC decline was recorded in KUS. However, the effect of drought stress (DS) was significant among treatments (Table III).

DS caused significant increase in proline content.
Considering the experimental groups, the highest content of proline was observed in KUS, whilst the lowest content was recorded in HUC. Corresponding to DS, the wider range of changes in proline content was observed between KGC and KGS. The rootstock contributed to the proline content in both cultivars and also the changes in proline content in response to cultivar (C), grafting (G), drought stress (DS), and the G × DS were significant (Table III).

Changes in aquaporin gene expression in grafted and ungrafted plants under drought stress
Corresponding to the expression levels of aquaporin genes, foliar PIP2;1, PIP2;2, and TIP2;1 aquaporins were down-regulated whilst foliar TIP1;1 aquaporin was up-regulated. Independent from tolerance levels, both cultivars exhibited similar responses with respect to the expression levels (Table IV).
PIP2;1 aquaporin exchange significantly decreased in response to drought stress. The highest relative gene expression of PIP2;1 was estimated in KGC, whereas the lowest expression level was quantified in KUS. Of the treatments, DS significantly affected expression level of PIP2;1. However, other factors were not statistically significant.
Regarding PIP2;2, the highest and lowest relative expression levels were estimated in KUC and KGS, respectively. Variance analysis revealed that C × G × DS interaction was significant for PIP2;2. Concerning TIP1;1, the highest and lowest expression levels were recorded in KUS and KGC, respectively. The G × DS and C × G × DS interactions were not significant for TIP1;1 aquaporin. However, all other interactions were significant at various levels. The TIP2;1 aquaporin gene was downregulated in response to foliar drought stress. The highest expression level of TIP2;1 was noted in HUC and the lowest expression level was recorded in KUS. Variance analysis showed that the C × DS and C × G × DS interactions were not significant whereas the other interactions were significant (Table IV). Drought is one of the damaging stressors that cause critical problems and deserves further investigation to reveal the mechanism of action on plants. After doing a basic search on SCOPUS with criteria inclusion "drought OR water stress" on January 11, 2023, about 264.397 documents were recorded. In spite of the high number of documents, the action mechanism of drought is not fully-elucidated due to the critical changes in severity of stress and frequency, being still very destructive on crop and non-crop species. Physiological and molecular basis responses of plants should be addressed and understood in order to cope with the stress and to propose solutions. In such analysis, different cultivars contrasting in tolerance are relatively significant to understand the response. For this reason, the present study was performed. Of the assayed parameters, RWC is closely related to leaf water potential, and simply and effectively determines the degree of drought stress tolerance in plants (Chaves, 1991). The cell structure collapses in response to a decrease in intracellular turgor, resulting in retarded physiological activity and loss of cell integrity. As the total cellular relative humidity declines to 75%, ATP and protein production are prevented (Lawlor and Cornic, 2002). As expected, significant decline in RWC of both cultivars was observed. The low rate of reduction in RWC was showed in drought-tolerant (Horozkarası) and both grafted cultivars. Buffering the decline in water status in tissues of the plants are critical in coping with the stress conditions. Of the devoted strategies, plants highly accumulate osmolytes to maintain cell turgor in droughtsuffering plants (Serraj and Sinclair, 2002). Among the stress indicators, proline is of the universal osmolytes estimated in stress-submitted plants, being a low molecular weight amino acid that is not cytotoxic (Ashraf and Fooland, 2007). Several studies clearly revealed that grapevine suffering from stress enhanced proline content (Şahin, 2009;Özden et al., 2009;Abdi et al., 2016). In the present work, critical increases in proline content were observed in drought-sensitive cultivar 'Kabarcık' grafted on 'Rupestris rootstock'.
Effective control of the water transport system can increase drought tolerance in grapevines. Expression levels of VvPIP2;1 and VvPIP2;2 were down-regulated in stress-submitted plants.
VvPIP2;2 aquaporin was more responsive in relation to VvPIP2;1 against stress and grafting. A study on 'Chardonnay' cultivar demonstrated a positive relationship between PIP2;1 and leaf hydraulic conductivity (Pou et al., 2013). Decrease in leaf water potential in response to drought stress was reported for 'Touriga Nacional' cultivar (Zorrouk et al., 2016). Concerning two cultivars ('Syrah' and 'Grenache') contrasting to drought stress, the expression levels of VvPIP2;1 did not change in 'Syrah' but decreased in isohydric 'Grenache' . Galmes et al. (2007) revealed an increase at the earlier developmental period of grapevine. The relevant increase was ascribed to the stomatal closure (Zorrouk et al., 2016). The link between abscisic acid (ABA) level and PIP aquaporin in stomatal closure was well-known (Grondin et al., 2015). Significant alteration in pH corresponding to the stomatal closure might trigger critical changes in gene expression levels of PIP aquaporin (Shelden et al., 2009). Orchestrated responses at physiological and physiological might leaf water integrity at the later stage of the stress.
TIPs play key roles in maintaining cell turgor pressure through osmotic adjustment. TIP1;1 maintains the osmotic balance of the cell in grapevine under arid conditions. In the present study, significant increases in TIP1;1 in both cultivars under drought stress were observed. VvTIP1;1 aquaporin increased in Vitis spp. leaves (Shelden et al., 2009;Pou et al., 2013;Zarrouk et al., 2015). For this reason, VvTIP1;1 may be considered as a significant stress indicator for grapevine. Changes in another TIP2;1 aquaporin isoform was also investigated. VvTIP2;1 was strongly downregulated in response to drought stress in 'Horozkarası' and 'Kabarcık' cultivars. TIP 2;1 might be expressed at higher levels in the roots and root tips than the leaves (Nguyen et al., 2013;Baiges et al., 2001). VvTIP1;1 might increase the permeability of aquaporins to small-molecule solvents. VvTIP2;1 may regulate water exchange in the vacuole.
Changes in aquaporin gene expression may be important criteria in the selection of grapevine cultivars tolerant to drought stress (Joshi et al., 2016). Regarding nucleotides and amino acids, VvTIP1;1 and VvTIP2;1 exhibited ~72% similarity. However, the similarity among PIP aquaporin genes may be as high as 99% (Shelden et al., 2009). The efficiency of PIP aquaporins can be increased by upregulation of duplicate genes (Chaumont et al., 2001). This approach may effectively identify different results in expression studies. Hence, the aquaporin family should be investigated from a more in-depth and broad perspective in platforms such as RNA sequencing.
Several theories have been postulated to explain the relationships between the scion and the rootstock of grapevine (Tsegay et al., 2014). Though there have been numerous studies on drought stress in grapevine, very few have been conducted on the responses of rootstocks and scions to drought stress. The present study showed the rootstock effects on the PIP2;2, TIP1;1, TIP2;1 aquaporin levels in the scion, and this effect varied with scion vigour. Rootstocks can affect the scion through phytohormones and signalling pathways and meet the water demand of the scion (Zhang et al., 2016). A previous study demonstrated that the genes regulating ABA biosynthesis in the roots of various graft combinations of 'Cabernet Savignon' cultivar were expressed at higher levels in the roots than in the leaves. Nevertheless, more ABA accumulated in the leaves than the roots (Prinsi et al., 2021). Rootstocks do not modulate the impact of drought stress resistance on physiological parameters such as fruit yield but transcriptionally modify secondary metabolism and reduce the severity of drought stress in the grape (Zombardo et al., 2020). Rootstocks might cause partial modulations in physiological and biochemical attributes of the scions. The phytohormones and other substances produced by the roots are translocated to the scion and protect it against drought and other abiotic stress.
The negative effects of drought on grape production are increasing, particularly in the Mediterranean region, in which viticulture is intense. For this reason, the selection of the appropriate cultivars/rootstocks is vital. 'Horozkarası' and 'Kabarcık' grape cultivars with different drought tolerances showed similar trends in the expression levels of their various foliar aquaporin isoforms, albeit at different levels of significance. Although rootstocks are effective at critical modulations of the scion, biochemical modulations are used more intensively depending on the vigour of the scion. Hence, it is crucial to consider the rootstock × scion × environment interaction in future endeavours to elucidate the drought stress response mechanism. In this way, more reasonable variety-rootstock combinations while maintain the viticultural sustainability in response to ongoing climate warming should be explored.

ACKNOWLEDGMENTS
This manuscript is derived from the doctoral dissertation of Mehmet Koc.